Evaluation of avian paramyxovirus serotypes 2 to 10 as vaccine vectors in chickens previously immunized against Newcastle disease virus.

PubMed

Tsunekuni, Ryota; Hikono, Hirokazu; Saito, Takehiko

2014-08-15

Newcastle disease virus (NDV), also known as avian paramyxovirus (APMV) serotype 1, is used as a vaccine vector to express the hemagglutinin protein of avian influenza (AI) virus. However, use of live NDV recombinant vaccines expressing AI virus hemagglutinin is not desirable in emergency vaccination programs to control severe AI outbreaks in chickens, because commercial chickens often possess pre-existing NDV immunity induced by routine vaccination. Therefore, a novel vaccine vector is required for emergency vaccination of chickens to control AI during outbreaks. We investigated whether candidate APMV strains could be used as vaccine vectors that could evade the pre-existing immunity acquired by chickens through NDV vaccination and that would replicate in the mucosal tissues where AI virus primarily replicates. To this end, we examined strains of APMV serotypes 2 to 10 for their immunogenicity and replication in chickens with pre-existing immunity to NDV. APMV serotypes 2, 6, and 10 were the least cross-reactive to antibodies to NDV in hemagglutination inhibition and/or virus neutralization tests. Virus replication in mucosal tissues, as well as antibody response after oculonasal inoculation, was observed when 7-week-old chickens were challenged with APMV of serotype 2, 6, or 10. The APMV also replicated in mucosal tissues and induced antibody responses in chickens that had been vaccinated twice with NDV before challenge. These results warrant further study to develop vaccine vectors based on APMV serotype 2, 6, or 10 for emergency vaccination of chickens against AI. Copyright © 2014 Elsevier B.V. All rights reserved.

Lytic Replication of Epstein-Barr Virus During Space Flight

NASA Technical Reports Server (NTRS)

Stowe, R. P.; Pierson, D. L.; Barrett, A. D. T.

1999-01-01

Reactivation of latent Epstein-Barr virus (EBV) may be an important threat to crew health during extended space missions. Cellular immunity, which is decreased during and after space flight, is responsible for controlling EBV replication in vivo. In this study, we investigated the effects of short-term space flight on latent EBV reactivation.

Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

PubMed

Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

PubMed Central

Tran, Anh T.; Cortens, John P.; Du, Qiujiang; Wilkins, John A.

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication. PMID:23135712

Noumeavirus replication relies on a transient remote control of the host nucleus

PubMed Central

Fabre, Elisabeth; Jeudy, Sandra; Santini, Sébastien; Legendre, Matthieu; Trauchessec, Mathieu; Couté, Yohann; Claverie, Jean-Michel; Abergel, Chantal

2017-01-01

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses. PMID:28429720

The roles of membranes and associated cytoskeleton in plant virus replication and cell-to-cell movement.

PubMed

Pitzalis, Nicolas; Heinlein, Manfred

2017-12-18

The infection of plants by viruses depends on cellular mechanisms that support the replication of the viral genomes, and the cell-to-cell and systemic movement of the virus via plasmodesmata (PD) and the connected phloem. While the propagation of some viruses requires the conventional endoplasmic reticulum (ER)-Golgi pathway, others replicate and spread between cells in association with the ER and are independent of this pathway. Using selected viruses as examples, this review re-examines the involvement of membranes and the cytoskeleton during virus infection and proposes potential roles of class VIII myosins and membrane-tethering proteins in controlling viral functions at specific ER subdomains, such as cortical microtubule-associated ER sites, ER-plasma membrane contact sites, and PD. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.

PubMed

Workenhe, Samuel T; Simmons, Graydon; Pol, Jonathan G; Lichty, Brian D; Halford, William P; Mossman, Karen L

2014-01-01

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.

Plant Virus-Insect Vector Interactions: Current and Potential Future Research Directions.

PubMed

Dietzgen, Ralf G; Mann, Krin S; Johnson, Karyn N

2016-11-09

Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus-insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.

Cancer Terminator Viruses and Approaches for Enhancing Therapeutic Outcomes

PubMed Central

Das, Swadesh K.; Sarkar, Siddik; Dash, Rupesh; Dent, Paul; Wang, Xiang-Yang; Sarkar, Devanand; Fisher, Paul B.

2015-01-01

No single or combinatorial therapeutic approach has proven effective in decreasing morbidity or engendering a cure of metastatic cancer. In principle, conditionally replication-competent adenoviruses that induce tumor oncolysis through cancer-specific replication hold promise for cancer therapy. However, a single-agent approach may not be adequate to completely eradicate cancer in a patient because most cancers arise from abnormalities in multiple genetic and signal transduction pathways and targeting disseminated metastases is difficult to achieve. Based on these considerations, a novel class of cancer destroying adenoviruses have been produced, cancer terminator viruses (CTVs), in which cancer-specific replication is controlled by the progression-elevated gene-3 promoter and replicating viruses produce a second transgene encoding an apoptosis-inducing and immunomodulatory cytokine, either melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) or interferon-γ. This review focuses on these viruses and ways to improve their delivery systemically and enhance their therapeutic efficacy. PMID:23021240

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

PubMed

Yang, Liping; Wang, Rong; Yang, Shixing; Ma, Zexu; Lin, Shaoli; Nan, Yuchen; Li, Qisheng; Tang, Qiyi; Zhang, Yan-Jin

2018-05-01

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV. IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics. Copyright © 2018 American Society for Microbiology.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

PubMed

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome.

PubMed

Tsukamoto, Tetsuo; Yamamoto, Hiroyuki; Okada, Seiji; Matano, Tetsuro

2016-09-01

Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4(+) T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4(+) T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dampened antiviral immunity to intravaginal exposure to RNA viral pathogens allows enhanced viral replication

PubMed Central

Woodruff, Erik M.; Trapecar, Martin; Fontaine, Krystal A.; Ezaki, Ashley; Ott, Melanie

2016-01-01

Understanding the host immune response to vaginal exposure to RNA viruses is required to combat sexual transmission of this class of pathogens. In this study, using lymphocytic choriomeningitis virus (LCMV) and Zika virus (ZIKV) in wild-type mice, we show that these viruses replicate in the vaginal mucosa with minimal induction of antiviral interferon and inflammatory response, causing dampened innate-mediated control of viral replication and a failure to mature local antigen-presenting cells (APCs). Enhancement of innate-mediated inflammation in the vaginal mucosa rescues this phenotype and completely inhibits ZIKV replication. To gain a better understanding of how this dampened innate immune activation in the lower female reproductive tract may also affect adaptive immunity, we modeled CD8 T cell responses using vaginal LCMV infection. We show that the lack of APC maturation in the vaginal mucosa leads to a delay in CD8 T cell activation in the draining lymph node and hinders the timely appearance of effector CD8 T cells in vaginal mucosa, thus further delaying viral control in this tissue. Our study demonstrates that vaginal tissue is exceptionally vulnerable to infection by RNA viruses and provides a conceptual framework for the male to female sexual transmission observed during ZIKV infection. PMID:27852793

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

PubMed

Hatta, Yasuko; Hershberger, Karen; Shinya, Kyoko; Proll, Sean C; Dubielzig, Richard R; Hatta, Masato; Katze, Michael G; Kawaoka, Yoshihiro; Suresh, M

2010-10-07

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression

PubMed Central

Jacobs, Evan S.; Abdel-Mohsen, Mohamed; Gibb, Stuart L.; Heitman, John W.; Inglis, Heather C.; Martin, Jeffrey N.; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C.; Young, Mary; Greenblatt, Ruth M.; Landay, Alan L.; Gange, Stephen J.; Deeks, Steven G.; Golub, Elizabeth T.; Pillai, Satish K.

2017-01-01

ABSTRACT A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. PMID:28053103

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

PubMed

Jacobs, Evan S; Keating, Sheila M; Abdel-Mohsen, Mohamed; Gibb, Stuart L; Heitman, John W; Inglis, Heather C; Martin, Jeffrey N; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C; Young, Mary; Greenblatt, Ruth M; Landay, Alan L; Gange, Stephen J; Deeks, Steven G; Golub, Elizabeth T; Pillai, Satish K; Norris, Philip J

2017-03-15

A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4 + T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4 + T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4 + T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4 + T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. Copyright © 2017 American Society for Microbiology.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome

PubMed Central

Hartley, Carol A.; Vaz, Paola K.; Diaz-Méndez, Andrés; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M.

2017-01-01

ABSTRACT Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. PMID:28939604

Plant Virus–Insect Vector Interactions: Current and Potential Future Research Directions

PubMed Central

Dietzgen, Ralf G.; Mann, Krin S.; Johnson, Karyn N.

2016-01-01

Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors. PMID:27834855

T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

PubMed Central

Plaisted, Warren C.; Weinger, Jason G.; Walsh, Craig M.; Lane, Thomas E.

2014-01-01

Neural precursor cells (NPCs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of NPCs remain unclear. This study demonstrates that NPCs support replication following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV infection leads to increased cell death and dampens IFN-γ-induced MHC class II expression. Importantly, cytokines secreted by CD4+ T cells inhibit JHMV replication in NPCs, and CD8+ T cells specifically target viral peptide-pulsed NPCs for lysis. Furthermore, treatment with IFN-γ inhibits JHMV replication in a dose-dependent manner. Together, these findings suggest that T cells play a critical role in controlling replication of a neurotropic virus in NPCs, a finding which has important implications when considering immune modulation for NPC-based therapies for treatment of human neurologic diseases. PMID:24418558

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge

PubMed Central

Goatley, Lynnette C.; Jabbar, Tamara; Sanchez-Cordon, Pedro J.; Netherton, Christopher L.; Chapman, David A. G.; Dixon, Linda K.

2017-01-01

ABSTRACT Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. PMID:28978700

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

PubMed

Reis, Ana L; Goatley, Lynnette C; Jabbar, Tamara; Sanchez-Cordon, Pedro J; Netherton, Christopher L; Chapman, David A G; Dixon, Linda K

2017-12-15

Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼10 6 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. Copyright © 2017 Reis et al.

Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques▿

PubMed Central

Queen, Suzanne E.; Mears, Brian M.; Kelly, Kathleen M.; Dorsey, Jamie L.; Liao, Zhaohao; Dinoso, Jason B.; Gama, Lucio; Adams, Robert J.; Zink, M. Christine; Clements, Janice E.; Kent, Stephen J.; Mankowski, Joseph L.

2011-01-01

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8+ T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8+ T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4+ T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8+ T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted. PMID:21715484

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana.

PubMed

Mendoza-Figueroa, J S; Kvarnheden, A; Méndez-Lozano, J; Rodríguez-Negrete, E-A; Arreguín-Espinosa de Los Monteros, R; Soriano-García, M

2018-02-01

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

PubMed

Martínez-Solís, María; Jakubowska, Agata K; Herrero, Salvador

2017-10-01

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

PubMed Central

2018-01-01

ABSTRACT A small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitness in vitro, cytopathicity, and infection progression in vivo. Therefore, we isolated full-length env genes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics. PMID:29636433

Depletion of CD4+ T cells abrogates post-peak decline of viremia in SIV-infected rhesus macaques

PubMed Central

Ortiz, Alexandra M.; Klatt, Nichole R.; Li, Bing; Yi, Yanjie; Tabb, Brian; Hao, Xing Pei; Sternberg, Lawrence; Lawson, Benton; Carnathan, Paul M.; Cramer, Elizabeth M.; Engram, Jessica C.; Little, Dawn M.; Ryzhova, Elena; Gonzalez-Scarano, Francisco; Paiardini, Mirko; Ansari, Aftab A.; Ratcliffe, Sarah; Else, James G.; Brenchley, Jason M.; Collman, Ronald G.; Estes, Jacob D.; Derdeyn, Cynthia A.; Silvestri, Guido

2011-01-01

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte–depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell– and B cell–mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines. PMID:22005304

Depletion of CD4⁺ T cells abrogates post-peak decline of viremia in SIV-infected rhesus macaques.

PubMed

Ortiz, Alexandra M; Klatt, Nichole R; Li, Bing; Yi, Yanjie; Tabb, Brian; Hao, Xing Pei; Sternberg, Lawrence; Lawson, Benton; Carnathan, Paul M; Cramer, Elizabeth M; Engram, Jessica C; Little, Dawn M; Ryzhova, Elena; Gonzalez-Scarano, Francisco; Paiardini, Mirko; Ansari, Aftab A; Ratcliffe, Sarah; Else, James G; Brenchley, Jason M; Collman, Ronald G; Estes, Jacob D; Derdeyn, Cynthia A; Silvestri, Guido

2011-11-01

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell- and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro.

PubMed

Zhao, Fu-Rong; Xie, Yin-Li; Liu, Ze-Zhong; Shao, Jun-Jun; Li, Shi-Fang; Zhang, Yong-Guang; Chang, Hui-Yun

2017-11-01

Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. FMD vaccine is the traditional way to protect against the disease, which can greatly reduce its occurrence. However, the use of FMD vaccines to protect early infection is limited. Therefore, the alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. As previously reported, LiCl has obviously inhibition effects on a variety of viruses such as transmissible gastroenteritis virus (TGEV), infectious bronchitis coronavirus (IBV), and pseudorabies herpesvirus and EV-A71 virus. In this study, our findings were the first to demonstrate that LiCl inhibition of the FMDV replication. In this study, BHK-21 cell was dose-dependent with LiCl at various stages of FMDV. Virus titration assay was calculated by the 50% tissue culture infected dose (TCID 50 ) with the Reed and Muench method. The cytotoxicity assay of LiCl was performed by the CCK8 kit. The expression level of viral mRNA was measured by RT-qPCR. The results revealed LiCl can inhibit FMDV replication, but it cannot affect FMDV attachment stage and entry stage in the course of FMDV life cycle. Further studies confirmed that the LiCl affect the replication stage of FMDV, especially the early stages of FMDV replication. So LiCl has potential as an effective anti-FMDV drug. Therefore, LiCl may be an effective drug for the control of FMDV. Based on that, the mechanism of the antiviral effect of LiCl on FMDV infection is need to in-depth research in vivo. © 2017 Wiley Periodicals, Inc.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

PubMed

Reynolds, Sara E; Earl, Patricia L; Minai, Mahnaz; Moore, Ian; Moss, Bernard

2017-01-15

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells. Published by Elsevier Inc.

Chapter One---Cancer terminator viruses and approaches for enhancing therapeutic outcomes.

PubMed

Das, Swadesh K; Sarkar, Siddik; Dash, Rupesh; Dent, Paul; Wang, Xiang-Yang; Sarkar, Devanand; Fisher, Paul B

2012-01-01

No single or combinatorial therapeutic approach has proven effective in decreasing morbidity or engendering a cure of metastatic cancer. In principle, conditionally replication-competent adenoviruses that induce tumor oncolysis through cancer-specific replication hold promise for cancer therapy. However, a single-agent approach may not be adequate to completely eradicate cancer in a patient because most cancers arise from abnormalities in multiple genetic and signal transduction pathways and targeting disseminated metastases is difficult to achieve. Based on these considerations, a novel class of cancer destroying adenoviruses have been produced, cancer terminator viruses (CTVs), in which cancer-specific replication is controlled by the progression-elevated gene-3 promoter and replicating viruses produce a second transgene encoding an apoptosis-inducing and immunomodulatory cytokine, either melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) or interferon-γ. This review focuses on these viruses and ways to improve their delivery systemically and enhance their therapeutic efficacy. Copyright © 2012 Elsevier Inc. All rights reserved.

Mathematical models of immune effector responses to viral infections: Virus control versus the development of pathology

NASA Astrophysics Data System (ADS)

Wodarz, Dominik

2005-12-01

This article reviews mathematical models which have investigated the importance of lytic and non-lytic immune responses for the control of viral infections. Lytic immune responses fight the virus by killing infected cells, while non-lytic immune responses fight the virus by inhibiting viral replication while leaving the infected cell alive. The models suggest which types or combinations of immune responses are required to resolve infections which vary in their characteristics, such as the rate of viral replication and the rate of virus-induced target cell death. This framework is then applied to persistent infections and viral evolution. It is investigated how viral evolution and antigenic escape can influence the relative balance of lytic and non-lytic responses over time, and how this might correlate with the transition from an asymptomatic infection to pathology. This is discussed in the specific context of hepatitis C virus infection.

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

Partial protection of SIV-infected rhesus monkeys against superinfection with a heterologous SIV isolate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korber, Bette

2009-01-01

Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virusmore » for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T-cell count, CD4(+) memory T-cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4(+) T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.« less

Function of BRCA1 at a DNA Replication Origin

DTIC Science & Technology

2004-07-01

origin of Epstein-Barr Virus DNA replication (Ori P). OriP replicates once and only once per cell cycle in synchrony with the cellular genome, and is...modifications, and to investigate its function at OriP in DNA replication and plasmid maintenance. We propose that these studies will provide valuable...information concerning the function of OriP at replication origins and in the control of DNA replication initiation and genome stability.

Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro.

PubMed

Medveczky, Maria M; Sherwood, Tracy A; Klein, Thomas W; Friedman, Herman; Medveczky, Peter G

2004-09-15

The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

PubMed

Loncoman, Carlos A; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Diaz-Méndez, Andrés; Browning, Glenn F; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M

2017-12-01

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1 ) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. Copyright © 2017 American Society for Microbiology.

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

PubMed Central

Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L

1988-01-01

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way. Images PMID:2458920

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

PubMed

Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L

1988-06-01

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

Promotion of Hendra Virus Replication by MicroRNA 146a

PubMed Central

Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

2013-01-01

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

Low-Pathogenic Avian Influenza Viruses in Wild House Mice

PubMed Central

Shriner, Susan A.; VanDalen, Kaci K.; Mooers, Nicole L.; Ellis, Jeremy W.; Sullivan, Heather J.; Root, J. Jeffrey; Pelzel, Angela M.; Franklin, Alan B.

2012-01-01

Background Avian influenza viruses are known to productively infect a number of mammal species, several of which are commonly found on or near poultry and gamebird farms. While control of rodent species is often used to limit avian influenza virus transmission within and among outbreak sites, few studies have investigated the potential role of these species in outbreak dynamics. Methodology/Principal Findings We trapped and sampled synanthropic mammals on a gamebird farm in Idaho, USA that had recently experienced a low pathogenic avian influenza outbreak. Six of six house mice (Mus musculus) caught on the outbreak farm were presumptively positive for antibodies to type A influenza. Consequently, we experimentally infected groups of naïve wild-caught house mice with five different low pathogenic avian influenza viruses that included three viruses derived from wild birds and two viruses derived from chickens. Virus replication was efficient in house mice inoculated with viruses derived from wild birds and more moderate for chicken-derived viruses. Mean titers (EID50 equivalents/mL) across all lung samples from seven days of sampling (three mice/day) ranged from 103.89 (H3N6) to 105.06 (H4N6) for the wild bird viruses and 102.08 (H6N2) to 102.85 (H4N8) for the chicken-derived viruses. Interestingly, multiple regression models indicated differential replication between sexes, with significantly (p<0.05) higher concentrations of avian influenza RNA found in females compared with males. Conclusions/Significance Avian influenza viruses replicated efficiently in wild-caught house mice without adaptation, indicating mice may be a risk pathway for movement of avian influenza viruses on poultry and gamebird farms. Differential virus replication between males and females warrants further investigation to determine the generality of this result in avian influenza disease dynamics. PMID:22720076

Lymph Node Cellular and Viral Dynamics in Natural Hosts and Impact for HIV Cure Strategies.

PubMed

Huot, Nicolas; Bosinger, Steven E; Paiardini, Mirko; Reeves, R Keith; Müller-Trutwin, Michaela

2018-01-01

Combined antiretroviral therapies (cARTs) efficiently control HIV replication leading to undetectable viremia and drastic increases in lifespan of people living with HIV. However, cART does not cure HIV infection as virus persists in cellular and anatomical reservoirs, from which the virus generally rebounds soon after cART cessation. One major anatomical reservoir are lymph node (LN) follicles, where HIV persists through replication in follicular helper T cells and is also trapped by follicular dendritic cells. Natural hosts of SIV, such as African green monkeys and sooty mangabeys, generally do not progress to disease although displaying persistently high viremia. Strikingly, these hosts mount a strong control of viral replication in LN follicles shortly after peak viremia that lasts throughout infection. Herein, we discuss the potential interplay between viral control in LNs and the resolution of inflammation, which is characteristic for natural hosts. We furthermore detail the differences that exist between non-pathogenic SIV infection in natural hosts and pathogenic HIV/SIV infection in humans and macaques regarding virus target cells and replication dynamics in LNs. Several mechanisms have been proposed to be implicated in the strong control of viral replication in natural host's LNs, such as NK cell-mediated control, that will be reviewed here, together with lessons and limitations of in vivo cell depletion studies that have been performed in natural hosts. Finally, we discuss the impact that these insights on viral dynamics and host responses in LNs of natural hosts have for the development of strategies toward HIV cure.

DEAD-box RNA helicase DDX3X inhibits DENV replication via regulating type one interferon pathway.

PubMed

Li, Guanghao; Feng, Tingting; Pan, Wen; Shi, Xiaohong; Dai, Jianfeng

2015-01-02

Dengue virus (DENV) is a mosquito-borne virus that threatens approximately 2.5 billion people worldwide. Vaccines against DENV are currently unavailable. DEAD-box RNA helicases (DDXs) have been reported to participate in viral replication and host innate immune response. In the present study, we analyzed the role of 40 DDX proteins during DENV replication. Among these proteins, DDX3X showed antiviral effect against DENV infection. Viral replication significantly increased in DDX3X-silenced cells compared with the controls. The interferon (IFN)-β transcription level decreased during the early stage of DENV infection in DDX3X-silenced cells compared with that in the controls. DDX3X could stimulate IFN-β transcription through the IRF3 and the NFκB branches in DENV-infected cells. Our data imply that DDX3X, a member of DEAD-box RNA helicase, is necessary for IFN production and could inhibit DENV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

PubMed Central

Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

PubMed

Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

2013-09-17

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication.

PubMed

Deepe, Raymond N; Kistner-Griffin, Emily; Martin, Jeffrey N; Deeks, Steven G; Pandey, Janardan P

2012-03-01

Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay.

PubMed

Henderson, C W; Johnson, C L; Lodhi, S A; Bilimoria, S L

2001-01-01

The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10(5)-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control.

Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

NASA Technical Reports Server (NTRS)

Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

2005-01-01

CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cockley, K.D.

1988-01-01

The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2)more » replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of ({sup 3}H)-labeled HSV-1-superinfected cells.« less

Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

PubMed Central

Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong

2013-01-01

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines. PMID:23576512

Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

PubMed

Kuo, Shu-Ming; Chen, Chi-Jene; Chang, Shih-Cheng; Liu, Tzu-Jou; Chen, Yi-Hsiang; Huang, Sheng-Yu; Shih, Shin-Ru

2017-06-13

Avian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2 627 E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2 627 K (human signature) and PB2 627 E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2 627 E than PB2 627 K in transfected human cells. Stronger binding of TUFM to avian-signature PB2 590 G/ 591 Q and PB2 627 E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2 627 E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2 627 E viruses, but not PB2 627 K viruses. In addition, enhanced levels of interaction between TUFM and PB2 627 E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2 627 E virus; however, autophagy remained consistent in PB2 627 K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts. Using a proteomic approach, we revealed a novel role for TUFM as a host restriction factor that exerts an inhibitory effect on avian-signature PB2 627 E influenza virus propagation in human cells. We further found that increased TUFM-dependent autophagy correlates with the inhibitory effect on avian-signature influenza virus replication and may serve as a key intrinsic mechanism to restrict avian influenza virus infection in humans. These findings provide new insight regarding the TUFM mitochondrial protein and may have important implications for the development of novel antiviral strategies. Copyright © 2017 Kuo et al.

Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gabriel, Benjamin; Fiebig, Uwe; Hohn, Oliver

Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequentmore » challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection. - Highlights: • A Nef-deleted RT-SHIV was used as a live attenuated vaccine in macaques. • Vaccine virus replication was shut down to investigate its role in protection. • Ongoing vaccine virus replication did not appear to be necessary for protection. • An analysis of T- and B-cell responses failed to identify a correlate of protection.« less

PA28γ is a novel corepressor of HTLV-1 replication and controls viral latency

PubMed Central

Ko, Nga Ling; Taylor, John M.; Bellon, Marcia; Bai, Xue Tao; Shevtsov, Sergey P.; Dundr, Miroslav

2013-01-01

The establishment of a latent reservoir by human tumor viruses is a vital step in initiating cellular transformation and represents a major shortcoming to current therapeutic strategies and the ability to eradicate virus-infected cells. Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong infection and is linked to adult T-cell leukemia lymphoma (ATLL). Here, we demonstrate that HTLV-1 p30 recruits the cellular proteasome activator PA28γ onto the viral tax/rex mRNA to prevent its nuclear export and suppress virus replication. Interaction of p30 with a PA28γ retaining fully functional proteasome activity is required for p30's ability to repress HTLV-1. Consistently, HTLV-1 molecular clones replicate better and produce more virus particles in PA28γ-deficient cells. These results define a unique and novel role for the cellular factor PA28γ in the control of nuclear RNA trafficking and HTLV-1–induced latency. Importantly, knockdown of PA28γ expression in ATLL cells latently infected with HTLV-1 reactivates expression of viral tax/rex RNA and the Tax protein. Because Tax is the most immunogenic viral antigen and triggers strong CTL responses, our results suggest that PA28γ-targeted therapy may reactivate virus expression from latently infected cells and allow their eradication from the host. PMID:23104922

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lloyd, Richard E., E-mail: rlloyd@bcm.edu

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizesmore » recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.« less

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

PubMed Central

Fuchs, Jonas; Hölzer, Martin; Schilling, Mirjam; Patzina, Corinna; Schoen, Andreas; Zimmer, Gert; Marz, Manja; Müller, Marcel A.

2017-01-01

ABSTRACT Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses. PMID:28490593

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses.

PubMed

Fuchs, Jonas; Hölzer, Martin; Schilling, Mirjam; Patzina, Corinna; Schoen, Andreas; Hoenen, Thomas; Zimmer, Gert; Marz, Manja; Weber, Friedemann; Müller, Marcel A; Kochs, Georg

2017-08-01

Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses. Copyright © 2017 American Society for Microbiology.

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

PubMed Central

Ramos, Hilario J.; Lanteri, Marion C.; Blahnik, Gabriele; Negash, Amina; Suthar, Mehul S.; Brassil, Margaret M.; Sodhi, Khushbu; Treuting, Piper M.; Busch, Michael P.; Norris, Philip J.; Gale, Michael

2012-01-01

West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8+ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons. PMID:23209411

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Role of Occult and Post-acute Phase Replication in Protective Immunity Induced with a Novel Live Attenuated SIV Vaccine

PubMed Central

Ham, Claire; Ferguson, Deborah; Tudor, Hannah; Mattiuzzo, Giada; Klaver, Bep; Page, Mark; Stebbings, Richard; Das, Atze T.; Berkhout, Ben; Almond, Neil; Cranage, Martin P.

2016-01-01

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity. PMID:28002473

Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

PubMed

Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

2011-12-09

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. Published by Elsevier Ltd.

Vaccine Induced Antibodies to the First Variable Loop of Human Immunodeficiency Virus Type 1 gp120, Mediate Antibody-Dependent Virus Inhibition in Macaques

PubMed Central

Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H.; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W.; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C.; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert; Landucci, Gary; Forthal, Donald N.; Franchini, Genoveffa

2011-01-01

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV89.6P replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as two weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by four weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R = -0.83, p 0.015), and ADCVI (R = -0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV89.6P variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV89.6 and virus levels (R = -0.72 p =0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV89.6P challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing FcγR-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. PMID:22037204

Viperin restricts chikungunya virus replication and pathology

PubMed Central

Teng, Terk-Shin; Foo, Suan-Sin; Simamarta, Diane; Lum, Fok-Moon; Teo, Teck-Hui; Lulla, Aleksei; Yeo, Nicholas K.W.; Koh, Esther G.L.; Chow, Angela; Leo, Yee-Sin; Merits, Andres; Chin, Keh-Chuang; Ng, Lisa F.P.

2012-01-01

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses. PMID:23160199

Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control.

PubMed

Simpson, Guy R; Han, Ziqun; Liu, Binlei; Wang, Yibing; Campbell, Gregor; Coffin, Robert S

2006-05-01

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5- to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous.

Inhibition of adenovirus 5 replication in COS-1 cells by antisense RNAs against the viral E1a region.

PubMed

Miroshnichenko, O I; Ponomareva, T I; Tikchonenko, T I

1989-12-07

To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements. The COS-1 cells, which contained up to 100 copies of the engineered plasmids, synthesized antiviral asRNAs, which provided 71 to over 95% inhibition of adenoviral replication, in comparison to the control cells not synthesizing asRNAs.

The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells

PubMed Central

Lin, Xiaochen; Tsai, Ming-Han; Shumilov, Anatoliy; Poirey, Remy; Bannert, Helmut; Middeldorp, Jaap M.; Feederle, Regina; Delecluse, Henri-Jacques

2015-01-01

The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host. PMID:26694854

Evidence for Protection against Chronic Hepatitis C Virus Infection in Chimpanzees by Immunization with Replicating Recombinant Vaccinia Virus▿

PubMed Central

Youn, Jin-Won; Hu, Yu-Wen; Tricoche, Nancy; Pfahler, Wolfram; Shata, Mohamed Tarek; Dreux, Marlene; Cosset, François-Loic; Folgori, Antonella; Lee, Dong-Hun; Brotman, Betsy; Prince, Alfred M.

2008-01-01

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance (P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection. PMID:18753204

Inhibition of mTORC1 inhibits lytic replication of Epstein-Barr virus in a cell-type specific manner.

PubMed

Adamson, Amy L; Le, Brandi T; Siedenburg, Brian D

2014-06-11

Epstein-Barr virus is a human herpesvirus that infects a majority of the human population. Primary infection of Epstein-Barr virus (EBV) causes the syndrome infectious mononucleosis. This virus is also associated with several cancers, including Burkitt's lymphoma, post-transplant lymphoproliferative disorder and nasopharyngeal carcinoma. As all herpesvirus family members, EBV initially replicates lytically to produce abundant virus particles, then enters a latent state to remain within the host indefinitely. Through a genetic screen in Drosophila, we determined that reduction of Drosophila Tor activity altered EBV immediate-early protein function. To further investigate this finding, we inhibited mTOR in EBV-positive cells and investigated subsequent changes to lytic replication via Western blotting, flow cytometry, and quantitative PCR. The student T-test was used to evaluate significance. mTOR, the human homolog of Drosophila Tor, is an important protein at the center of a major signaling pathway that controls many aspects of cell biology. As the EBV immediate-early genes are responsible for EBV lytic replication, we examined the effect of inhibition of mTORC1 on EBV lytic replication in human EBV-positive cell lines. We determined that treatment of cells with rapamycin, which is an inhibitor of mTORC1 activity, led to a reduction in the ability of B cell lines to undergo lytic replication. In contrast, EBV-positive epithelial cell lines underwent higher levels of lytic replication when treated with rapamycin. Overall, the responses of EBV-positive cell lines vary when treated with mTOR inhibitors, and this may be important when considering such inhibitors as anti-cancer therapeutic agents.

Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication

USDA-ARS?s Scientific Manuscript database

Several studies have demonstrated that administration of type I, II, or III interferons (IFN) delivered using a replication defective human adenovirus 5 (Ad5) vector is effective to control Foot-and-Mouth Disease (FMD) in cattle and swine during experimental infections. However, high doses are requi...

Dynamics of melanoma tumor therapy with vesicular stomatitis virus: explaining the variability in outcomes using mathematical modeling.

PubMed

Rommelfanger, D M; Offord, C P; Dev, J; Bajzer, Z; Vile, R G; Dingli, D

2012-05-01

Tumor selective, replication competent viruses are being tested for cancer gene therapy. This approach introduces a new therapeutic paradigm due to potential replication of the therapeutic agent and induction of a tumor-specific immune response. However, the experimental outcomes are quite variable, even when studies utilize highly inbred strains of mice and the same cell line and virus. Recognizing that virotherapy is an exercise in population dynamics, we utilize mathematical modeling to understand the variable outcomes observed when B16ova malignant melanoma tumors are treated with vesicular stomatitis virus in syngeneic, fully immunocompetent mice. We show how variability in the initial tumor size and the actual amount of virus delivered to the tumor have critical roles on the outcome of therapy. Virotherapy works best when tumors are small, and a robust innate immune response can lead to superior tumor control. Strategies that reduce tumor burden without suppressing the immune response and methods that maximize the amount of virus delivered to the tumor should optimize tumor control in this model system.

Spatiotemporally restricted arenavirus replication induces immune surveillance and type I interferon-dependent tumour regression

PubMed Central

Kalkavan, Halime; Sharma, Piyush; Kasper, Stefan; Helfrich, Iris; Pandyra, Aleksandra A.; Gassa, Asmae; Virchow, Isabel; Flatz, Lukas; Brandenburg, Tim; Namineni, Sukumar; Heikenwalder, Mathias; Höchst, Bastian; Knolle, Percy A.; Wollmann, Guido; von Laer, Dorothee; Drexler, Ingo; Rathbun, Jessica; Cannon, Paula M.; Scheu, Stefanie; Bauer, Jens; Chauhan, Jagat; Häussinger, Dieter; Willimsky, Gerald; Löhning, Max; Schadendorf, Dirk; Brandau, Sven; Schuler, Martin; Lang, Philipp A.; Lang, Karl S.

2017-01-01

Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses. PMID:28248314

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Architecture and biogenesis of plus-strand RNA virus replication factories

PubMed Central

Paul, David; Bartenschlager, Ralf

2013-01-01

Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virus-induced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories. PMID:24175228

Functional analysis of human foamy virus accessory reading frames.

PubMed Central

Baunach, G; Maurer, B; Hahn, H; Kranz, M; Rethwilm, A

1993-01-01

Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses. Images PMID:8394455

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication

PubMed Central

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses. PMID:28056087

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

PubMed

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

Role of MAPK/MNK1 signaling in virus replication.

PubMed

Kumar, Ram; Khandelwal, Nitin; Thachamvally, Riyesh; Tripathi, Bhupendra Nath; Barua, Sanjay; Kashyap, Sudhir Kumar; Maherchandani, Sunil; Kumar, Naveen

2018-06-01

Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

PubMed Central

Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

1999-01-01

The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

Assessing Human Immunodeficiency Virus Type 1 Tropism: Comparison of Assays Using Replication-Competent Virus versus Plasma-Derived Pseudotyped Virions ▿

PubMed Central

Hosoya, Noriaki; Su, Zhaohui; Wilkin, Timothy; Gulick, Roy M.; Flexner, Charles; Hughes, Michael D.; Skolnik, Paul R.; Giguel, Françoise; Greaves, Wayne L.; Coakley, Eoin; Kuritzkes, Daniel R.

2009-01-01

Detection of CXCR4-using human immunodeficiency virus by the Trofile assay was compared to that by assays using virus isolates or replication-competent recombinants. Concordance with the Trofile assay was good, but assays using replicating viruses did not increase substantially the ability to detect the presence of CXCR4-using virus. PMID:19494074

HIV vaccine research and discovery in the nonhuman primates model: a unified theory in acquisition prevention and control of SIV infection.

PubMed

Lynch, Rebecca M; Yamamoto, Takuya; McDermott, Adrian B

2013-07-01

Here we highlight the latest advances in HIV vaccine concepts that will expand our knowledge on how to elicit effective acquisition-prevention and/or control of simian immunodeficiency virus (SIV) replication in the nonhuman primate (NHP) model. In the context of the promising analyses from the RV144 Thai Trial and the effective control of SIV replication exerted by rhCMV-(SIV) elicited EM CD8 T cells, the HIV field has recently shifted toward vaccine concepts that combine protection from acquisition with effective control of SIV replication. Current studies in the NHP model have demonstrated the efficacy of HIV-neutralizing antibodies via passive transfer, the potential importance of the CD4 Tfh subset, the ability to effectively model the RV144 vaccine trial and the capacity of an Ad26 prime and modified vaccinia Ankara virus boost to elicit Env-specific antibody and cellular responses that both limit acquisition and control heterologous SIVmac251 challenge. The latest work in the NHP model suggests that the next generation HIV-1 vaccines should aim to provoke a comprehensive adaptive immune response for both prevention of SIV acquisition as well as control of replication in breakthrough infection.

Cytomegalovirus and polyomavirus BK posttransplant.

PubMed

Egli, Adrian; Binggeli, Simone; Bodaghi, Sohrab; Dumoulin, Alexis; Funk, Georg A; Khanna, Nina; Leuenberger, David; Gosert, Rainer; Hirsch, Hans H

2007-09-01

Virus replication and progression to disease in transplant patients is determined by patient-, graft- and virus-specific factors. This complex interaction is modulated by the net state of immunosuppression and its impact on virus-specific cellular immunity. Due to the increasing potency of immunosuppressive regimens, graft rejections have decreased, but susceptibility to infections has increased. Therefore, cytomegalovirus (CMV) remains the most important viral pathogen posttransplant despite availability of effective antiviral drugs and validated strategies for prophylactic, preemptive and therapeutic intervention. CMV replication can affect almost every organ system, with frequent recurrences and increasing rates of antiviral resistance. Together with indirect long-term effects, CMV significantly reduces graft and patient survival after solid organ and hematopoietic stem cell transplantation. The human polyomavirus called BK virus (BKV), on the other hand, only recently surfaced as pathogen with organ tropism largely limited to the reno-urinary tract, manifesting as polyomavirus-associated nephropathy in kidney transplant and hemorrhagic cystitis in hematopoetic stem cell transplant patients. No licensed anti-polyoma viral drugs are available, and treatment relies mainly on improving immune functions to regain control over BKV replication. In this review, we discuss diagnostic and therapeutic aspects of CMV and BKV replication and disease posttransplantation.

Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

PubMed

Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

2017-12-13

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus. Copyright © 2017 American Society for Microbiology.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

PubMed

Fuller, Michael J; Callendret, Benoit; Zhu, Baogong; Freeman, Gordon J; Hasselschwert, Dana L; Satterfield, William; Sharpe, Arlene H; Dustin, Lynn B; Rice, Charles M; Grakoui, Arash; Ahmed, Rafi; Walker, Christopher M

2013-09-10

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

PubMed Central

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Vesicular Egress of Non-Enveloped Lytic Parvoviruses Depends on Gelsolin Functioning

PubMed Central

Bär, Séverine; Daeffler, Laurent; Rommelaere, Jean; Nüesch, Jürg P. F.

2008-01-01

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of “actin patches.” This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIα, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process. PMID:18704167

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans

PubMed Central

Schountz, Tony; Baker, Michelle L.; Butler, John; Munster, Vincent

2017-01-01

Bats are reservoir hosts of many important viruses that cause substantial disease in humans, including coronaviruses, filoviruses, lyssaviruses, and henipaviruses. Other than the lyssaviruses, they do not appear to cause disease in the reservoir bats, thus an explanation for the dichotomous outcomes of infections of humans and bat reservoirs remains to be determined. Bats appear to have a few unusual features that may account for these differences, including evidence of constitutive interferon (IFN) activation and greater combinatorial diversity in immunoglobulin genes that do not undergo substantial affinity maturation. We propose these features may, in part, account for why bats can host these viruses without disease and how they may contribute to the highly pathogenic nature of bat-borne viruses after spillover into humans. Because of the constitutive IFN activity, bat-borne viruses may be shed at low levels from bat cells. With large naive antibody repertoires, bats may control the limited virus replication without the need for rapid affinity maturation, and this may explain why bats typically have low antibody titers to viruses. However, because bat viruses have evolved in high IFN environments, they have enhanced countermeasures against the IFN response. Thus, upon infection of human cells, where the IFN response is not constitutive, the viruses overwhelm the IFN response, leading to abundant virus replication and pathology. PMID:28959255

Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges

PubMed Central

Kolliopoulou, Anna; Taning, Clauvis N. T.; Smagghe, Guy; Swevers, Luc

2017-01-01

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed. PMID:28659820

The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, Winnie; Zhou, Helen; Kemble, George

2008-10-25

We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 deg. C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 deg. C during amore » single-step replication, however, vRNA synthesis was reduced and the nuclear-cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 deg. C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.« less

Elimination of A-type inclusion formation enhances cowpox virus replication in mice: implications for orthopoxvirus evolution.

PubMed

Kastenmayer, Robin J; Maruri-Avidal, Liliana; Americo, Jeffrey L; Earl, Patricia L; Weisberg, Andrea S; Moss, Bernard

2014-03-01

Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein. Published by Elsevier Inc.

Reactivation and Lytic Replication of Kaposi’s Sarcoma-Associated Herpesvirus: An Update

PubMed Central

Aneja, Kawalpreet K.; Yuan, Yan

2017-01-01

The life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed a sophisticated regulation network that controls the important events in KSHV life cycle. PMID:28473805

A universal mammalian vaccine cell line substrate.

PubMed

Murray, Jackelyn; Todd, Kyle V; Bakre, Abhijeet; Orr-Burks, Nichole; Jones, Les; Wu, Weilin; Tripp, Ralph A

2017-01-01

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.

PubMed

Grabowski, Jeffrey M; Tsetsarkin, Konstantin A; Long, Dan; Scott, Dana P; Rosenke, Rebecca; Schwan, Tom G; Mlera, Luwanika; Offerdahl, Danielle K; Pletnev, Alexander G; Bloom, Marshall E

2017-08-22

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission. IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

Influence of IFNL3 and HLA-DPB1 genotype on postpartum control of hepatitis C virus replication and T-cell recovery.

PubMed

Honegger, Jonathan R; Tedesco, Dana; Kohout, Jennifer A; Prasad, Mona R; Price, Aryn A; Lindquist, Tera; Ohmer, Samantha; Moore-Clingenpeel, Melissa; Grakoui, Arash; Walker, Christopher M

2016-09-20

Chronic hepatitis C virus (HCV) infection is characterized by exhaustion of virus-specific T-cells and stable viremia. Pregnancy is an exception. Viremia gradually climbs during gestation but sometimes declines sharply in the months following delivery. Here, we demonstrated that postpartum HCV control was associated with enhanced virus-specific T-cell immunity. Women with viral load declines of at least 1 log10 between the third trimester and 3-mo postpartum exhibited HCV-specific T-cell responses of greater breadth (P = 0.0052) and magnitude (P = 0.026) at 3-mo postpartum than women who failed to control viremia. Moreover, viral dynamics were consistent in women after consecutive pregnancies, suggesting genetic underpinnings. We therefore searched for genetic associations with human leukocyte antigen (HLA) alleles and IFN-λ3 gene (IFNL3) polymorphisms that influence HCV infection outcome. Postpartum viral control was associated with the IFNL3 rs12979860 genotype CC (P = 0.045 at 6 mo) that predicts a positive response to IFN-based therapy. Suppression of virus replication after pregnancy was also strongly influenced by the HLA class II DPB1 locus. HLA-DPB1 alleles are classified by high and low patterns of expression. Carriage of at least one high-expression HLA-DPB1 allele predicted resurgent virus-specific T-cell immunity and viral control at 3-mo postpartum (P = 0.0002). When considered together in multivariable analysis, IFNL3 and HLA-DPB1 independently affected viral control at 3- and 6-mo postpartum. Together, these findings support a model where spontaneous control of HCV such as sometimes follows pregnancy is governed by genetic polymorphisms that affect type III IFN signaling and virus-specific cellular immune responses.

Rewiring of cellular membrane homeostasis by picornaviruses.

PubMed

Belov, George A; Sztul, Elizabeth

2014-09-01

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals.

PubMed

El-Shesheny, Rabeh; Feeroz, Mohammed M; Krauss, Scott; Vogel, Peter; McKenzie, Pamela; Webby, Richard J; Webster, Robert G

2018-04-25

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

PubMed Central

Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H.; Wahl-Jensen, Victoria

2012-01-01

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. PMID:21699921

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Fangke; Chen, Yun; Shi, Jintong

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold weremore » observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5 h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies. - Highlights: • This is the first description of the replication cycle of DHAV-1. • Firstly find that DHAV-1 adsorption saturated at 90 min post-infection. • The replication lasted around 13 h after early protein synthesis for about 5 h. • The release of DHAV-1 was in steady state after 32 h.« less

Lipids and RNA virus replication.

PubMed

Konan, Kouacou V; Sanchez-Felipe, Lorena

2014-12-01

Most viruses rely heavily on their host machinery to successfully replicate their genome and produce new virus particles. Recently, the interaction of positive-strand RNA viruses with the lipid biosynthetic and transport machinery has been the subject of intense investigation. In this review, we will discuss the contribution of various host lipids and related proteins in RNA virus replication and maturation. Copyright © 2014 Elsevier B.V. All rights reserved.

Nuclear Proteins Hijacked by Mammalian Cytoplasmic Plus Strand RNA Viruses

PubMed Central

Lloyd, Richard E.

2015-01-01

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. PMID:25818028

Direct-acting Antivirals and Host-targeting Agents against the Hepatitis A Virus

PubMed Central

Kanda, Tatsuo; Nakamoto, Shingo; Wu, Shuang; Nakamura, Masato; Jiang, Xia; Haga, Yuki; Sasaki, Reina; Yokosuka, Osamu

2015-01-01

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis and occasionally leads to acute liver failure in both developing and developed countries. Although effective vaccines for HAV are available, the development of new antivirals against HAV may be important for the control of HAV infection in developed countries where no universal vaccination program against HAV exists, such as Japan. There are two forms of antiviral agents against HAV: direct-acting antivirals (DAAs) and host-targeting agents (HTAs). Studies using small interfering ribonucleic acid (siRNA) have suggested that the HAV internal ribosomal entry site (IRES) is an attractive target for the control of HAV replication and infection. Among the HTAs, amantadine and interferon-lambda 1 (IL-29) inhibit HAV IRES-mediated translation and HAV replication. Janus kinase (JAK) inhibitors inhibit La protein expression, HAV IRES activity, and HAV replication. Based on this review, both DAAs and HTAs may be needed to control effectively HAV infection, and their use should continue to be explored. PMID:26623267

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Interaction between Flavivirus and Cytoskeleton during Virus Replication

PubMed Central

Foo, Kar Yue; Chee, Hui-Yee

2015-01-01

Flaviviruses are potentially human pathogens that cause major epidemics worldwide. Flavivirus interacts with host cell factors to form a favourable virus replication site. Cell cytoskeletons have been observed to have close contact with flaviviruses, which expands the understanding of cytoskeleton functions during virus replication, although many detailed mechanisms are still unclear. The interactions between the virus and host cytoskeletons such as actin filaments, microtubules, and intermediate filaments have provided insight into molecular alterations during the virus infection, such as viral entry, in-cell transport, scaffold assembly, and egress. This review article focuses on the utilization of cytoskeleton by Flavivirus and the respective functions during virus replication. PMID:26347881

Journal of Virology

Science.gov Websites

Herpes Simplex Virus 1 Improved Control of Simian/Human Immunodeficiency Virus in Macaques following Hemisphere Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human -associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. Temporal

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro

PubMed Central

van de Water, Sandra G. P.; Potgieter, Christiaan A.; van Rijn, Piet A.

2016-01-01

ABSTRACT The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro. Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses. PMID:27903804

Understanding the causes and consequences of measles virus persistence

PubMed Central

Griffin, Diane E.; Lin, Wen-Hsuan W.; Nelson, Ashley N.

2018-01-01

Measles is an acute systemic viral disease with initial amplification of infection in lymphoid tissue and subsequent spread over 10–14 days to multiple organs. Failure of the innate response to control initial measles virus (MeV) replication is associated with the ability of MeV to inhibit the induction of type I interferon and interferon-stimulated antiviral genes. Rather, the innate response is characterized by the expression of proteins regulated by nuclear factor kappa B and the inflammasome. With eventual development of the adaptive response, the rash appears with immune cell infiltration into sites of virus replication to initiate the clearance of infectious virus. However, MeV RNA is cleared much more slowly than recoverable infectious virus and remains present in lymphoid tissue for at least 6 months after infection. Persistence of viral RNA and protein suggests persistent low-level replication in lymphoid tissue that may facilitate maturation of the immune response, resulting in lifelong protection from reinfection, while persistence in other tissues (for example, the nervous system) may predispose to development of late disease such as subacute sclerosing panencephalitis. Further studies are needed to identify mechanisms of viral clearance and to understand the relationship between persistence and development of lifelong immunity. PMID:29560260

Understanding the causes and consequences of measles virus persistence.

PubMed

Griffin, Diane E; Lin, Wen-Hsuan W; Nelson, Ashley N

2018-01-01

Measles is an acute systemic viral disease with initial amplification of infection in lymphoid tissue and subsequent spread over 10-14 days to multiple organs. Failure of the innate response to control initial measles virus (MeV) replication is associated with the ability of MeV to inhibit the induction of type I interferon and interferon-stimulated antiviral genes. Rather, the innate response is characterized by the expression of proteins regulated by nuclear factor kappa B and the inflammasome. With eventual development of the adaptive response, the rash appears with immune cell infiltration into sites of virus replication to initiate the clearance of infectious virus. However, MeV RNA is cleared much more slowly than recoverable infectious virus and remains present in lymphoid tissue for at least 6 months after infection. Persistence of viral RNA and protein suggests persistent low-level replication in lymphoid tissue that may facilitate maturation of the immune response, resulting in lifelong protection from reinfection, while persistence in other tissues (for example, the nervous system) may predispose to development of late disease such as subacute sclerosing panencephalitis. Further studies are needed to identify mechanisms of viral clearance and to understand the relationship between persistence and development of lifelong immunity.

Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

PubMed Central

Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

2014-01-01

ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines. PMID:24807715

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

PubMed

Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

2017-11-01

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen. Copyright © 2017 American Society for Microbiology.

Virus reactivation: a panoramic view in human infections

PubMed Central

Traylen, Christopher M; Patel, Hersh R; Fondaw, Wylder; Mahatme, Sheran; Williams, John F; Walker, Lia R; Dyson, Ossie F; Arce, Sergio; Akula, Shaw M

2011-01-01

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is ‘quiescent’ (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus. PMID:21799704

Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

PubMed

Neumann, Friederike; Czech-Sioli, Manja; Dobner, Thomas; Grundhoff, Adam; Schreiner, Sabrina; Fischer, Nicole

2016-11-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

PubMed Central

Ducasse, D A; Mushegian, A R; Shepherd, R J

1995-01-01

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID:7543587

A CRISPR toolbox to study virus–host interactions

PubMed Central

Puschnik, Andreas S.; Majzoub, Karim; Ooi, Yaw Shin; Carette, Jan E.

2018-01-01

Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR–Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR–Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR–Cas make it a powerful tool for probing virus–host interactions and for identifying new antiviral targets. PMID:28420884

Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue.

PubMed

Harouaka, Djamila; Engle, Ronald E; Wollenberg, Kurt; Diaz, Giacomo; Tice, Ashley B; Zamboni, Fausto; Govindarajan, Sugantha; Alter, Harvey; Kleiner, David E; Farci, Patrizia

2016-02-02

Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

PubMed

van de Sandt, Carolien E; Pronk, Mark R; van Baalen, Carel A; Fouchier, Ron A M; Rimmelzwaan, Guus F

2018-06-01

Influenza virus-specific CD8 + T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M1 58-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M1 58-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M1 58-66 -specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M1 58-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity. IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8 + T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8 + T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8 + T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity. Copyright © 2018 American Society for Microbiology.

Ultrastructure of the replication sites of positive-strand RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection.

PubMed

Saxena, Kapil; Simon, Lukas M; Zeng, Xi-Lei; Blutt, Sarah E; Crawford, Sue E; Sastri, Narayan P; Karandikar, Umesh C; Ajami, Nadim J; Zachos, Nicholas C; Kovbasnjuk, Olga; Donowitz, Mark; Conner, Margaret E; Shaw, Chad A; Estes, Mary K

2017-01-24

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

PubMed Central

Saxena, Kapil; Simon, Lukas M.; Zeng, Xi-Lei; Blutt, Sarah E.; Crawford, Sue E.; Sastri, Narayan P.; Karandikar, Umesh C.; Ajami, Nadim J.; Zachos, Nicholas C.; Kovbasnjuk, Olga; Donowitz, Mark; Conner, Margaret E.; Shaw, Chad A.; Estes, Mary K.

2017-01-01

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine. PMID:28069942

Hepatitis Delta Virus: Replication Strategy and Upcoming Therapeutic Options for a Neglected Human Pathogen

PubMed Central

Lempp, Florian A.; Urban, Stephan

2017-01-01

The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15–20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B. PMID:28677645

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV).

PubMed

Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall

2011-06-01

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential Impact of In Vivo CD8+ T Lymphocyte Depletion in Controller versus Progressor Simian Immunodeficiency Virus-Infected Macaques.

PubMed

Chowdhury, Ankita; Hayes, Timothy L; Bosinger, Steven E; Lawson, Benton O; Vanderford, Thomas; Schmitz, Joern E; Paiardini, Mirko; Betts, Michael; Chahroudi, Ann; Estes, Jacob D; Silvestri, Guido

2015-09-01

Numerous studies have demonstrated that CD8(+) T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8(+) T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIVmac239. At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8(+) T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8(+) T-bet(+) lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4(+) central memory T lymphocytes (TCM) and CD4(+) effector memory T lymphocytes (TEM) in progressor RMs but decreased in the CD4(+) TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4(+) T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8(+) T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8(+) T lymphocytes. In this study, we further dissect the impact of CD8(+) T lymphocytes on HIV/SIV replication during SIV infection. CD8(+) T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8(+) T lymphocyte depletion resulted in a more prominent increase in viral loads and CD4(+) T lymphocyte activation in controllers than in progressors. Interestingly, we found T-bet expression on CD8(+) T lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV DNA increase postdepletion in both CD4(+) TCM and TEM in progressor RMs but decrease in the CD4(+) TCM of controllers. The findings described in this study provide key insights into the differential functions of CD8(+) T lymphocytes in controller and progressor RMs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Differential Impact of In Vivo CD8+ T Lymphocyte Depletion in Controller versus Progressor Simian Immunodeficiency Virus-Infected Macaques

PubMed Central

Chowdhury, Ankita; Hayes, Timothy L.; Bosinger, Steven E.; Lawson, Benton O.; Vanderford, Thomas; Schmitz, Joern E.; Paiardini, Mirko; Betts, Michael; Chahroudi, Ann; Estes, Jacob D.

2015-01-01

ABSTRACT Numerous studies have demonstrated that CD8+ T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8+ T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIVmac239. At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8+ T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8+ T-bet+ lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4+ central memory T lymphocytes (TCM) and CD4+ effector memory T lymphocytes (TEM) in progressor RMs but decreased in the CD4+ TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4+ T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8+ T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8+ T lymphocytes. IMPORTANCE In this study, we further dissect the impact of CD8+ T lymphocytes on HIV/SIV replication during SIV infection. CD8+ T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8+ T lymphocyte depletion resulted in a more prominent increase in viral loads and CD4+ T lymphocyte activation in controllers than in progressors. Interestingly, we found T-bet expression on CD8+ T lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV DNA increase postdepletion in both CD4+ TCM and TEM in progressor RMs but decrease in the CD4+ TCM of controllers. The findings described in this study provide key insights into the differential functions of CD8+ T lymphocytes in controller and progressor RMs. PMID:26063417

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti.

PubMed

van den Hurk, Andrew F; Hall-Mendelin, Sonja; Pyke, Alyssa T; Frentiu, Francesca D; McElroy, Kate; Day, Andrew; Higgs, Stephen; O'Neill, Scott L

2012-01-01

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10(4) times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.

Impact of Wolbachia on Infection with Chikungunya and Yellow Fever Viruses in the Mosquito Vector Aedes aegypti

PubMed Central

van den Hurk, Andrew F.; Hall-Mendelin, Sonja; Pyke, Alyssa T.; Frentiu, Francesca D.; McElroy, Kate; Day, Andrew; Higgs, Stephen; O'Neill, Scott L.

2012-01-01

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 104 times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression. PMID:23133693

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

PubMed

Wirth, Jennifer Fulton; Snyder, Jamie C; Hochstein, Rebecca A; Ortmann, Alice C; Willits, Deborah A; Douglas, Trevor; Young, Mark J

2011-06-20

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication. Copyright © 2011. Published by Elsevier Inc.

CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

PubMed

Ayala, Victor I; Deleage, Claire; Trivett, Matthew T; Jain, Sumiti; Coren, Lori V; Breed, Matthew W; Kramer, Joshua A; Thomas, James A; Estes, Jacob D; Lifson, Jeffrey D; Ott, David E

2017-06-01

Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the T FH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on T FH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus. Copyright © 2017 American Society for Microbiology.

Influenza virus replication in macrophages: balancing protection and pathogenesis

PubMed Central

Beck, Donald; Bianchini, Elizabeth

2017-01-01

Macrophages are essential for protection against influenza A virus infection, but are also implicated in the morbidity and mortality associated with severe influenza disease, particularly during infection with highly pathogenic avian influenza (HPAI) H5N1 virus. While influenza virus infection of macrophages was once thought to be abortive, it is now clear that certain virus strains can replicate productively in macrophages. This may have important consequences for the antiviral functions of macrophages, the course of disease and the outcome of infection for the host. In this article, we review findings related to influenza virus replication in macrophages and the impact of productive replication on macrophage antiviral functions. A clear understanding of the interactions between influenza viruses and macrophages may lead to new antiviral therapies to relieve the burden of severe disease associated with influenza viruses. PMID:28884667

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

PubMed Central

Londrigan, Sarah L.; Short, Kirsty R.; Ma, Joel; Gillespie, Leah; Rockman, Steven P.; Brooks, Andrew G.

2015-01-01

ABSTRACT Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. PMID:26423941

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle.

PubMed

Londrigan, Sarah L; Short, Kirsty R; Ma, Joel; Gillespie, Leah; Rockman, Steven P; Brooks, Andrew G; Reading, Patrick C

2015-12-01

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Innate immune responses against foot-and-mouth disease virus: current understanding and future directions.

PubMed

Summerfield, Artur; Guzylack-Piriou, Laurence; Harwood, Lisa; McCullough, Kenneth C

2009-03-15

Foot-and-mouth disease (FMD) represents one of the most economically important diseases of farm animals. The basis for the threat caused by this virus is the high speed of replication, short incubation time, high contagiousness, and high mutation rate resulting in constant antigenic changes. Thus, although protective immune responses against FMD virus (FMDV) can be efficacious, the rapidity of virus replication and spread can outpace immune defence development and overrun the immune system. FMDV can also evade innate immune responses through its ability to shut down cellular protein synthesis, including IFN type I, in susceptible epithelial cells. This is important for virus evolution, as FMDV is quite sensitive to the action of IFN. Despite this, innate immune responses are probably induced in vivo, although detailed studies on this subject are lacking. Accordingly, this interaction of FMDV with cells of the innate immune system is of particular interest. Dendritic cells (DC) can be infected by FMDV and support viral RNA replication, and viral protein synthesis but the latter is inefficient or abortive, leading most often to incomplete replication and progeny virus release. As a result DC can be activated, and particularly in the case of plasmacytoid DC (pDC), this is manifest in terms of IFN-alpha release. Our current state of knowledge on innate immune responses induced by FMDV is still only at a relatively early stage of understanding. As we progress, the investigations in this area will help to improve the design of current vaccines and the development of novel control strategies against FMD.

Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

PubMed

Susta, Leonardo; Diel, Diego G; Courtney, Sean; Cardenas-Garcia, Stivalis; Sundick, Roy S; Miller, Patti J; Brown, Corrie C; Afonso, Claudio L

2015-08-08

In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (10(3.46) EID50 /0.1 ml) and drastically decreased at day 5 pi (10(0.9) EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 10(5.35) EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. Increased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4-5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

PubMed

Dietrich, Isabelle; Jansen, Stephanie; Fall, Gamou; Lorenzen, Stephan; Rudolf, Martin; Huber, Katrin; Heitmann, Anna; Schicht, Sabine; Ndiaye, El Hadji; Watson, Mick; Castelli, Ilaria; Brennan, Benjamin; Elliott, Richard M; Diallo, Mawlouth; Sall, Amadou A; Failloux, Anna-Bella; Schnettler, Esther; Kohl, Alain; Becker, Stefanie C

2017-01-01

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus , Bunyaviridae ) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster . We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems

PubMed Central

Jansen, Stephanie; Fall, Gamou; Lorenzen, Stephan; Rudolf, Martin; Huber, Katrin; Heitmann, Anna; Schicht, Sabine; Ndiaye, El Hadji; Watson, Mick; Castelli, Ilaria; Elliott, Richard M.; Diallo, Mawlouth; Sall, Amadou A.; Failloux, Anna-Bella; Schnettler, Esther

2017-01-01

ABSTRACT The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies. PMID:28497117

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17.

PubMed

Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

2012-12-01

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.

[Efficacy of siRNA on feline leukemia virus replication in vitro].

PubMed

Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

2015-01-01

Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication

PubMed Central

Olsen, Michelle E.; Filone, Claire Marie; Rozelle, Dan; Mire, Chad E.; Agans, Krystle N.; Hensley, Lisa

2016-01-01

ABSTRACT Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates. EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein. PMID:27460797

Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus

PubMed Central

Messaoudi, Ilhem; Amarasinghe, Gaya K.; Basler, Christopher F.

2016-01-01

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people. The Ebola virus epidemic in West Africa, which was first recognized in early 2014, highlights the threat posed by these deadly viruses. Filovirus disease is characterized by uncontrolled virus replication and the activation of damaging host pathways. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon (IFN) response, which allows high levels of replication. Here we review the mechanisms deployed by filoviruses to block host innate immunity and discuss aspects of virus replication that promote disease. PMID:26439085

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

PubMed

Advani, S J; Weichselbaum, R R; Whitley, R J; Roizman, B

2002-09-01

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation.

PubMed

Huang, Lu; Yang, Mengtian; Yuan, Yan; Li, Xiaojuan; Kuang, Ersheng

2017-02-01

Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

Roles of the phosphorylation of specific serines and threonines in the NS1 protein of human influenza A viruses.

PubMed

Hsiang, Tien-Ying; Zhou, Ligang; Krug, Robert M

2012-10-01

We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C α (PKCα) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKCα. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus.

Multifarious immunotherapeutic approaches to cure HIV-1 infection.

PubMed

Imami, Nesrina; Herasimtschuk, Anna A

2015-01-01

Immunotherapy in the context of treated HIV-1 infection aims to improve immune responses to achieve better control of the virus. To date, multifaceted immunotherapeutic approaches have been shown to reduce immune activation and increase CD4 T-lymphocyte counts, further to the effects of antiretroviral therapy alone, in addition to improving HIV-1-specific T-cell responses. While sterilizing cure of HIV-1 would involve elimination of all replication-competent virus, a functional cure in which the host has long-lasting control of viral replication may be more feasible. In this commentary, we discuss novel strategies aimed at targeting the latent viral reservoir with cure of HIV-1 infection being the ultimate goal, an achievement that would have considerable impact on worldwide HIV-1 infection.

Targeting Nucleotide Biosynthesis: A Strategy for Improving the Oncolytic Potential of DNA Viruses

PubMed Central

Irwin, Chad R.; Hitt, Mary M.; Evans, David H.

2017-01-01

The rapid growth of tumors depends upon elevated levels of dNTPs, and while dNTP concentrations are tightly regulated in normal cells, this control is often lost in transformed cells. This feature of cancer cells has been used to advantage to develop oncolytic DNA viruses. DNA viruses employ many different mechanisms to increase dNTP levels in infected cells, because the low concentration of dNTPs found in non-cycling cells can inhibit virus replication. By disrupting the virus-encoded gene(s) that normally promote dNTP biosynthesis, one can assemble oncolytic versions of these agents that replicate selectively in cancer cells. This review covers the pathways involved in dNTP production, how they are dysregulated in cancer cells, and the various approaches that have been used to exploit this biology to improve the tumor specificity of oncolytic viruses. In particular, we compare and contrast the ways that the different types of oncolytic virus candidates can directly modulate these processes. We limit our review to the large DNA viruses that naturally encode homologs of the cellular enzymes that catalyze dNTP biogenesis. Lastly, we consider how this knowledge might guide future development of oncolytic viruses. PMID:29018771

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

PubMed Central

Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

2000-01-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

African Green Monkeys Recapitulate the Clinical Experience with Replication of Live Attenuated Pandemic Influenza Virus Vaccine Candidates

PubMed Central

Matsuoka, Yumiko; Suguitan, Amorsolo; Orandle, Marlene; Paskel, Myeisha; Boonnak, Kobporn; Gardner, Donald J.; Feldmann, Friederike; Feldmann, Heinz; Marino, Michael; Jin, Hong; Kemble, George

2014-01-01

ABSTRACT Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. IMPORTANCE Ferrets and mice are commonly used for preclinical evaluation of influenza vaccines. However, we observed significant inconsistencies between observations in humans and in these animal models. We used African green monkeys (AGMs) as a nonhuman primate (NHP) model for a comprehensive and comparative evaluation of pairs of wild-type and pandemic live attenuated influenza virus vaccines (pLAIV) representing four subtypes of avian influenza viruses and found that pLAIVs replicate similarly in AGMs and humans and that AGMs can be useful for evaluation of the protective efficacy of pLAIV. PMID:24807726

Evaluation of seasonal influenza vaccines for H1N1pdm09 and type B viruses based on a replication-incompetent PB2-KO virus.

PubMed

Ui, Hiroki; Yamayoshi, Seiya; Uraki, Ryuta; Kiso, Maki; Oishi, Kohei; Murakami, Shin; Mimori, Shigetaka; Kawaoka, Yoshihiro

2017-04-04

Vaccination is the first line of protection against influenza virus infection in humans. Although inactivated and live-attenuated vaccines are available, each vaccine has drawbacks in terms of immunogenicity and safety. To overcome these issues, our group has developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2-expressing cells. Here we generated PB2-KO viruses possessing the hemagglutinin (HA) and neuraminidase (NA) segments from H1N1pdm09 or type B viruses and tested their vaccine potential. The two PB2-KO viruses propagated efficiently in PB2-expressing cells, and expressed chimeric HA as expected. Virus-specific IgG and IgA antibodies were detected in mice immunized with the viruses, and the immunized mice showed milder clinical signs and/or lower virus replication levels in the respiratory tract upon virus challenge. Our results indicate that these PB2-KO viruses have potential as vaccine candidates. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

PubMed

Li, Na; Yan, Yunhuan; Zhang, Angke; Gao, Jiming; Zhang, Chong; Wang, Xue; Hou, Gaopeng; Zhang, Gaiping; Jia, Jinbu; Zhou, En-Min; Xiao, Shuqi

2016-12-13

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Single-cycle replicable Rift Valley fever virus mutants as safe vaccine candidates

PubMed Central

Terasaki, Kaori; Tercero, Breanna R.; Makino, Shinji

2015-01-01

Rift Valley fever virus (RVFV) is an arbovirus circulating between ruminants and mosquitoes to maintain its enzootic cycle. Humans are infected with RVFV through mosquito bites or direct contact with materials of infected animals. The virus causes Rift Valley fever, which was first recognized in the Great Rift Valley of Kenya in 1931. RVFV is characterized by a febrile illness resulting in a high rate of abortions in ruminants and an acute febrile illness, followed by fatal hemorrhagic fever and encephalitis in humans. Initially, the virus was restricted to the eastern region of Africa, but the disease has now spread to southern and western Africa, as well as outside of the African continent, e.g., Madagascar, Saudi Arabia and Yemen. There is a serious concern that the virus may spread to other areas, such as North America and Europe. As vaccination is an effective tool to control RVFV epidemics, formalin-inactivated vaccines and live-attenuated RVFV vaccines have been used in endemic areas. The formalin-inactivated vaccines require boosters for effective protection, whereas the live-attenuated vaccines enable the induction of protective immunity by a single vaccination. However, the use of live-attenuated RVFV vaccines for large human populations having a varied health status is of concern, because of these vaccines’ residual neuro-invasiveness and neurovirulence. Recently, novel vaccine candidates have been developed using replication-defective RVFV that can undergo only a single round of replication in infected cells. The single-cycle replicable RVFV does not cause systemic infection in immunized hosts, but enables the conferring of protective immunity. This review summarizes the properties of various RVFV vaccines and recent progress on the development of the single-cycle replicable RVFV vaccines. PMID:26022573

Single-cycle replicable Rift Valley fever virus mutants as safe vaccine candidates.

PubMed

Terasaki, Kaori; Tercero, Breanna R; Makino, Shinji

2016-05-02

Rift Valley fever virus (RVFV) is an arbovirus circulating between ruminants and mosquitoes to maintain its enzootic cycle. Humans are infected with RVFV through mosquito bites or direct contact with materials of infected animals. The virus causes Rift Valley fever (RVF), which was first recognized in the Great Rift Valley of Kenya in 1931. RVF is characterized by a febrile illness resulting in a high rate of abortions in ruminants and an acute febrile illness, followed by fatal hemorrhagic fever and encephalitis in humans. Initially, the virus was restricted to the eastern region of Africa, but the disease has now spread to southern and western Africa, as well as outside of the African continent, e.g., Madagascar, Saudi Arabia and Yemen. There is a serious concern that the virus may spread to other areas, such as North America and Europe. As vaccination is an effective tool to control RVFV epidemics, formalin-inactivated vaccines and live-attenuated RVFV vaccines have been used in endemic areas. The formalin-inactivated vaccines require boosters for effective protection, whereas the live-attenuated vaccines enable the induction of protective immunity by a single vaccination. However, the use of live-attenuated RVFV vaccines for large human populations having a varied health status is of concern, because of these vaccines' residual neuro-invasiveness and neurovirulence. Recently, novel vaccine candidates have been developed using replication-defective RVFV that can undergo only a single round of replication in infected cells. The single-cycle replicable RVFV does not cause systemic infection in immunized hosts, but enables the conferring of protective immunity. This review summarizes the properties of various RVFV vaccines and recent progress on the development of the single-cycle replicable RVFV vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1.

PubMed

Agarwal, Shubhra; Baccam, Prasith; Aggarwal, Rakesh; Veerapu, Naga Suresh

2018-02-15

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 -3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Copyright © 2018 American Society for Microbiology.

Methadone enhances human influenza A virus replication.

PubMed

Chen, Yun-Hsiang; Wu, Kuang-Lun; Tsai, Ming-Ta; Chien, Wei-Hsien; Chen, Mao-Liang; Wang, Yun

2017-01-01

Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs. © 2015 Society for the Study of Addiction.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

PubMed

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Experimentally Guided Models Reveal Replication Principles that Shape the Mutation Distribution of RNA Viruses

DTIC Science & Technology

2015-01-30

intracel- lular replication. Two classic replication modes have been described for single-stranded RNA viruses: the ‘stamping machine’ mode ( Stent ...Journal of Theoretical Biology 218:309–321. doi: 10.1006/jtbi.2002.3078. Stent GS. 1963. Molecular Biology of Bacterial Viruses. San Francisco, Calif: W H

Susceptibility of Skeletal Muscle to Coxsackie A2 Virus Infection: Effects of Botulinum Toxin and Denervation

NASA Astrophysics Data System (ADS)

Andrew, Clifford G.; Drachman, Daniel B.; Pestronk, Alan; Narayan, Opendra

1984-02-01

Coxsackie A viruses can infect denervated but not innervated mature skeletal muscles. The role of synaptic transmission in preventing susceptibility to Coxsackievirus infection was studied by surgically denervating leg muscles of mice or injecting the muscles with botulinum toxin to block quantal release of acetylcholine. Control muscles were injected with heat-inactivated toxin. Subsequent injection of Coxsackie A2 virus resulted in extensive virus replication and tissue destruction in the denervated and botulinum toxin-treated muscles, while the control muscles showed only minimal changes. This suggests that the susceptibility of skeletal muscle to Coxsackievirus infection is regulated by synaptic transmission.

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction

PubMed Central

Cruz-Cano, Raul; Chew, David S.H.; Kwok-Pui, Choi; Ming-Ying, Leung

2010-01-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications. PMID:20729987

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction.

PubMed

Cruz-Cano, Raul; Chew, David S H; Kwok-Pui, Choi; Ming-Ying, Leung

2010-06-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications.

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1992-01-01

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression. Images PMID:1475192

CD11c controls herpes simplex virus 1 responses to limit virus replication during primary infection.

PubMed

Allen, Sariah J; Mott, Kevin R; Chentoufi, Aziz A; BenMohamed, Lbachir; Wechsler, Steven L; Ballantyne, Christie M; Ghiasi, Homayon

2011-10-01

CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c(+) DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8(+) T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-β, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-β, and CD8(+) T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8(+) T cell responses.

The role of virulence for in vivo superinfection fitness of a vertebrate RNA virus

USGS Publications Warehouse

Kell, Alison M.; Wargo, Andrew R.; Kurath, Gael

2013-01-01

We have developed a novel, in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural, vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a co-evolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 hours to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal virulence and unequal virulence genotype pairs, the frequency of superinfection was the same regardless of which genotype was used in the primary exposure. However, the ability to replicate in the context of superinfection did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection, while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV, and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural, vertebrate host.

Expression of CD154 by a Simian Immunodeficiency Virus Vector Induces Only Transitory Changes in Rhesus Macaques

PubMed Central

Hodara, Vida L.; Velasquillo, M. Cristina; Parodi, Laura M.; Giavedoni, Luis D.

2005-01-01

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154. PMID:15795254

Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

PubMed Central

Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

2012-01-01

Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.

PubMed

Badia, Roger; Angulo, Guillem; Riveira-Muñoz, Eva; Pujantell, Maria; Puig, Teresa; Ramirez, Cristina; Torres-Torronteras, Javier; Martí, Ramón; Pauls, Eduardo; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2016-02-01

Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM). MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect. SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Tolerance of Sulfolobus SMV1 virus to the immunity of I-A and III-B CRISPR-Cas systems in Sulfolobus islandicus.

PubMed

Guo, Tong; Han, Wenyuan; She, Qunxin

2018-04-09

Sulfolobus islandicus Rey15A encodes one type I-A and two type III-B systems, all of which are active in mediating nucleic acids interference. However, the effectiveness of each CRISPR system against virus infection was not tested in this archaeon. Here we constructed S. islandicus strains that constitutively express the antiviral immunity from either I-A, or III-B, or I-A plus III-B systems against SMV1 and tested the response of each host to SMV1 infection. We found that, although both CRISPR immunities showed a strongly inhibition to viral DNA replication at an early stage of incubation, the host I-A CRISPR immunity gradually lost the control on virus proliferation, allowing accumulation of cellular viral DNA and release of a large number of viral particles. In contrast, the III-B CRISPR immunity showed a tight control on both viral DNA replication and virus particle formation. Furthermore, the SMV1 tolerance to the I-A CRISPR immunity did not result from the occurrence of escape mutations, suggesting the virus probably encodes an anti-CRISPR protein (Acr) to compromise the host I-A CRISPR immunity. Together, this suggests that the interplay between viral Acrs and CRISPR-Cas systems in thermophilic archaea could have shaped the stable virus-host relationship currently seen for many archaeal viruses.

Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

PubMed Central

Casciano, Jessica C.; Duchemin, Nicholas J.; Lamontagne, R. Jason; Steel, Laura F.; Bouchard, Michael J.

2017-01-01

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases. PMID:28151934

Crimean-Congo haemorrhagic fever virus replication in adult Hyalomma truncatum and Amblyomma variegatum ticks.

PubMed

Gonzalez, J P; Cornet, J P; Wilson, M L; Camicas, J L

1991-01-01

The kinetics of the replication of the Crimean-Congo haemorrhagic fever virus (CCHFV) was studied in intra-anally inoculated adult Hyalomma truncatum and Amblyomma variegatum ticks. The virus was re-isolated by suckling mouse inoculation and revealed by antigen capture with ground ticks and indirect immunofluorescence of haemolymph. The virus was detected in ticks in the first hours post-inoculation (p.i.) and its replication was observed from 36 h p.i. onwards. Virus titre reached a maximum within 3-5 days then decreased slowly to a level of at 2 log LD50/ml for several months until the end of observations. Several specific, non-identified factors seem to favour CCHFV replication in H. truncatum. Long-term virus persistence seems to occur in CCHFV-infected adult ticks.

Two interferon-independent double-stranded RNA-induced host defense strategies suppress the common cold virus at warm temperature.

PubMed

Foxman, Ellen F; Storer, James A; Vanaja, Kiran; Levchenko, Andre; Iwasaki, Akiko

2016-07-26

Most strains of rhinovirus (RV), the common cold virus, replicate better at cool temperatures found in the nasal cavity (33-35 °C) than at lung temperature (37 °C). Recent studies found that although 37 °C temperature suppressed RV growth largely by engaging the type 1 IFN response in infected epithelial cells, a significant temperature dependence to viral replication remained in cells devoid of IFN induction or signaling. To gain insight into IFN-independent mechanisms limiting RV replication at 37 °C, we studied RV infection in human bronchial epithelial cells and H1-HeLa cells. During the single replication cycle, RV exhibited temperature-dependent replication in both cell types in the absence of IFN induction. At 37 °C, earlier signs of apoptosis in RV-infected cells were accompanied by reduced virus production. Furthermore, apoptosis of epithelial cells was enhanced at 37 °C in response to diverse stimuli. Dynamic mathematical modeling and B cell lymphoma 2 (BCL2) overexpression revealed that temperature-dependent host cell death could partially account for the temperature-dependent growth observed during RV amplification, but also suggested additional mechanisms of virus control. In search of a redundant antiviral pathway, we identified a role for the RNA-degrading enzyme RNAseL. Simultaneous antagonism of apoptosis and RNAseL increased viral replication and dramatically reduced temperature dependence. These findings reveal two IFN-independent mechanisms active in innate defense against RV, and demonstrate that even in the absence of IFNs, temperature-dependent RV amplification is largely a result of host cell antiviral restriction mechanisms operating more effectively at 37 °C than at 33 °C.

Identification of amino acid residues in infectious hematopoietic necrosis virus (IHNV) NV protein necessary for viral replication and pathogenicity.

PubMed

Wu, Yang; Wang, Li; Guo, Tiantian; Jiang, Yanping; Qiao, Xinyuan; Sun, Li; Liu, Min; Tang, Lijie; Xu, Yigang; Li, Yijing

2018-05-18

Our previous studies demonstrated that the nonstructural NV protein of infectious hematopoietic necrosis virus (IHNV) was essential for efficient viral replication and pathogenicity, and that the amino acid residues 32 EGDL 35 of the NV protein were responsible for nuclear localization, and played important roles in suppressing IFN and inhibiting NF-κB activity. However, little is known about the influence of 32 EGDL 35 on IHNV replication and pathogenicity. In the present study, two recombinant IHNV strains with deletions of NV 32 EGDL 35 were generated and the effect on IHNV replication and pathogenicity was explored. Our results showed that both mutants stably replicated in Chinook salmon embryo cells for 15 consecutive passages, and had similar host-tropism as wild-type (wt) IHNV; however, titers of the mutants were lower than those of wt IHNV in CHSE-214 cells. Infection of rainbow trout showed wt IHNV produced 90% cumulative mortality, while the mutants produced 55% and 60% cumulative mortality, respectively. Histopathological evaluation showed that tissues from the liver, brain, kidney, and heart of fish infected with wt IHNV exhibited pathological changes, but significant lesions were found only in the liver and heart of fish infected with the recombinant viruses. In addition, the recombinant viruses induced higher expression levels of IFN1, Mx-1, and IL-6 compared with those induced by wt IHNV. These results indicated that the 32 EGDL 35 residues were essential for the efficient anti-IFN and NF-κB-inhibiting activity of NV. Our results provide a basis for understanding the roles of 32 EGDL 35 in IHNV replication and pathogenicity, and may prove beneficial in the prevention and control of IHNV infections of fish. Copyright © 2018. Published by Elsevier Ltd.

Cathepsin B & L are not required for ebola virus replication.

PubMed

Marzi, Andrea; Reinheckel, Thomas; Feldmann, Heinz

2012-01-01

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation.

PubMed

Park, Mi-Ri; Kwon, Sun-Jung; Choi, Hong-Soo; Hemenway, Cynthia L; Kim, Kook-Hyung

2008-08-15

The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses.

PubMed

Ferlin, Juliette; Farhat, Rayan; Belouzard, Sandrine; Cocquerel, Laurence; Bertin, Antoine; Hober, Didier; Dubuisson, Jean; Rouillé, Yves

2018-06-20

GBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and Arf5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or Arf5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication.

Pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection

PubMed Central

2013-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive problems are characterized by embryonic death, late-term abortions, early farrowing and increase in number of dead and mummified fetuses, and weak-born piglets. The virus recovery from fetal tissues illustrates transplacental infection, but despite many studies on the subject, the means by which PRRSV spreads from mother to fetus and the exact pathophysiological basis of the virus-induced reproductive failure remain unexplained. Recent findings from our group indicate that the endometrium and placenta are involved in the PRRSV passage from mother to fetus and that virus replication in the endometrial/placental tissues can be the actual reason for fetal death. The main purpose of this review is to clarify the role that PRRSV replication and PRRSV-induced changes in the endometrium/placenta play in the pathogenesis of PRRSV-induced reproductive failure in pregnant sows. In addition, strategies to control placental and transplacental PRRSV infection are discussed. PMID:24099529

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

PubMed Central

Adeyemi, Richard O.

2012-01-01

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G2/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK. PMID:22623787

Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

PubMed

Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

2015-08-01

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the Study of Liver Diseases.

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perera, Rushika M.; Riley, Catherine; Isaac, Georgis

Dengue virus causes {approx}50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complexmore » membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.« less

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

PubMed Central

Perera, Rushika; Moore, Ronald J.; Weitz, Karl W.; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Adamec, Jiri; Kuhn, Richard J.

2012-01-01

Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture. PMID:22457619

M Protein-Deficient Respiratory Syncytial Virus (RSV) Vaccine Protects Infant Baboons Against RSV Challenge

PubMed Central

Welliver, Robert C; Oomens, Antonius; Wolf, Roman; Papin, James; Ivanov, Vadim; Preno, Alisha; Staats, Rachel; Piedra, Pedro; Yu, Zhongxin

2017-01-01

Abstract Background RSV bronchiolitis is the most common cause of hospitalization of infants in the US, and may lead to the development of long-term airway disease. Inactivated vaccines may lead to enhanced disease, while replicating vaccines have caused unacceptable degrees of illness, and may revert back to wild type. We developed an RSV vaccine lacking the gene for the M protein (Mnull RSV). The M protein is responsible for reassembly of the virus after it infects cells and expresses its proteins. Infant baboons vaccinated intranasally (IN) with Mnull RSV develop serum neutralizing antibody (NA) responses, but the virus does not replicate. Methods 2-week-old baboons (n = 12) were primed IN with 107 vaccine units of Mnull RSV or a control preparation, and a similar booster dose was given 4 weeks later. Mnull RSV vaccination did not cause tachypnea, airway inflammation or other signs of illness when compared with sham-vaccinated controls. Two weeks after boosting, all infants were challenged intratracheally with human RSV A2. We continuously monitored respiratory rates and levels of overall activity. On various days following challenge, we obtained BAL fluids for leukocyte counts and degree of virus replication, and evaluated alveolar-arterial oxygen gradients (A-a O2). Results Vaccinated animals (vs. unvaccinated controls) had lower respiratory rates (P = 0.0014), improved A-a O2 (P = 0.0063) and reduced viral replication (P = 0.0014). Activity scores were higher in vaccine recipients than in unvaccinated animals. Vaccine recipients also were primed for earlier serum and secretory neutralizing antibody responses, and greater airway lymphocyte responses. Airway lymphocyte numbers (but not antibody responses) were associated with lower respiratory rates and reduced viral replication (P < 0.01). Conclusion Vaccination intranasally with Mnull RSV protected infant baboons against an RSV challenge without causing respiratory disease or enhanced illness, and is a promising candidate for use in human infants. Lymphocyte responses to vaccination may play an equal or greater role in protection against RSV infection than antibody responses. Disclosures All authors: No reported disclosures.

A novel subviral agent associated with a geminivirus: The first report of a DNA satellite

PubMed Central

Dry, Ian B.; Krake, Leslie R.; Rigden, Justin E.; Rezaian, M. Ali

1997-01-01

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. PMID:9192696

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

PubMed

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

2016-11-18

Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

PubMed

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

2018-02-14

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

PubMed Central

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H.; Veits, Jutta; Gischke, Marcel

2018-01-01

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host. PMID:29443887

Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

PubMed

Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

2010-12-01

Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

PubMed Central

Lee, Jinhwa; Yu, Hai; Li, Yonghai; Ma, Jingjiao; Lang, Yuekun; Duff, Michael; Henningson, Jamie; Liu, Qinfang; Li, Yuhao; Nagy, Abdou; Bawa, Bhupinder; Li, Zejun; Tong, Guangzhi; Richt, Juergen A.; Ma, Wenjun

2017-01-01

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses. PMID:28142079

Replication of H9 influenza viruses in the human ex vivo respiratory tract, and the influence of neuraminidase on virus release.

PubMed

Chan, Renee W Y; Chan, Louisa L Y; Mok, Chris K P; Lai, Jimmy; Tao, Kin P; Obadan, Adebimpe; Chan, Michael C W; Perez, Daniel R; Peiris, J S Malik; Nicholls, John M

2017-07-24

H9N2 viruses are the most widespread influenza viruses in poultry in Asia. We evaluated the infection and tropism of human and avian H9 influenza virus in the human respiratory tract using ex vivo respiratory organ culture. H9 viruses infected the upper and lower respiratory tract and the majority of H9 viruses had a decreased ability to release virus from the bronchus rather than the lung. This may be attributed to a weak neuraminidase (NA) cleavage of carbon-6-linked sialic acid (Sia) rather than carbon-3-linked Sia. The modified cleavage of N-acetlylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) by NA in H9 virus replication was observed by reverse genetics, and recombinant H9N2 viruses with amino acids (38KQ) deleted in the NA stalk, and changing the amino acid at position 431 from Proline-to-Lysine. Using recombinant H9 viruses previously evaluated in the ferret, we found that viruses which replicated well in the ferret did not replicate to the same extent in the human ex vivo cultures. The existing risk assessment models for H9N2 viruses in ferrets may not always have a strong correlation with the replication in the human upper respiratory tract. The inclusion of the human ex vivo cultures would further strengthen the future risk-assessment strategies.

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals

PubMed Central

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-01-01

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans. PMID:27094903

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

PubMed

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-04-20

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

Vaccination with a Porcine Reproductive and Respiratory Syndrome (PRRS) Modified Live Virus Vaccine Followed by Challenge with PRRS Virus and Porcine Circovirus Type 2 (PCV2) Protects against PRRS but Enhances PCV2 Replication and Pathogenesis Compared to Results for Nonvaccinated Cochallenged Controls.

PubMed

Niederwerder, Megan C; Bawa, Bhupinder; Serão, Nick V L; Trible, Benjamin R; Kerrigan, Maureen A; Lunney, Joan K; Dekkers, Jack C M; Rowland, Raymond R R

2015-12-01

Coinfections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) contribute to a group of disease syndromes known as porcine circovirus-associated disease (PCVAD). Presumably, PRRSV infection enhances PCV2 replication as a result of modulation of host immunity. The purpose of this study was to evaluate PCV2 replication and pathogenesis in pigs vaccinated with a PRRS modified live virus (MLV) vaccine and subsequently challenged with a combination of PRRSV and PCV2. During the early postchallenge period, the number of pigs with PRRSV-associated clinical signs was decreased, and average daily gain (ADG) was increased, in the vaccinated group, demonstrating the protective effect of PRRS vaccination. However, during the later postchallenge period, more pigs in the vaccinated group showed increased PCV2 viremia, decreased ADG, increased PCVAD clinical signs, and increased mortality. In this disease model, the early benefits of PRRSV vaccination were outweighed by the later amplification of PCVAD. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Vaccination with a Porcine Reproductive and Respiratory Syndrome (PRRS) Modified Live Virus Vaccine Followed by Challenge with PRRS Virus and Porcine Circovirus Type 2 (PCV2) Protects against PRRS but Enhances PCV2 Replication and Pathogenesis Compared to Results for Nonvaccinated Cochallenged Controls

PubMed Central

Bawa, Bhupinder; Serão, Nick V. L.; Trible, Benjamin R.; Kerrigan, Maureen A.; Lunney, Joan K.; Dekkers, Jack C. M.; Rowland, Raymond R. R.

2015-01-01

Coinfections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) contribute to a group of disease syndromes known as porcine circovirus-associated disease (PCVAD). Presumably, PRRSV infection enhances PCV2 replication as a result of modulation of host immunity. The purpose of this study was to evaluate PCV2 replication and pathogenesis in pigs vaccinated with a PRRS modified live virus (MLV) vaccine and subsequently challenged with a combination of PRRSV and PCV2. During the early postchallenge period, the number of pigs with PRRSV-associated clinical signs was decreased, and average daily gain (ADG) was increased, in the vaccinated group, demonstrating the protective effect of PRRS vaccination. However, during the later postchallenge period, more pigs in the vaccinated group showed increased PCV2 viremia, decreased ADG, increased PCVAD clinical signs, and increased mortality. In this disease model, the early benefits of PRRSV vaccination were outweighed by the later amplification of PCVAD. PMID:26446422

Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes. Copyright © 2018 American Society for Microbiology.

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

PubMed Central

Foy, Niall J.; Akhrymuk, Maryna; Shustov, Alexander V.; Frolova, Elena I.

2013-01-01

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs. PMID:23637407

Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents

PubMed Central

Rosenfeld, Paul; Turner, Patricia V.; MacInnes, Janet I.; Nagy, Éva; Yoo, Dongwan

2009-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. PMID:20046635

Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: whole-body assay and immunohistochemistry

USGS Publications Warehouse

Yamamoto, T.; Batts, W.N.; Arakawa, C.K.; Winton, J.R.

1990-01-01

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.

Zika Virus RNA Replication and Persistence in Brain and Placental Tissue

PubMed Central

Rabeneck, Demi B.; Martines, Roosecelis B.; Reagan-Steiner, Sarah; Ermias, Yokabed; Estetter, Lindsey B.C.; Suzuki, Tadaki; Ritter, Jana; Keating, M. Kelly; Hale, Gillian; Gary, Joy; Muehlenbachs, Atis; Lambert, Amy; Lanciotti, Robert; Oduyebo, Titilope; Meaney-Delman, Dana; Bolaños, Fernando; Saad, Edgar Alberto Parra; Shieh, Wun-Ju; Zaki, Sherif R.

2017-01-01

Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections. PMID:27959260

Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

Treesearch

James M. Slavicek; Nancy Hayes-Plazolles

1991-01-01

Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice

PubMed Central

Brostoff, Terza; Pesavento, Patricia A.; Barker, Christopher M.; Kenney, Joan L.; Dietrich, Elizabeth A.; Duggal, Nisha K.; Bosco-Lauth, Angela M.; Brault, Aaron C.

2017-01-01

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (~75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3′-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93–100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development. PMID:27637937

MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice.

PubMed

Brostoff, Terza; Pesavento, Patricia A; Barker, Christopher M; Kenney, Joan L; Dietrich, Elizabeth A; Duggal, Nisha K; Bosco-Lauth, Angela M; Brault, Aaron C

2016-10-17

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (∼75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development. Published by Elsevier Ltd.

CRISPR/Cas13a targeting of RNA virus in plants.

PubMed

Chaudhary, Kulbhushan

2018-05-19

This approach is quite promising to control plant viral diseases and create synthetic networks to better understand the structure/function relationship in RNA and proteins. Plant viruses are obligate intracellular parasites which causes enormous losses in crop yield worldwide. These viruses replicate into infected cells by highjacking host cellular machinery. Over the last two decades, diverse approaches such as conventional breeding, transgenic approach and gene silencing strategies have been used to control RNA viruses, but escaped due to high rate of mutation. Recently, a novel CRISPR enzyme, called Cas13a, has been used engineered to confer RNA viruses resistance in plants. Here, we summarize the recent breakthrough of CRISPR/Cas13a and its applications in RNA biology.

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

PubMed Central

Komoda, Keisuke; Naito, Satoshi; Ishikawa, Masayuki

2004-01-01

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses. PMID:14769932

Satellite RNAs of plant viruses: structures and biological effects.

PubMed Central

Roossinck, M J; Sleat, D; Palukaitis, P

1992-01-01

Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered. PMID:1620065

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication

USGS Publications Warehouse

Debing, Yannick; Winton, James; Neyts, Johan; Dallmeier, Kai

2013-01-01

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2′-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

Cell fusing agent virus and dengue virus mutually interact in Aedes aegypti cell lines.

PubMed

Zhang, Guangmei; Asad, Sultan; Khromykh, Alexander A; Asgari, Sassan

2017-07-31

The genus Flavivirus contains more than 70 single-stranded, positive-sense arthropod-borne RNA viruses. Some flaviviruses are particularly medically important to humans and other vertebrates including dengue virus (DENV), West Nile virus, and yellow fever virus. These viruses are transmitted to vertebrates by mosquitoes and other arthropod species. Mosquitoes are also infected by insect-specific flaviviruses (ISFs) that do not appear to be infective to vertebrates. Cell fusing agent virus (CFAV) was the first described ISF, which was discovered in an Aedes aegypti cell culture. We found that while CFAV infection could be significantly reduced by application of RNAi against the NS5 gene, removal of the treatment led to quick restoration of CFAV replication. Interestingly, we found that CFAV infection significantly enhanced replication of DENV, and vice versa, DENV infection significantly enhanced replication of CFAV in mosquito cells. We have shown that CFAV infection leads to increase in the expression of ribonuclease kappa (RNASEK), which is known to promote infection of viruses that rely on endocytosis and pH-dependent entry. Knockdown of RNASEK by dsRNA resulted in reduced DENV replication. Thus, increased expression of RNASEK induced by CFAV is likely to contribute to enhanced DENV replication in CFAV-infected cells.

Engineering T Cells to Functionally Cure HIV-1 Infection.

PubMed

Leibman, Rachel S; Riley, James L

2015-07-01

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients' lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1-resistant cells, redirecting HIV-1-specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1-specific lymphocytes within a patient's own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy-mediated functional cure.

Short Communication: Potential Risk of Replication-Competent Virus in HIV-1 Env-Pseudotyped Virus Preparations.

PubMed

Bilska, Miroslawa; Tang, Haili; Montefiori, David C

2017-04-01

Env-pseudotyped viruses are valuable reagents for studies of HIV-1 neutralizing antibodies. It is often assumed that all pseudovirus particles are capable of only a single round of infection, making them a safe alternative to work with live HIV-1. In this study, we show that some Env-pseudotyped virus preparations give rise to low levels of replication-competent virus. These levels did not compromise results in the TZM-bl neutralization assay; however, their presence highlights a need to adhere to the same level of biosafety when working with Env-pseudotyped viruses that are required for work with replication competent HIV-1.

The Bean Pod Mottle Virus RNA2-Encoded 58-Kilodalton Protein P58 Is Required in cis for RNA2 Accumulation

PubMed Central

Lin, Junyan; Guo, Jiangbo; Finer, John; Dorrance, Anne E.; Redinbaugh, Margaret G.

2014-01-01

ABSTRACT Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction. IMPORTANCE Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus. PMID:24390330

Activation of the N-Ras-PI3K-Akt-mTOR Pathway by Hepatitis C Virus: Control of Cell Survival and Viral Replication

PubMed Central

Mannová, Petra; Beretta, Laura

2005-01-01

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV. PMID:15994768

Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses.

PubMed

Royle, Jamie; Donald, Claire L; Merits, Andres; Kohl, Alain; Varjak, Margus

2017-12-01

The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

You, Jae-Hwan; Reed, Mark L.; Hiscox, Julian A.

The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in themore » C-terminal region of the protein.« less

Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1.

PubMed

Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

2016-07-15

The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species. Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Suppression of Poxvirus Replication by Resveratrol.

PubMed

Cao, Shuai; Realegeno, Susan; Pant, Anil; Satheshkumar, Panayampalli S; Yang, Zhilong

2017-01-01

Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV), the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

Replication of swine and human influenza viruses in juvenile and layer turkey hens.

PubMed

Ali, Ahmed; Yassine, Hadi; Awe, Olusegun O; Ibrahim, Mahmoud; Saif, Yehia M; Lee, Chang-Won

2013-04-12

Since the first reported isolation of swine influenza viruses (SIVs) in turkeys in the 1980s, transmission of SIVs to turkeys was frequently documented. Recently, the 2009 pandemic H1N1 virus, that was thought to be of swine origin, was detected in turkeys with a severe drop in egg production. In this study, we assessed the infectivity of different mammalian influenza viruses including swine, pandemic H1N1 and seasonal human influenza viruses in both juvenile and layer turkeys. In addition, we investigated the potential influenza virus dissemination in the semen of experimentally infected turkey toms. Results showed that all mammalian origin influenza viruses tested can infect turkeys. SIVs were detected in respiratory and digestive tracts of both juvenile and layer turkeys. Variations in replication efficiencies among SIVs were observed especially in the reproductive tract of layer turkeys. Compared to SIVs, limited replication of seasonal human H1N1 and no detectable replication of recent human-like swine H1N2, pandemic H1N1 and seasonal human H3N2 viruses was noticed. All birds seroconverted to all tested viruses regardless of their replication level. In turkey toms, we were able to detect swine H3N2 virus in semen and reproductive tract of infected toms by real-time RT-PCR although virus isolation was not successful. These data suggest that turkey hens could be affected by diverse influenza strains especially SIVs. Moreover, the differences in the replication efficiency we demonstrated among SIVs and between SIV and human influenza viruses in layer turkeys suggest a possible use of turkeys as an animal model to study host tropism and pathogenesis of influenza viruses. Our results also indicate a potential risk of venereal transmission of influenza viruses in turkeys. Copyright © 2012 Elsevier B.V. All rights reserved.

The progressive adaptation of a georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

PubMed

Krug, Peter W; Holinka, Lauren G; O'Donnell, Vivian; Reese, Bo; Sanford, Brenton; Fernandez-Sainz, Ignacio; Gladue, Douglas P; Arzt, Jonathan; Rodriguez, Luis; Risatti, Guillermo R; Borca, Manuel V

2015-02-01

African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Progressive Adaptation of a Georgian Isolate of African Swine Fever Virus to Vero Cells Leads to a Gradual Attenuation of Virulence in Swine Corresponding to Major Modifications of the Viral Genome

PubMed Central

Krug, Peter W.; Holinka, Lauren G.; O'Donnell, Vivian; Reese, Bo; Sanford, Brenton; Fernandez-Sainz, Ignacio; Gladue, Douglas P.; Arzt, Jonathan; Rodriguez, Luis; Risatti, Guillermo R.

2014-01-01

ABSTRACT African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCE The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection. PMID:25505073

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line.

PubMed

Yang, Darong; Zuo, Chaohui; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin; Liu, Nianli; Yu, Rong; Qin, Yuwen; Gao, Yimin; Wang, Qiuping; Hu, Jun; Wang, Ling; Zhou, Zebin; Liu, Bing; Tan, Deming; Guan, Yang; Zhu, Haizhen

2014-04-01

The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors' knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

PubMed

Kimball, Kristopher J; Rivera, Angel A; Zinn, Kurt R; Icyuz, Mert; Saini, Vaibhav; Li, Jing; Zhu, Zeng B; Siegal, Gene P; Douglas, Joanne T; Curiel, David T; Alvarez, Ronald D; Borovjagin, Anton V

2009-01-01

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Analysis of BZLF1 mRNA detection in saliva as a marker for active replication of Epstein-Barr virus.

PubMed

Fagin, Ursula; Nerbas, Linda; Vogl, Bastian; Jabs, Wolfram J

2017-06-01

Monitoring replicative Epstein-Barr virus (EBV) infection still remains a challenge in modern laboratory routine. The immediate-early protein BZLF1 mediates the switch between latent and replicate forms of EBV infection. The aim of this study was to analyze the feasibility of BZLF1 mRNA detection in saliva as a marker for active replication of the virus. Various specimens (saliva, plasma, PBMC) from 17 patients with EBV-induced infectious mononucleosis (IM) and 4 control patients were examined for expression of viral BZLF1 mRNA by means of real-time PCR. BZLF1 expression was correlated to the amount of viral DNA in either compartment. Digestion of plasma and saliva samples with DNase I allowed distinguishing between encapsidated and naked viral DNA. BZLF1 transcripts were found in all different types of specimens in varying frequencies. BZLF1 expression in saliva, PBMC, and plasma correlated with viral load in each compartment. Interestingly, those patients with detectable BZLF1 expression in saliva had a more severe course of infection with longer duration of hospitalization. In conclusion, this study demonstrates the feasibility of BZLF1 mRNA detection in saliva specimens during replicative EBV infection. Its significance for the diagnosis of reactivated EBV infection, particularly under immunosuppression, has to be elucidated in further studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

PubMed

Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

2017-08-15

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

NASA Technical Reports Server (NTRS)

Butel, J. S.

2000-01-01

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.

Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question

PubMed Central

Koonin, Eugene V.; Starokadomskyy, Petro

2016-01-01

The question whether or not “viruses are alive” has caused considerable debate over many years. Yet, the question is effectively without substance because the answer depends entirely on the definition of life or the state of “being alive” that is bound to be arbitrary. In contrast, the status of viruses among biological entities is readily defined within the replicator paradigm. All biological replicators form a continuum along the selfishness-cooperativity axis, from the completely selfish to fully cooperative forms. Within this range, typical, lytic viruses represent the selfish extreme whereas temperate viruses and various mobile elements occupy positions closer to the middle of the range. Selfish replicators not only belong to the biological realm but are intrinsic to any evolving system of replicators. No such system can evolve without the emergence of parasites, and moreover, parasites drive the evolution of biological complexity at multiple levels. The history of life is a story of parasite-host coevolution that includes both the incessant arms race and various forms of cooperation. All organisms are communities of interacting, coevolving replicators of different classes. A complete theory of replicator coevolution remains to be developed, but it appears likely that not only the differentiation between selfish and cooperative replicators but the emergence of the entire range of replication strategies, from selfish to cooperative, is intrinsic to biological evolution. PMID:26965225

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

PubMed Central

Hyodo, Kiwamu; Taniguchi, Takako; Manabe, Yuki; Kaido, Masanori; Mise, Kazuyuki; Sugawara, Tatsuya; Taniguchi, Hisaaki; Okuno, Tetsuro

2015-01-01

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate. PMID:26020241

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

PubMed Central

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C.; Miller, W. Allen

2016-01-01

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits. PMID:27869713

Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

PubMed

Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

2010-09-01

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

PubMed Central

Papior, Peer; Arteaga-Salas, José M.; Günther, Thomas; Grundhoff, Adam

2012-01-01

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle. PMID:22891264

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

PubMed Central

Liu, XueQiao

2014-01-01

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication. PMID:24227856

VZV Replication Assays

PubMed Central

Griffiths, Samantha J.; Haas, Jürgen

2017-01-01

Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the development of disease. This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. As VZV is highly cell-associated in tissue culture, the reporter virus inoculum described here is a preparation of infected cells. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication. PMID:29085851

The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route.

PubMed

Fujimoto, Yoshikazu; Ito, Hiroshi; Ono, Etsuro; Kawaoka, Yoshihiro; Ito, Toshihiro

2016-04-01

Influenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine. Neuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Influenza A Virus Polymerase Is a Site for Adaptive Changes during Experimental Evolution in Bat Cells

PubMed Central

Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.

2014-01-01

ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity. PMID:25142579

MicroRNA regulation of human protease genes essential for influenza virus replication.

PubMed

Meliopoulos, Victoria A; Andersen, Lauren E; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J Keegan; Tompkins, S Mark; Tripp, Ralph A

2012-01-01

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

A stem–loop structure in the 59 untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought ...

Mechanisms of DNA replication termination.

PubMed

Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

Viral Activation of Cellular Metabolism

PubMed Central

Sanchez, Erica L.; Lagunoff, Michael

2015-01-01

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

PubMed Central

Tawaratsumida, Kazuki; Phan, Van; Hrincius, Eike R.; High, Anthony A.; Webby, Richard; Redecke, Vanessa

2014-01-01

ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense. PMID:24899174

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

PubMed

Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

2015-01-01

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication.

PubMed

Debing, Yannick; Winton, James; Neyts, Johan; Dallmeier, Kai

2013-10-01

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2'-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae. Copyright © 2013 Elsevier B.V. All rights reserved.

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

PubMed

Zhang, Shishuo; Wang, Ruifang; Su, Huijuan; Wang, Biaoxiong; Sizhu, Suolang; Lei, Zhixin; Jin, Meilin; Chen, Huanchun; Cao, Jiyue; Zhou, Hongbo

2017-04-03

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

Molecular Basis of Latency in Pathogenic Human Viruses

NASA Astrophysics Data System (ADS)

Garcia-Blanco, Mariano A.; Cullen, Bryan R.

1991-11-01

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of Foot-and-Mouth Disease Virus in host cells.

USDA-ARS?s Scientific Manuscript database

Foot-and-Mouth Disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergenc...

Viruses within the Flaviviridae Decrease CD4 Expression and Inhibit HIV Replication in Human CD4+ Cells1

PubMed Central

Xiang, Jinhua; McLinden, James H.; Rydze, Robert A.; Chang, Qing; Kaufman, Thomas M.; Klinzman, Donna; Stapleton, Jack T.

2013-01-01

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbes in coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people (dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A) and the DV NS5 protein in CD4+ T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replication is conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitis C virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4+ T cells. All NS5 proteins inhibited HIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression. Infection of CD4+ T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 gene expression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumps infection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteins studied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replication inhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategies for HIV and immunological diseases. PMID:19923460

Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves.

PubMed

Eskelin, K; Suntio, T; Hyvärinen, S; Hafren, A; Mäkinen, K

2010-03-01

A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renillareniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Antiviral Activities of Sulfated Polysaccharides Isolated from Sphaerococcus coronopifolius (Rhodophytha, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales)

PubMed Central

Bouhlal, Rhimou; Haslin, Camille; Chermann, Jean-Claude; Colliec-Jouault, Sylvia; Sinquin, Corinne; Simon, Gaelle; Cerantola, Stephane; Riadi, Hassane; Bourgougnon, Nathalie

2011-01-01

Water-soluble sulfated polysaccharides isolated from two red algae Sphaerococcus coronopifolius (Gigartinales, Sphaerococcaceae) and Boergeseniella thuyoides (Ceramiales, Rhodomelaceae) collected on the coast of Morocco inhibited in vitro replication of the Human Immunodeficiency Virus (HIV) at 12.5 μg/mL. In addition, polysaccharides were capable of inhibiting the in vitro replication of Herpes simplex virus type 1 (HSV-1) on Vero cells values of EC50 of 4.1 and 17.2 μg/mL, respectively. The adsorption step of HSV-1 to the host cell seems to be the specific target for polysaccharide action. While for HIV-1, these results suggest a direct inhibitory effect on HIV-1 replication by controlling the appearance of the new generations of virus and potential virucidal effect. The polysaccharides from S. coronopifolius (PSC) and B. thuyoides (PBT) were composed of galactose, 3,6-anhydrogalactose, uronics acids, sulfate in ratios of 33.1, 11.0, 7.7 and 24.0% (w/w) and 25.4, 16.0, 3.2, 7.6% (w/w), respectively. PMID:21822410

Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells.

PubMed

Rajan, Devi; Chinnadurai, Raghavan; Keefe, Evan O; Boyoglu-Barnum, Seyhan; Todd, Sean O; Hartert, Tina V; Galipeau, Jacques; Anderson, Larry J

2017-12-01

RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication

PubMed Central

Reynard, Olivier; Nguyen, Xuan-Nhi; Alazard-Dany, Nathalie; Barateau, Véronique; Cimarelli, Andrea; Volchkov, Viktor E.

2015-01-01

The current outbreak of Ebola virus (EBOV) in West Africa has claimed the lives of more than 15,000 people and highlights an urgent need for therapeutics capable of preventing virus replication. In this study we screened known nucleoside analogues for their ability to interfere with EBOV replication. Among them, the cytidine analogue β-d-N4-hydroxycytidine (NHC) demonstrated potent inhibitory activities against EBOV replication and spread at non-cytotoxic concentrations. Thus, NHC constitutes an interesting candidate for the development of a suitable drug treatment against EBOV. PMID:26633464

Computer Virus Protection

ERIC Educational Resources Information Center

Rajala, Judith B.

2004-01-01

A computer virus is a program--a piece of executable code--that has the unique ability to replicate. Like biological viruses, computer viruses can spread quickly and are often difficult to eradicate. They can attach themselves to just about any type of file, and are spread by replicating and being sent from one individual to another. Simply having…

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Rabies Control and Treatment: From Prophylaxis to Strategies with Curative Potential

PubMed Central

Zhu, Shimao; Guo, Caiping

2016-01-01

Rabies is an acute, fatal, neurological disease that affects almost all kinds of mammals. Vaccination (using an inactivated rabies vaccine), combined with administration of rabies immune globulin, is the only approved, effective method for post-exposure prophylaxis against rabies in humans. In the search for novel rabies control and treatment strategies, live-attenuated viruses have recently emerged as a practical and promising approach for immunizing and controlling rabies. Unlike the conventional, inactivated rabies vaccine, live-attenuated viruses are genetically modified viruses that are able to replicate in an inoculated recipient without causing adverse effects, while still eliciting robust and effective immune responses against rabies virus infection. A number of viruses with an intrinsic capacity that could be used as putative candidates for live-attenuated rabies vaccine have been intensively evaluated for therapeutic purposes. Additional novel strategies, such as a monoclonal antibody-based approach, nucleic acid-based vaccines, or small interfering RNAs (siRNAs) interfering with virus replication, could further add to the arena of strategies to combat rabies. In this review, we highlight current advances in rabies therapy and discuss the role that they might have in the future of rabies treatment. Given the pronounced and complex impact of rabies on a patient, a combination of these novel modalities has the potential to achieve maximal anti-rabies efficacy, or may even have promising curative effects in the future. However, several hurdles regarding clinical safety considerations and public awareness should be overcome before these approaches can ultimately become clinically relevant therapies. PMID:27801824

CD8+ lymphocytes control viral replication in SIVmac239-infected rhesus macaques without decreasing the lifespan of productively infected cells.

PubMed

Klatt, Nichole R; Shudo, Emi; Ortiz, Alex M; Engram, Jessica C; Paiardini, Mirko; Lawson, Benton; Miller, Michael D; Else, James; Pandrea, Ivona; Estes, Jacob D; Apetrei, Cristian; Schmitz, Joern E; Ribeiro, Ruy M; Perelson, Alan S; Silvestri, Guido

2010-01-29

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

PubMed Central

Klatt, Nichole R.; Shudo, Emi; Ortiz, Alex M.; Engram, Jessica C.; Paiardini, Mirko; Lawson, Benton; Miller, Michael D.; Else, James; Pandrea, Ivona; Estes, Jacob D.; Apetrei, Cristian; Schmitz, Joern E.; Ribeiro, Ruy M.; Perelson, Alan S.; Silvestri, Guido

2010-01-01

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication. PMID:20126441

Interfering With Lipid Raft Association: A Mechanism to Control Influenza Virus Infection By Sambucus Nigra

PubMed Central

Shahsavandi, Shahla; Ebrahimi, Mohammad Majid; Hasaninejad Farahani, Ameneh

2017-01-01

Sambucus nigra (elder) are broadly used species to treat microbial infections. The potential antiviral activity and mechanism action of elder fruit (EF) in human epithelium cell (A549) cultures infected with H9N2 influenza virus were determined. The effect of various concentrations of EF on influenza virus replication was examined by using virus titration, quantitative real time RT-PCR, fusion and lipid raft assays following two treatment procedures: A) pre-treated H9N2 virus with each concentration of EF extract and transfection of A549 cell cultures, and B) each concentrations of EF was added to H9N2 virus infected-cell cultures following virus adsorption. In both treatments with lower doses of EF increased viral titer as well as synthesized viral nucleoprotein as indicating the herb had no inhibitory effects on virus replication. In (B) trial with higher doses, 40 and 80 μg/mL of EF, a significant decrease in virus titer and viral protein synthesis were shown in EF treated cells indicating the herb affect either entry of viruses or inhibition virus particle release. The results suggest that EF treatment of the influenza virus infected-human epithelial cells may involve in lipid raft association which function as platform for formation of viral membrane fusion and budding. Differencesin treatment time and dose of EF extract in infected cells with influenza virus have a marked effect on the efficacy of the herb. PMID:29201101

Avian Influenza H7N9/13 and H7N7/13: a Comparative Virulence Study in Chickens, Pigeons, and Ferrets

PubMed Central

Kalthoff, Donata; Bogs, Jessica; Grund, Christian; Tauscher, Kerstin; Teifke, Jens P.; Starick, Elke; Harder, Timm

2014-01-01

ABSTRACT Human influenza cases caused by a novel avian H7N9 virus in China emphasize the zoonotic potential of that subtype. We compared the infectivity and pathogenicity of the novel H7N9 virus with those of a recent European avian H7N7 strain in chickens, pigeons, and ferrets. Neither virus induced signs of disease despite substantial replication in inoculated chickens and rapid transmission to contact chickens. Evidence of the replication of both viruses in pigeons, albeit at lower levels of RNA excretion, was also detected. No clear-cut differences between the two H7 isolates emerged regarding replication and antibody development in avian hosts. In ferrets, in contrast, greater replication of the avian H7N9 virus than of the H7N7 strain was observed with significant differences in viral presence, e.g., in nasal wash, lung, and cerebellum samples. Importantly, both viruses showed the potential to spread to the mammal brain. We conclude that efficient asymptomatic viral replication and shedding, as shown in chickens, facilitate the spread of H7 viruses that may harbor zoonotic potential. Biosafety measures are required for the handling of poultry infected with avian influenza viruses of the H7 subtype, independently of their pathogenicity for gallinaceous poultry. IMPORTANCE This study is important to the field since it provides data about the behavior of the novel H7N9 avian influenza virus in chickens, pigeons, and ferrets in comparison with that of a recent low-pathogenicity H7N7 strain isolated from poultry. We clearly show that chickens, but not pigeons, are highly permissive hosts of both H7 viruses, allowing high-titer replication and virus shedding without any relevant clinical signs. In the ferret model, the potential of both viruses to infect mammals could be demonstrated, including infection of the brain. However, the replication efficiency of the H7N9 virus in ferrets was higher than that of the H7N7 strain. In conclusion, valuable data for the risk analysis of low-pathogenicity avian influenza viruses of the H7 subtype are provided that could also be used for the risk assessment of zoonotic potentials and necessary biosafety measures. PMID:24899194

Avian influenza H7N9/13 and H7N7/13: a comparative virulence study in chickens, pigeons, and ferrets.

PubMed

Kalthoff, Donata; Bogs, Jessica; Grund, Christian; Tauscher, Kerstin; Teifke, Jens P; Starick, Elke; Harder, Timm; Beer, Martin

2014-08-01

Human influenza cases caused by a novel avian H7N9 virus in China emphasize the zoonotic potential of that subtype. We compared the infectivity and pathogenicity of the novel H7N9 virus with those of a recent European avian H7N7 strain in chickens, pigeons, and ferrets. Neither virus induced signs of disease despite substantial replication in inoculated chickens and rapid transmission to contact chickens. Evidence of the replication of both viruses in pigeons, albeit at lower levels of RNA excretion, was also detected. No clear-cut differences between the two H7 isolates emerged regarding replication and antibody development in avian hosts. In ferrets, in contrast, greater replication of the avian H7N9 virus than of the H7N7 strain was observed with significant differences in viral presence, e.g., in nasal wash, lung, and cerebellum samples. Importantly, both viruses showed the potential to spread to the mammal brain. We conclude that efficient asymptomatic viral replication and shedding, as shown in chickens, facilitate the spread of H7 viruses that may harbor zoonotic potential. Biosafety measures are required for the handling of poultry infected with avian influenza viruses of the H7 subtype, independently of their pathogenicity for gallinaceous poultry. This study is important to the field since it provides data about the behavior of the novel H7N9 avian influenza virus in chickens, pigeons, and ferrets in comparison with that of a recent low-pathogenicity H7N7 strain isolated from poultry. We clearly show that chickens, but not pigeons, are highly permissive hosts of both H7 viruses, allowing high-titer replication and virus shedding without any relevant clinical signs. In the ferret model, the potential of both viruses to infect mammals could be demonstrated, including infection of the brain. However, the replication efficiency of the H7N9 virus in ferrets was higher than that of the H7N7 strain. In conclusion, valuable data for the risk analysis of low-pathogenicity avian influenza viruses of the H7 subtype are provided that could also be used for the risk assessment of zoonotic potentials and necessary biosafety measures. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Identification of SIV Nef CD8(+) T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model.

PubMed

Nomura, Takushi; Yamamoto, Hiroyuki; Takahashi, Naofumi; Naruse, Taeko K; Kimura, Akinori; Matano, Tetsuro

2014-07-25

Virus-specific CD8(+) T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag206-216 and Gag241-249 epitope-specific CD8(+) T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8(+) T-cell responses induced in all the 90-120-Ia(+) macaques on SIV replication remains unknown. Here, we identified three CD8(+) T-cell epitopes, Nef9-19, Nef89-97, and Nef193-203, associated with 90-120-Ia. Nef9-19 and Nef193-203 epitope-specific CD8(+) T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8(+) T cells, indicating that these CD8(+) T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia. Copyright © 2014 Elsevier Inc. All rights reserved.

p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

NASA Astrophysics Data System (ADS)

Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

1995-02-01

Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.

PubMed

Zhang, Zhenfeng; Filzmayer, Christina; Ni, Yi; Sültmann, Holger; Mutz, Pascal; Hiet, Marie-Sophie; Vondran, Florian W R; Bartenschlager, Ralf; Urban, Stephan

2018-07-01

Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication. HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs. Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2 NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I (DDX58) or TLR3 knock-down, but were almost completely abolished upon MDA5 (IFIH1) depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in HuH7.5 NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2 NTCP and HepaRG NTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication. Active replication of HDV induces an IFN-β/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN-activated state. In contrast to hepatitis B virus, infection with hepatitis D virus induces a strong IFN-β/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of hepatitis D virus replicative intermediates. An IFN-activated state did not prevent hepatitis D virus replication in vitro, indicating that hepatitis D virus is resistant to self-induced innate immune responses and therapeutic IFN treatment. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

PubMed Central

Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

2012-01-01

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

Indirect Transmission of Influenza A Virus between Pig Populations under Two Different Biosecurity Settings

PubMed Central

Allerson, Matt W.; Cardona, Carol J.; Torremorell, Montserrat

2013-01-01

Respiratory disease due to influenza virus is common in both human and swine populations around the world with multiple transmission routes capable of transmitting influenza virus, including indirect routes. The objective of this study was to evaluate the role of fomites in influenza A virus (IAV) transmission between pig populations separated by two different biosecurity settings. Thirty-five pigs were divided into four experimental groups: 10 pigs (1 replicate) were assigned to the infected group (I), 10 pigs (2 replicates of 5 pigs) were assigned to the low biosecurity sentinel group (LB), 10 pigs (2 replicates of 5 pigs) were assigned to the medium biosecurity sentinel group (MB), and 5 pigs (1 replicate) were assigned to the negative control group (NC). Eight of 10 pigs in the infected group were inoculated with IAV and 36 hours following inoculation, personnel movement events took place in order to move potentially infectious clothing and personal protective equipment (PPE) to sentinel pig rooms. Following contact with the infected group, personnel moved to the MB group after designated hygiene measures while personnel moved directly to the LB group. Nasal swabs and blood samples were collected from pigs to assess IAV infection status and fomites were sampled and tested via RRT-PCR. All experimentally inoculated pigs were infected with IAV and 11 of the 144 fomite samples collected following contact with infected pigs were low level positive for IAV genome. One replicate of each sentinel groups LB and MB became infected with IAV and all five pigs were infected over time. This study provides evidence that fomites can serve as an IAV transmission route from infected to sentinel pigs and highlights the need to focus on indirect routes as well as direct routes of transmission for IAV. PMID:23805306

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

PubMed

Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

2016-02-02

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain. Published by Elsevier B.V.

Ferret airway epithelial cell cultures support efficient replication of influenza B virus but not mumps virus.

PubMed

Elderfield, Ruth A; Parker, Lauren; Stilwell, Peter; Roberts, Kim L; Schepelmann, Silke; Barclay, Wendy S

2015-08-01

Ferrets have become the model animal of choice for influenza pathology and transmission experiments as they are permissive and susceptible to human influenza A viruses. However, inoculation of ferrets with mumps virus (MuV) did not lead to successful infections. We evaluated the use of highly differentiated ferret tracheal epithelium cell cultures, FTE, for predicting the potential of ferrets to support respiratory viral infections. FTE cultures supported productive replication of human influenza A and B viruses but not of MuV, whereas analogous cells generated from human airways supported replication of all three viruses. We propose that in vitro strategies using these cultures might serve as a method of triaging viruses and potentially reducing the use of ferrets in viral studies.

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

PubMed

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

PubMed

Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

2013-12-01

Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

Beclin 1 is involved in regulation of apoptosis and autophagy during replication of ectromelia virus in permissive L929 cells.

PubMed

Martyniszyn, Lech; Szulc, Lidia; Boratyńska, Anna; Niemiałtowski, Marek G

2011-12-01

Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au; Centre for Cancer Biology, SA Pathology, Adelaide; Hampton-Smith, Rachel J.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using amore » customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.« less

A thiazepino[4,5-a]benzimidazole derivative hampers the RNA replication of Eurasian serotypes of foot-and-mouth disease virus.

PubMed

Lefebvre, David J; De Vleeschauwer, Annebel R; Goris, Nesya; Van Borm, Steven; Chimirri, Alba; Monforte, Anna Maria; Valdazo-Gonzalez, Begona; King, Donald P; Neyts, Johan; De Clercq, Kris

2014-12-12

The stamping-out policy for the control of foot-and-mouth disease virus (FMDV) in countries that are free from FMD without vaccination has a dramatic socio-economic impact, huge animal welfare issues and may result in the loss of farm animal genetic resources. As an alternative to pre-emptive culling or emergency vaccination we further explore the possibility to use antiviral drugs in the event of an FMD outbreak. In the present study, we tested the in vitro cytotoxicity and anti-FMDV activity of 1,2,4,5-tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole. The molecule was shown to inhibit the replication of reference strains of the Eurasian FMDV serotypes O, A, C and Asia but not the FMDV serotypes from the South African Territories (SAT) neither a related picornavirus, i.e. swine vesicular disease virus. The molecule can be added until 2h post inoculation in a 'single replication cycle experiment' without losing its antiviral activity. The genetic characterization of progressively selected resistant FMD viruses shows that the molecule presumably interacts with the non-structural 2C protein of FMDV. Further studies are required on the use of this molecule in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection and analysis of bovine foamy virus infection by an indicator cell line.

PubMed

Ma, Zhe; Qiao, Wen-tao; Xuan, Cheng-hao; Xie, Jin-hui; Chen, Qi-min; Geng, Yun-qi

2007-07-01

To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. BFV infection was induced by the co-culture method or the transient transfection of the infectious BFV plasmid [pCMV (cytomegalovirus) - BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein-positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. The titer of BFV in fetal bovine lung cells was 4-5-folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co-culture method, and in the meantime was significantly higher than that produced by the infectious clone pCMV-BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. BICL can be used for the titration of the BFV viral infection in non-cytopathic condition. In addition, we provide important evidence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus.

PubMed

Phongphaew, Wallaya; Kobayashi, Shintaro; Sasaki, Michihito; Carr, Michael; Hall, William W; Orba, Yasuko; Sawa, Hirofumi

2017-01-15

Valosin-containing protein (VCP) is classified as a member of the type II AAA + ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

PubMed

Al-Mulla, Hawaa M N; Turrell, Lauren; Smith, Nicola M; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C; Siddell, Stuart G; Neuman, Benjamin W

2014-04-01

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers.

PubMed

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W; Axthelm, Michael K; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M; Edlefsen, Paul T; Piatak, Michael; Estes, Jacob D; Lifson, Jeffrey D; Picker, Louis J

2015-02-01

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Wolbachia wStri Blocks Zika Virus Growth at Two Independent Stages of Viral Replication.

PubMed

Schultz, M J; Tan, A L; Gray, C N; Isern, S; Michael, S F; Frydman, H M; Connor, J H

2018-05-22

Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia w Stri, isolated from Laodelphax striatellus , was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that w Stri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. w Stri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by w Stri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into w Stri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia -infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in w Stri-infected cells. This study's findings increase the potential for application of w Stri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection. IMPORTANCE Dengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont, Wolbachia w Stri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use of Wolbachia w Stri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy. Copyright © 2018 Schultz et al.

HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1–Infected Children Who Received Early Treatment

PubMed Central

Luzuriaga, Katherine; Tabak, Barbara; Garber, Manuel; Chen, Ya Hui; Ziemniak, Carrie; McManus, Margaret M.; Murray, Danielle; Strain, Matthew C.; Richman, Douglas D.; Chun, Tae-Wook; Cunningham, Coleen K.; Persaud, Deborah

2014-01-01

Background. Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)–infected infants controls HIV-1 replication and reduces mortality. Methods. Plasma viremia (lower limit of detection, <2 copies/mL), T-cell activation, HIV-1–specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1–infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth. Results. Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1–specific immune responses. Conclusions. Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies. PMID:24850788

Studies on Sam68 a cell factor involved in the life cycle of foot-and-mouth disease virus

USDA-ARS?s Scientific Manuscript database

As with other RNA viruses, Foot-and-Mouth Disease Virus (FMDV) recruits various host cell factors to assist in translation and replication of the virus genome. While FMDV translation has been thoroughly investigated, much remains unknown regarding replication of the positive-sense RNA genome. In th...

Eilat virus host range restriction is present at multiple levels of the virus life cycle.

PubMed

Nasar, Farooq; Gorchakov, Rodion V; Tesh, Robert B; Weaver, Scott C

2015-01-15

Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described, and in contrast to all other mosquito-borne viruses, it is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis virus (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3' untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV nonstructural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both nonstructural protein (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses. Our work explores the nature of host range restriction of the first "mosquito-only alphavirus," EILV. EILV is related to pathogenic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezuelan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

PubMed Central

Morosky, Stefanie; Lennemann, Nicholas J.

2016-01-01

ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. PMID:26962226

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

PubMed

Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

2016-05-15

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dynamics of virus shedding and in situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with Fibropapillomatosis

USGS Publications Warehouse

Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schettle, Nelli; Ackermann, Mathias

2015-01-01

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

Dynamics of Virus Shedding and In Situ Confirmation of Chelonid Herpesvirus 5 in Hawaiian Green Turtles With Fibropapillomatosis.

PubMed

Work, T M; Dagenais, J; Balazs, G H; Schettle, N; Ackermann, M

2015-11-01

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5. © The Author(s) 2014.

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

PubMed Central

Viglianti, G A; Rubinstein, E P; Graves, K L

1992-01-01

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions. Images PMID:1629957

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2016-06-07

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2016-01-01

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation.

PubMed

Ambrose, R L; Mackenzie, J M

2015-07-01

The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidencemore » that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.« less

MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

PubMed Central

Meliopoulos, Victoria A.; Andersen, Lauren E.; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J. Keegan; Tompkins, S. Mark; Tripp, Ralph A.

2012-01-01

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies. PMID:22606348

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Replication of Heliothis virescens ascovirus in insect cell lines.

PubMed

Asgari, S

2006-09-01

Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

Replication-Competent Influenza A Viruses Expressing Reporter Genes.

PubMed

Breen, Michael; Nogales, Aitor; Baker, Steven F; Martínez-Sobrido, Luis

2016-06-23

Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo.

Replication-Competent Influenza A Viruses Expressing Reporter Genes

PubMed Central

Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

2016-01-01

Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA.

PubMed

Miorin, Lisa; Romero-Brey, Inés; Maiuri, Paolo; Hoppe, Simone; Krijnse-Locker, Jacomine; Bartenschlager, Ralf; Marcello, Alessandro

2013-06-01

Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595

Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

PubMed

Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

2017-06-01

Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

Intestinal replication of influenza A viruses in two mammalian species. Brief report.

PubMed

Kawaoka, Y; Bordwell, E; Webster, R G

1987-01-01

The sites of replication of influenza A viruses in ferrets and pigs were studied. The majority of the swine, equine, and avian influenza A viruses tested were recovered from the intestinal tract of ferrets as well as from the respiratory tract; most of the human influenza viruses studied were recovered only from the respiratory tract. In contrast with ferrets, only Hong Kong/1/68 (H 3 N 2) influenza virus was recovered from the intestinal tract of pigs. Despite the large biological variability found in ferrets and in pigs, the results do establish that the majority of influenza viruses have the potential to replicate in the intestinal tissues of some mammals. Additionally, the study suggests that there are differences among the influenza A viruses in tissue tropism in different mammals. Both viral and host genetic factors determine the tissue tropism of influenza viruses in mammals.

A nuclear factor-κB signaling pathway via protein kinase C δ regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells

PubMed Central

Masaki, Tomoyuki; Kojima, Takashi; Okabayashi, Tamaki; Ogasawara, Noriko; Ohkuni, Tsuyoshi; Obata, Kazufumi; Takasawa, Akira; Murata, Masaki; Tanaka, Satoshi; Hirakawa, Satoshi; Fuchimoto, Jun; Ninomiya, Takafumi; Fujii, Nobuhiro; Tsutsumi, Hiroyuki; Himi, Tetsuo; Sawada, Norimasa

2011-01-01

Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma, and severe lower respiratory tract disease in infants and young children. The airway epithelium, which has a well-developed barrier regulated by tight junctions, is the first line of defense during respiratory virus infection. In upper airway human nasal epithelial cells (HNECs), however, the primary site of RSV infection, the mechanisms of replication and budding of RSV, and the epithelial cell responses, including the tight junctional barrier, remain unknown. To investigate the detailed mechanisms of replication and budding of RSV in HNECs and the epithelial cell responses, we established an RSV-infected model using human telomerase reverse transcriptase–-transfected HNECs. We first found that the expression and barrier function of tight junction molecules claudin-4 and occludin were markedly induced together with production of proinflammatory cytokines interleukin 8 and tumor necrosis factor-α in HNECs after RSV infection, and the induction of tight junction molecules possibly contributed to budding of RSV. Furthermore, the replication and budding of RSV and the epithelial cell responses in HNECs were regulated via a protein kinase C δ/hypoxia-inducible factor-1α/nuclear factor-κB pathway. The control of this pathway in HNECs may be useful not only for prevention of replication and budding of RSV, but also in therapy for RSV-induced respiratory pathogenesis. PMID:21562222

Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho

USGS Publications Warehouse

Purcell, M.K.; Garver, K.A.; Conway, C.; Elliott, D.G.; Kurath, G.

2009-01-01

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control. ?? 2009 Blackwell Publishing Ltd.

Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho.

PubMed

Purcell, M K; Garver, K A; Conway, C; Elliott, D G; Kurath, G

2009-07-01

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control.

Viral Interference and Persistence in Mosquito-Borne Flaviviruses.

PubMed

Salas-Benito, Juan Santiago; De Nova-Ocampo, Mónica

2015-01-01

Mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. However, the epidemiological impact remains to be determined. Mosquito-borne flaviviruses are primarily transmitted through Aedes and Culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. This establishment requires a balance between virus replication and the antiviral host response. Viral interference is a phenomenon whereby one virus inhibits the replication of other viruses, and this condition is frequently associated with persistent infections. Viral interference and persistent infection are determined by several factors, such as defective interfering particles, competition for cellular factors required for translation/replication, and the host antiviral response. The interaction between two flaviviruses typically results in viral interference, indicating that these viruses share common features during the replicative cycle in the vector. The potential mechanisms involved in these processes are reviewed here.

Evaluation of an edible blue-green alga, Aphanothece sacrum, for its inhibitory effect on replication of herpes simplex virus type 2 and influenza virus type A.

PubMed

Ogura, Fumie; Hayashi, Kyoko; Lee, Jung-Bum; Kanekiyo, Kenji; Hayashi, Toshimitsu

2010-01-01

A hot-water extract of Aphanothece sacrum, an edible aquacultured blue-green alga, was found to show a remarkable inhibitory effect on the replication of enveloped viruses including herpes simplex virus type 2 (HSV-2) and influenza virus type A (IFV-A, H1N1) in vitro. The main active components were suggested to be sulfated polysaccharides in non-dialyzable portion (ASWPH). ASWPH was found to inhibit the viral adsorption to the receptor of the host cells involved in the replication process of HSV-2 and IFV-A. In addition, while the penetration stage of HSV-2 was also significantly suppressed with ASWPH, no such effect was observed in the replication of IFV-A. These results suggest that ASWPH might be useful in the prevention of infectious diseases caused by HSV-2 as well as IFV-A.

Recent H1N1 viruses (A/USSR/90/77, A/Fiji/15899/83, A/Firenze/13/83) replicate poorly in ferret bronchial epithelium. Brief report.

PubMed

Sweet, C; Bird, R A; Coates, D M; Overton, H A; Smith, H

1985-01-01

Three recent wild-type H1N1 influenza virus isolates (A/USSR/90/77, A/Fiji/15899/83 and A/Firenze/13/83) replicated poorly in organ cultures of ferret bronchial tissue compared with the replication of an H3N2 wild-type virus (A/England/939/69). All four viruses replicated well in nasal turbinate tissue. Examination of one H1N1 virus (A/USSR/90/77) in vivo showed heavy infection in the upper respiratory tract of ferrets but little in the lower respiratory tract. These results raise the possibility that the mildness of human influenza arising from the H1N1 strains may be due to lack of capacity to attack the lower respiratory tract as well as the presence of antibody in previously exposed persons.

Cooperative vaccinia infection demonstrated at the single-cell level using FluidFM.

PubMed

Stiefel, Philipp; Schmidt, Florian I; Dörig, Pablo; Behr, Pascal; Zambelli, Tomaso; Vorholt, Julia A; Mercer, Jason

2012-08-08

The mechanisms used by viruses to enter and replicate within host cells are subjects of intense investigation. These studies are ultimately aimed at development of new drugs that interfere with these processes. Virus entry and infection are generally monitored by dispensing bulk virus suspensions on layers of cells without accounting for the fate of each virion. Here, we take advantage of the recently developed FluidFM to deposit single vaccinia virions onto individual cells in a controlled manner. While the majority of virions were blocked prior to early gene expression, infection of individual cells increased in a nondeterministic fashion with respect to the number of viruses placed. Microscopic analyses of several stages of the virus lifecycle indicated that this was the result of cooperativity between virions during early stages of infection. These findings highlight the importance of performing controlled virus infection experiments at the single cell level.

Function of duck RIG-I in induction of antiviral response against IBDV and avian influenza virus on chicken cells.

PubMed

Shao, Qiang; Xu, Wenpin; Yan, Li; Liu, Jinhua; Rui, Lei; Xiao, Xiao; Yu, Xiaoxue; Lu, Yanan; Li, Zandong

2014-10-13

The avian influenza (AI) H9N2 virus and IBDV are two major problems in the poultry industry. They have been prevalent among domestic poultry in Asia for many years and have caused considerable economic losses. Retinoic-acid-induced gene I (RIG-I) is a cytoplasmic sensor of dsRNA and ssRNA. It can detect Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) in human cells, influenza virus in duck leads to production of IFN-β and IFN-stimulated antiviral genes and reductions in the replication of RNA virus. Chickens, which lack RIG-I, are more sensitive to influenza virus than ducks. However, little is known about the roles of duck RIG-I (dRIG-I) in the detection of IBDV and AI H9N2 in chicken cells DF-1. The purpose of this study was to examine the function of dRIG-I in the recognition of IBDV Ts strain and H9N2 A/Chicken/Shandong/ZB/2007(ZB07) and in the induction of antiviral gene expression to gain an understanding of antiviral ability of dRIG-I in chicken cells against dsRNA virus IBDV and ssRNA virus ZB07. After challenge with the IBDV Ts strain and ZB07 the expression levels of Type I IFN (IFN-β and IFN-α) and IFN-induced antiviral genes (Mx and PKR) were significantly up-regulated in dRIG-I-transfected DF-1cells compared with the empty-vector-transfected control. dRIG-I knockdown experiments further proved that dRIG-I is essential to sensing IBDV and ZB07 in duck embryo fibroblasts (DEF). Growth curves showed that dRIG-I repressed the replication of IBDV and almost blunted the growth of ZB07 in DF-1. Apoptosis analysis revealed that dRIG-I increase the number of the survival cells after IBDV Ts strain or ZB07 infection relative to the empty-vector-transfected control. These results indicate that dRIG-I can up-regulates type I IFN and reduce viral gene expression and viral replication and protect chicken cells from virus-induced apoptosis during ZB07 and IBDV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Replication of tobacco mosaic virus RNA.

PubMed Central

Buck, K W

1999-01-01

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA. PMID:10212941

Early function of the Abutilon mosaic virus AC2 gene as a replication brake.

PubMed

Krenz, Björn; Deuschle, Kathrin; Deigner, Tobias; Unseld, Sigrid; Kepp, Gabi; Wege, Christina; Kleinow, Tatjana; Jeske, Holger

2015-04-01

The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Molecular Sleds and More: Novel Antiviral Agents via Single-Molecule Biology (441st Brookhaven Lecture)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangel, Wally

2008-10-15

Vaccines are effective against viruses such as polio and measles, but vaccines against other important viruses, such as HIV and flu viruses, may be impossible to obtain. These viruses change their genetic makeup each time they replicate so that the immune system cannot recognize all their variations. Hence it is important to develop new antiviral agents that inhibit virus replication. During this lecture, Dr. Mangel will discuss his group's work with a model system, the human adenovirus, which causes, among other ailments, pink eye, blindness and obesity. Mangel's team has developed a promising drug candidate that works by inihibiting adenovirusmore » proteinase, an enzyme necessary for viral replication.« less

Viral subversion of host functions for picornavirus translation and RNA replication

PubMed Central

Chase, Amanda J; Semler, Bert L

2012-01-01

Picornavirus infections lead to symptoms that can have serious health and economic implications. The viruses in this family (Picornaviridae) have a small genomic RNA and must rely on host proteins for efficient viral gene expression and RNA replication. To ensure their effectiveness as pathogens, picornaviruses have evolved to utilize and/or alter host proteins for the benefit of the virus life cycle. This review discusses the host proteins that are subverted during infection to aid in virus replication. It will also describe proteins and functions that are altered during infection for the benefit of the virus. PMID:23293659

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral position is available in the Viral Recombination Section (VRS), HIV Dynamics and Replication Program, CCR.  The VRS studies retroviral replication using human immunodeficiency viruses and other retroviruses, with a particular emphasis on the mechanisms of viral RNA biology, specific RNA packaging, virus assembly, and HIV replication.  Molecular tools and

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals

PubMed Central

Banga, Riddhima; Procopio, Francesco A.; Ruggiero, Alessandra; Noto, Alessandra; Ohmiti, Khalid; Cavassini, Matthias; Corpataux, Jean-Marc; Paxton, William A.; Pollakis, Georgios; Perreau, Matthieu

2018-01-01

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+ CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+ CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals. PMID:29459864

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals.

PubMed

Banga, Riddhima; Procopio, Francesco A; Ruggiero, Alessandra; Noto, Alessandra; Ohmiti, Khalid; Cavassini, Matthias; Corpataux, Jean-Marc; Paxton, William A; Pollakis, Georgios; Perreau, Matthieu

2018-01-01

We recently demonstrated that lymph nodes (LNs) PD-1 + /T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1 + CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1 + CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

PubMed Central

Hanson, Laura K.; Slater, Jacquelyn S.; Karabekian, Zaruhi; Virgin, Herbert W.; Biron, Christine A.; Ruzek, Melanie C.; van Rooijen, Nico; Ciavarra, Richard P.; Stenberg, Richard M.; Campbell, Ann E.

1999-01-01

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection. PMID:10364349

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

PubMed Central

Pappas, Claudia; Aguilar, Patricia V.; Basler, Christopher F.; Solórzano, Alicia; Zeng, Hui; Perrone, Lucy A.; Palese, Peter; García-Sastre, Adolfo; Katz, Jacqueline M.; Tumpey, Terrence M.

2008-01-01

The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the 1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain. PMID:18287069

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses. PMID:26041291

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

PubMed Central

Truyen, U; Parrish, C R

1992-01-01

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts. Images PMID:1323703

Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets

PubMed Central

Sutton, Troy C.; Lamirande, Elaine W.; Czako, Rita

2017-01-01

ABSTRACT The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound “avian-like” α2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain “human-like” α2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with ≥14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against ≥15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness. IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind “avian-like” sialic acids, although several isolates also displayed binding to “human-like” sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain. PMID:28701401

Applications of Replicating-Competent Reporter-Expressing Viruses in Diagnostic and Molecular Virology.

PubMed

Li, Yongfeng; Li, Lian-Feng; Yu, Shaoxiong; Wang, Xiao; Zhang, Lingkai; Yu, Jiahui; Xie, Libao; Li, Weike; Ali, Razim; Qiu, Hua-Ji

2016-05-06

Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. Notably, the development of replicating-competent reporter-expressing viruses (RCREVs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. This article reviews the applications of RCREVs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. However, there remain various challenges associated with RCREVs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. Despite all these limitations, RCREVs have become powerful tools for both basic and applied virology with the development of new technologies for generating RCREVs, the inventions of novel reporters and the better understanding of regulation of viral replication.

Activation of DNA damage repair pathways by murine polyomavirus.

PubMed

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L

2016-10-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

PubMed Central

Le Boeuf, Fabrice; Lemay, Chantal; De Silva, Naomi; Diallo, Jean-Simon; Cox, Julie; Becker, Michelle; Choi, Youngmin; Ananth, Abhirami; Sellers, Clara; Breton, Sophie; Roy, Dominic; Falls, Theresa; Brun, Jan; Hemminki, Akseli; Hinkkanen, Ari; Bell, John C.

2013-01-01

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. PMID:23221568

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.

PubMed Central

Vanin, E F; Kaloss, M; Broscius, C; Nienhuis, A W

1994-01-01

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution. Images PMID:8207799

A Novel Agonist of the TRIF Pathway Induces a Cellular State Refractory to Replication of Zika, Chikungunya, and Dengue Viruses

PubMed Central

Pryke, Kara M.; Abraham, Jinu; Sali, Tina M.; Gall, Bryan J.; Archer, Iris; Liu, Andrew; Bambina, Shelly; Baird, Jason; Gough, Michael; Chakhtoura, Marita; Haddad, Elias K.; Kirby, Ilsa T.; Nilsen, Aaron; Streblow, Daniel N.; Hirsch, Alec J.; Smith, Jessica L.

2017-01-01

ABSTRACT The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy’s potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity. PMID:28465426

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.

PubMed

Aagaard, Lars; Mikkelsen, Jacob Giehm; Warming, Søren; Duch, Mogens; Pedersen, Finn Skou

2002-02-01

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

PubMed Central

Vereecke, Lars; Mc Guire, Conor; Sze, Mozes; Schuijs, Martijn J.; Willart, Monique; Itati Ibañez, Lorena; Hammad, Hamida; Lambrecht, Bart N.; Beyaert, Rudi; Saelens, Xavier; van Loo, Geert

2016-01-01

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection. PMID:26815999

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

PubMed

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

PubMed

Watashi, Koichi; Ishii, Naoto; Hijikata, Makoto; Inoue, Daisuke; Murata, Takayuki; Miyanari, Yusuke; Shimotohno, Kunitada

2005-07-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice.

PubMed

Long, Kelly R; Lomonosova, Elena; Li, Qilan; Ponzar, Nathan L; Villa, Juan A; Touchette, Erin; Rapp, Stephen; Liley, R Matt; Murelli, Ryan P; Grigoryan, Alexandre; Buller, R Mark; Wilson, Lisa; Bial, John; Sagartz, John E; Tavis, John E

2018-01-01

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development. Copyright © 2017 Elsevier B.V. All rights reserved.

Influenza B virus: alpha-amanitin sensitivity of replication and primer-dependence of in vitro transcription.

PubMed

Mowshowitz, S L; Deval, J

1980-01-01

The replication of influenza B/Lee/40 virus in MDCK (canine kidney) cells was sensitive to alpha-amanitin and actinomycin D. In vitro, virion transcriptase activity was stimulated by dinucleotide primers such as ApG. The above characteristics are shared by A/WSN virus.

Activation of DNA damage repair pathways by murine polyomavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

PubMed

Chotiwan, Nunya; Andre, Barbara G; Sanchez-Vargas, Irma; Islam, M Nurul; Grabowski, Jeffrey M; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D; Belisle, John T; Hill, Catherine A; Kuhn, Richard J; Perera, Rushika

2018-02-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes

PubMed Central

Chotiwan, Nunya; Andre, Barbara G.; Sanchez-Vargas, Irma; Islam, M. Nurul; Grabowski, Jeffrey M.; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D.; Hill, Catherine A.; Kuhn, Richard J.

2018-01-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. PMID:29447265

The IFITMs Inhibit Zika Virus Replication.

PubMed

Savidis, George; Perreira, Jill M; Portmann, Jocelyn M; Meraner, Paul; Guo, Zhiru; Green, Sharone; Brass, Abraham L

2016-06-14

Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses. Copyright © 2016. Published by Elsevier Inc.

Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector

PubMed Central

Liu, Si; Xie, Jiatao; Cheng, Jiasen; Li, Bo; Chen, Tao; Fu, Yanping; Li, Guoqing; Wang, Manqun; Jin, Huanan; Wan, Hu; Jiang, Daohong

2016-01-01

Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a nonhost fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1–like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage. PMID:27791095

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

PubMed Central

Prasanth, K. Reddisiva; Barajas, Daniel

2014-01-01

ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. PMID:25540361

Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity

PubMed Central

Naidoo, Vanessa L.; Mann, Jaclyn K.; Noble, Christie; Adland, Emily; Carlson, Jonathan M.; Thomas, Jake; Brumme, Chanson J.; Thobakgale-Tshabalala, Christina F.; Brumme, Zabrina L.; Goulder, Philip J. R.

2017-01-01

ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that “consensus-like” virus sequences correspond to lower in vitro replication abilities yet appear to be preferentially transmitted suggests that viral characteristics favoring transmission are decoupled from those that enhance replicative capacity. PMID:28637761

Resistance to alpha/beta interferon is a determinant of West Nile virus replication fitness and virulence.

PubMed

Keller, Brian C; Fredericksen, Brenda L; Samuel, Melanie A; Mock, Richard E; Mason, Peter W; Diamond, Michael S; Gale, Michael

2006-10-01

The emergence of West Nile virus (WNV) in the Western Hemisphere is marked by the spread of pathogenic lineage I strains, which differ from typically avirulent lineage II strains. To begin to understand the virus-host interactions that may influence the phenotypic properties of divergent lineage I and II viruses, we compared the genetic, pathogenic, and alpha/beta interferon (IFN-alpha/beta)-regulatory properties of a lineage II isolate from Madagascar (MAD78) with those of a new lineage I isolate from Texas (TX02). Full genome sequence analysis revealed that MAD78 clustered, albeit distantly, with other lineage II strains, while TX02 clustered with emergent North American isolates, more specifically with other Texas strains. Compared to TX02, MAD78 replicated at low levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro, and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling, including the activation of Tyk2 and downstream phosphorylation and nuclear translocation of STAT1 and STAT2. However, replication of MAD78 was rescued in cells with a nonfunctional IFN-alpha/beta receptor (IFNAR). Consistent with this finding, the virulence of MAD78 was unmasked upon infection of mice lacking IFNAR. Thus, control of the innate host response and IFN actions is a key feature of WNV pathogenesis and replication fitness.

Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells

PubMed Central

Hidalgo, Paloma; Anzures, Lourdes; Hernández-Mendoza, Armando; Guerrero, Adán; Wood, Christopher D.; Valdés, Margarita; Dobner, Thomas

2016-01-01

ABSTRACT Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCE RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle. PMID:26764008

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

Possible Increased Pathogenicity of Pandemic (H1N1) 2009 Influenza Virus upon Reassortment

PubMed Central

Schrauwen, Eefje J.A.; Herfst, Sander; Chutinimitkul, Salin; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Kuiken, Thijs

2011-01-01

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses—seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009—have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets. PMID:21291589

Phosphorodiamidate morpholino targeting the 5' untranslated region of the ZIKV RNA inhibits virus replication.

PubMed

Popik, Waldemar; Khatua, Atanu; Hildreth, James E K; Lee, Benjamin; Alcendor, Donald J

2018-06-01

Zika virus (ZIKV) infection has been associated with microcephaly in infants. Currently there is no treatment or vaccine. Here we explore the use of a morpholino oligonucleotide targeted to the 5' untranslated region (5'-UTR) of the ZIKV RNA to prevent ZIKV replication. Morpholino DWK-1 inhibition of ZIKV replication in human glomerular podocytes was examined by qRT-PCR, reduction in ZIKV genome copy number, western blot analysis, immunofluorescence and proinflammatory cytokine gene expression. Podocytes pretreated with DWK-1 showed reduced levels of both viral mRNA and ZIKV E protein expression compared to controls. We observed suppression in proinflammatory gene expression for IFN-β (interferon β) RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1α (macrophage inflammatory protein-1α), TNF-α (tumor necrosis factor-α) and IL1-α (interleukin 1-α) in ZIKV-infected podocytes pretreated with DWK-1. Morpholino DWK-1 targeting the ZIKV 5'-UTR effectively inhibits ZIKV replication and suppresses ZIKV-induced proinflammatory gene expression. Copyright © 2018 Elsevier Inc. All rights reserved.

Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles

PubMed Central

Andresen, Vibeke; Pise-Masison, Cynthia A.; Sinha-Datta, Uma; Bellon, Marcia; Valeri, Valerio; Washington Parks, Robyn; Cecchinato, Valentina; Fukumoto, Risaku; Nicot, Christophe

2011-01-01

Disease development in human T-cell leukemia virus type 1 (HTLV-1)–infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication. PMID:21677314

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2007-10-01

Crimean Congo Hemorrhagic Fever Virus PRINCIPAL INVESTIGATOR: Adolfo García-Sastre, Ph.D. CONTRACTING...Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus 5b. GRANT NUMBER W81XWH-04-1-0876 5c. PROGRAM ELEMENT...localization and antigenic characterization of Crimean - Congo hemorrhagic fever virus glycoproteins. J.Virol. 79: 6152-61. Ahmed, A., McFalls,

The matrix gene segment destabilizes the acid and thermal stability of the hemagglutinin of pandemic live attenuated influenza virus vaccines.

PubMed

O'Donnell, Christopher D; Vogel, Leatrice; Matsuoka, Yumiko; Jin, Hong; Subbarao, Kanta

2014-11-01

The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults. There is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccine (pLAIV) viruses and observed that the pLAIV viruses consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIV viruses may account for their restricted replication in clinical trials. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

PubMed Central

O'Donnell, Christopher D.; Vogel, Leatrice; Matsuoka, Yumiko; Jin, Hong

2014-01-01

ABSTRACT The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults. IMPORTANCE There is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccine (pLAIV) viruses and observed that the pLAIV viruses consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIV viruses may account for their restricted replication in clinical trials. PMID:25122789

N-Myc Interactor Inhibits Prototype Foamy Virus by Sequestering Viral Tas Protein in the Cytoplasm

PubMed Central

Hu, Xiaomei; Yang, Wei; Liu, Ruikang; Geng, Yunqi; Qiao, Wentao

2014-01-01

ABSTRACT Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its depletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection. IMPORTANCE From this study, we report that the N-Myc interactor (Nmi), an interferon-induced protein, can interact with the regulatory protein Tas of the prototype foamy virus and sequester it in the cytoplasm. The results of this study suggest that Nmi plays an important role in maintaining foamy virus latency and may reveal a new pathway in the interferon-mediated antiviral barrier against viruses. These findings are important for understanding virus-host relationships not only with FVs but potentially for other retroviruses as well. PMID:24719420

[Research Progress on Antiviral Activity of Interferon-induced Transmembrane Proteins].

PubMed

Chen, Yongkun; Zhu, Wenfei; Shu, Yuelong

2016-03-01

Interferon-induced Transmembrane Proteins (IFITMs) were identified through small interference RNA (siRNA) screening method in 1980s. The antiviral properties of the IFITMs were firstly discovered in 1996. Recently, its antiviral effect and mechanism have become a research hotspot. Many studies have shown that IFITM can inhibit the replication of multiple pathogenic viruses, including influenza A virus (IAV), Human Immunodeficiency Virus (HIV-1), hepatitis C virus (HCV), Ebola virus (EBOV), West Nile virus and so on. IFITMs inhibit the replication of virus in the early stage of the viral life cycle, which occurred before the release of viral genomes into the cytosol. Recent studies indicate that IFITM proteins could block viral replication by mediate viral membrane fusion. However, the mechanism is still under investigation. Here we review the discovery and characterization of the IFITM proteins, elucidate their antiviral activities and the potential mechanisms.

Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus.

PubMed

Sánchez-Navarro, J A; Reusken, C B; Bol, J F; Pallás, V

1997-12-01

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) are tripartite positive-strand RNA plant viruses that encode functionally similar translation products. Although the two viruses are phylogenetically closely related, they infect a very different range of natural hosts. The coat protein (CP) gene, the movement protein (MP) gene or both genes in AMV RNA 3 were replaced by the corresponding genes of PNRSV. The chimeric viruses were tested for heterologous encapsidation, replication in protoplasts from plants transformed with AMV replicase genes P1 and P2 (P12 plants) and for cell-to-cell transport in P12 plants. The chimeric viruses exhibited basic competence for encapsidation and replication in P12 protoplasts and for a low level of cell-to-cell movement in P12 plants. The potential involvement of the MP gene in determining host specificity in ilarviruses is discussed.

Mink parvoviruses and interferons: in vitro studies.

PubMed Central

Wiedbrauk, D L; Bloom, M E; Lodmell, D L

1986-01-01

Although interferons can inhibit the replication of a number of viruses, little is known about their ability to inhibit parvovirus replication. Therefore, in vitro experiments were done to determine if Aleutian disease virus and mink enteritis virus, two autonomously replicating mink parvoviruses, induced interferon, were sensitive to the effects of interferon, or inhibited the production of interferon. The results indicated that these parvoviruses neither induced nor were sensitive to the effects of interferon. Furthermore, preexisting parvovirus infections did not inhibit poly(I).poly(C)-induced interferon production. This independence from the interferon system may, therefore, be a general property of the autonomously replicating parvoviruses. PMID:2431162

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

PubMed

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R; Li, Melody M H; Rice, Charles M; MacDonald, Margaret R

2016-01-06

DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

PubMed Central

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.

2016-01-01

ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development. PMID:26739057

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum.

PubMed

Verchot, Jeanmarie

2016-11-19

The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus-host interactions will provide new targets for crop improvement.

Experimental infection of mandarin duck with highly pathogenic avian influenza A (H5N8 and H5N1) viruses.

PubMed

Kang, Hyun-Mi; Lee, Eun-Kyoung; Song, Byung-Min; Heo, Gyeong-Beom; Jung, Joojin; Jang, Il; Bae, You-Chan; Jung, Suk Chan; Lee, Youn-Jeong

2017-01-01

A highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in poultry and wild birds in South Korea in January 2014. Here, we determined the pathogenicity and transmissibility of three different clades of H5 viruses in mandarin ducks to examine the potential for wild bird infection. H5N8 (clade 2.3.4.4) replicated more efficiently in the upper and lower respiratory tract of mandarin ducks than two previously identified H5N1 virus clades (clades 2.2 and 2.3.2.1). However, none of the mandarin ducks infected with H5N8 and H5N1 viruses showed severe clinical signs or mortality, and gross lesions were only observed in a few tissues. Viral replication and shedding were greater in H5N8-infected ducks than in H5N1-infected ducks. Recovery of all viruses from control duck in contact with infected ducks indicated that the highly pathogenic H5 viruses spread horizontally through contact. Taken together, these results suggest that H5N8 viruses spread efficiently in mandarin ducks. Further studies of pathogenicity in wild birds are required to examine possible long-distance dissemination via migration routes. Copyright © 2016 Elsevier B.V. All rights reserved.

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

PubMed

Gong, Xiao-Qian; Sun, Ying-Feng; Ruan, Bao-Yang; Liu, Xiao-Min; Wang, Qi; Yang, Hai-Ming; Wang, Shuai-Yong; Zhang, Peng; Wang, Xiu-Hui; Shan, Tong-Ling; Tong, Wu; Zhou, Yan-Jun; Li, Guo-Xin; Zheng, Hao; Tong, Guang-Zhi; Yu, Hai

2017-06-01

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors. PMID:25653442

Autophagic machinery activated by dengue virus enhances virus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Y.-R.; Lei, H.-Y.; Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

2008-05-10

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that ismore » favorable for viral replication.« less

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

PubMed

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

Downregulation of MicroRNA miR-526a by Enterovirus Inhibits RIG-I-Dependent Innate Immune Response

PubMed Central

Xu, Changzhi; He, Xiang; Zheng, Zirui; Zhang, Zhe; Wei, Congwen; Guan, Kai; Hou, Lihua; Zhang, Buchang; Zhu, Lin; Cao, Yuan; Zhang, Yanhong; Cao, Ye; Ma, Shengli; Wang, Penghao; Zhang, Pingping; Xu, Quanbin; Ling, Youguo

2014-01-01

ABSTRACT Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71. PMID:25056901

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

PubMed

Ali, Md Emran; Ishii, Yuko; Taniguchi, Jyun-Ichi; Waliullah, Sumyya; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

2018-05-01

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Further evaluation of alternative air-filtration systems for reducing the transmission of Porcine reproductive and respiratory syndrome virus by aerosol

PubMed Central

Deen, John; Cano, Jean Paul; Batista, Laura; Pijoan, Carlos

2006-01-01

Abstract The purpose of this study was to compare 4 methods for the reduction of aerosol transmission of Porcine reproductive and respiratory syndrome virus (PRRSV): high-efficiency particulate air (HEPA) filtration, 2×-low-cost filtration, bag filtration, and use of a filter tested against particles derived from dioctylphthalate (DOP). The HEPA-filtration system used a prefilter screen, a bag filter (Eurovent [EU] 8 rating), and a HEPA filter (EU13 rating). The low-cost-filtration system contained mosquito netting (prefilter), 2 fiberglass furnace filters, and 2 electrostatic furnace filters. Bag filtration involved the use of a filter rated EU8 and a minimum efficiency reporting value (MERV) of 14. The 95%-DOP, 0.3-μm-filtration system involved a pleat-in-pleat V-bank disposable filter with a 95% efficiency rating for particles 0.3 μm or greater in diameter and ratings of EU9 and MERV 15. No form of intervention was used in the control group. The experimental facilities consisted of 2 chambers connected by a 1.3-m-long duct containing the treatments. Recipient pigs, housed in chamber 2, were exposed to artificial aerosols created by a mechanically operated mister containing modified live PRRSV vaccine located in chamber 1. Aerosol transmission of PRRSV occurred in 0 of the 10 HEPA-filtration replicates, 2 of the 10 bag-filtration replicates, 4 of the 10 low-cost-filtration replicates, 0 of the 10 95%-DOP, 0.3-μm-filtration replicates, and all 10 of the control replicates. Using a similar approach, we further evaluated the HEPA- and 95%-DOP, 0.3-μm-filtration systems. Infection was not observed in any of the 76 HEPA-filtration replicates but was observed in 2 of the 76 95%-DOP, 0.3-μm replicates and 42 of the 50 control replicates. Although the difference between the 95%-DOP, 0.3-μm and control replicates was significant (P < 0.0005), so was the level of failure of the 95%-DOP, 0.3-μm system (P = 0.02). In conclusion, under the conditions of this study, some methods of air filtration were significantly better than others in reducing aerosol transmission of PRRSV, and HEPA filtration was the only system that completely prevented transmission. PMID:16850938

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

PubMed Central

Taylor, R. Travis; Lubick, Kirk J.; Robertson, Shelly J.; Broughton, James P.; Bloom, Marshall E.; Bresnahan, Wade A.; Best, Sonja M.

2011-01-01

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses. PMID:21925107

Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

PubMed

Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

2017-05-22

Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

PubMed Central

Mondal, Arindam; Potts, Gregory K.; Dawson, Anthony R.; Coon, Joshua J.; Mehle, Andrew

2015-01-01

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle. PMID:25867750

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

PubMed

Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

2016-10-01

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Immunization against Small Ruminant Lentiviruses

PubMed Central

Reina, Ramsés; de Andrés, Damián; Amorena, Beatriz

2013-01-01

Multisystemic disease caused by Small Ruminant Lentiviruses (SRLV) in sheep and goats leads to production losses, to the detriment of animal health and welfare. This, together with the lack of treatments, has triggered interest in exploring different strategies of immunization to control the widely spread SRLV infection and, also, to provide a useful model for HIV vaccines. These strategies involve inactivated whole virus, subunit vaccines, DNA encoding viral proteins in the presence or absence of plasmids encoding immunological adjuvants and naturally or artificially attenuated viruses. In this review, we revisit, comprehensively, the immunization strategies against SRLV and analyze this double edged tool individually, as it may contribute to either controlling or enhancing virus replication and/or disease. PMID:23917352

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

PubMed

Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

2017-02-14

Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature. Copyright © 2017 Jose et al.

ICAM-3 influences human immunodeficiency virus type 1 replication in CD4+ T-cells independent of DC-SIGN-mediated transmission

PubMed Central

Biggins, Julia E.; Biesinger, Tasha; Yu Kimata, Monica T.; Arora, Reetakshi; Kimata, Jason T.

2007-01-01

We investigated the role of ICAM-3 in DC-SIGN-mediated human immunodeficiency virus (HIV) infection of CD4+ T cells. Our results demonstrate that ICAM-3 does not appear to play a role in DC-SIGN-mediated infection of CD4+ T cells as virus is transmitted equally to ICAM-3+ or ICAM-3− Jurkat T cells. However, HIV-1 replication is enhanced in ICAM-3− cells, suggesting that ICAM-3 may limit HIV-1 replication. Similar results were obtained when SIV replication was examined in ICAM-3+ and ICAM-3− CEMx174 cells. Furthermore, while ICAM-3 has been proposed to play a co-stimulatory role in T cell activation, DC-SIGN expression on antigen presenting cells did not enhance antigen-dependent activation of T cells. Together, these data indicate that while ICAM-3 may influence HIV-1 replication, it does so independent of DC-SIGN mediated virus transmission or activation of CD4+ T cells. PMID:17434553

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp.

PubMed

Ongvarrasopone, Chalermporn; Chomchay, Ekapol; Panyim, Sakol

2010-10-01

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp. Copyright © 2010 Elsevier B.V. All rights reserved.

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

PubMed Central

Hsu, Shih-Feng; Su, Wen-Chi; Jeng, King-Song

2015-01-01

ABSTRACT Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection. PMID:25589657

Unraveling the Armor of a Killer: Evasion of Host Defenses by African Swine Fever Virus.

PubMed

Reis, Ana Luisa; Netherton, Chris; Dixon, Linda K

2017-03-15

African swine fever is an acute hemorrhagic disease of pigs. Extensive recent spread in the Russian Federation and Eastern Europe has increased the risk to global pig production. The virus is a large DNA virus and is the only member of the Asfarviridae family. In pigs, the virus replicates predominantly in macrophages. We review how the virus overcomes the barriers to replication in the macrophage and the virus mechanism to inhibit key host defense pathways. Copyright © 2017 American Society for Microbiology.

Infection of Mosquito Cells (C6/36) by Dengue-2 Virus Interferes with Subsequent Infection by Yellow Fever Virus.

PubMed

Abrao, Emiliana Pereira; da Fonseca, Benedito Antônio Lopes

2016-02-01

Dengue is one of the most important diseases caused by arboviruses in the world. Yellow fever is another arthropod-borne disease of great importance to public health that is endemic to tropical regions of Africa and the Americas. Both yellow fever and dengue viruses are flaviviruses transmitted by Aedes aegypti mosquitoes, and then, it is reasonable to consider that in a given moment, mosquito cells could be coinfected by both viruses. Therefore, we decided to evaluate if sequential infections of dengue and yellow fever viruses (and vice-versa) in mosquito cells could affect the virus replication patterns. Using immunofluorescence and real-time PCR-based replication assays in Aedes albopictus C6/36 cells with single or sequential infections with both viruses, we demonstrated the occurrence of viral interference, also called superinfection exclusion, between these two viruses. Our results show that this interference pattern is particularly evident when cells were first infected with dengue virus and subsequently with yellow fever virus (YFV). Reduction in dengue virus replication, although to a lower extent, was also observed when C6/36 cells were initially infected with YFV followed by dengue virus infection. Although the importance that these findings have on nature is unknown, this study provides evidence, at the cellular level, of the occurrence of replication interference between dengue and yellow fever viruses and raises the question if superinfection exclusion could be a possible explanation, at least partially, for the reported lack of urban yellow fever occurrence in regions where a high level of dengue transmission occurs.

RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

PubMed

Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

2014-01-01

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

A virocentric perspective on the evolution of life

PubMed Central

Koonin, Eugene V.; Dolja, Valerian V.

2015-01-01

Viruses and/or virus-like selfish elements are associated with all cellular life forms and are the most abundant biological entities on Earth, with the number of virus particles in many environments exceeding the number of cells by one to two orders of magnitude. The genetic diversity of viruses is commensurately enormous and might substantially exceed the diversity of cellular organisms. Unlike cellular organisms with their uniform replication-expression scheme, viruses possess either RNA or DNA genomes and exploit all conceivable replication-expression strategies. Although viruses extensively exchange genes with their hosts, there exists a set of viral hallmark genes that are shared by extremely diverse groups of viruses to the exclusion of cellular life forms. Coevolution of viruses and host defense systems is a key aspect in the evolution of both viruses and cells, and viral genes are often recruited for cellular functions. Together with the fundamental inevitability of the emergence of genomic parasites in any evolving replicator system, these multiple lines of evidence reveal the central role of viruses in the entire evolution of life. PMID:23850169

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus▿

PubMed Central

Jones, Daniel M.; Patel, Arvind H.; Targett-Adams, Paul; McLauchlan, John

2009-01-01

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release. PMID:19073716

Cyclophilin B facilitates the replication of Orf virus.

PubMed

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-06-15

Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model

PubMed Central

Sreenivasan, Chithra; Thomas, Milton; Sheng, Zizhang; Hause, Ben M.; Collin, Emily A.; Knudsen, David E. B.; Pillatzki, Angela; Nelson, Eric; Wang, Dan; Kaushik, Radhey S.

2015-01-01

ABSTRACT Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. IMPORTANCE Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV. PMID:26378161

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

PubMed

Sreenivasan, Chithra; Thomas, Milton; Sheng, Zizhang; Hause, Ben M; Collin, Emily A; Knudsen, David E B; Pillatzki, Angela; Nelson, Eric; Wang, Dan; Kaushik, Radhey S; Li, Feng

2015-12-01

Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families.

PubMed

Pinto, Amelia K; Williams, Graham D; Szretter, Kristy J; White, James P; Proença-Módena, José Luiz; Liu, Gai; Olejnik, Judith; Brien, James D; Ebihara, Hideki; Mühlberger, Elke; Amarasinghe, Gaya; Diamond, Michael S; Boon, Adrianus C M

2015-09-01

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5'-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine

PubMed Central

Solórzano, Alicia; Foni, Emanuela; Córdoba, Lorena; Baratelli, Massimiliano; Razzuoli, Elisabetta; Bilato, Dania; Martín del Burgo, María Ángeles; Perlin, David S.; Martínez, Jorge; Martínez-Orellana, Pamela; Fraile, Lorenzo; Chiapponi, Chiara; Amadori, Massimo; del Real, Gustavo

2015-01-01

ABSTRACT Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. IMPORTANCE Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs. PMID:26311894

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

PubMed

Solórzano, Alicia; Foni, Emanuela; Córdoba, Lorena; Baratelli, Massimiliano; Razzuoli, Elisabetta; Bilato, Dania; Martín del Burgo, María Ángeles; Perlin, David S; Martínez, Jorge; Martínez-Orellana, Pamela; Fraile, Lorenzo; Chiapponi, Chiara; Amadori, Massimo; del Real, Gustavo; Montoya, María

2015-11-01

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

PubMed

Subramanian, Gayatri; Kuzmanovic, Teodora; Zhang, Ying; Peter, Cara Beate; Veleeparambil, Manoj; Chakravarti, Ritu; Sen, Ganes C; Chattopadhyay, Saurabh

2018-01-01

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Apoptosis induced in an early step of African swine fever virus entry into vero cells does not require virus replication.

PubMed

Carrascosa, Angel L; Bustos, María J; Nogal, María L; González de Buitrago, Gonzalo; Revilla, Yolanda

2002-03-15

Permissive Vero cells develop apoptosis, as characterized by DNA fragmentation, caspases activation, cytosolic release of mitochondrial cytochrome c, and flow cytometric analysis of DNA content, upon infection with African swine fever virus (ASFV). To determine the step in virus replication that triggers apoptosis, we used UV-inactivated virus, inhibitors of protein and nucleic acid synthesis, and lysosomotropic drugs that block virus uncoating. ASFV-induced apoptosis was accompanied by caspase-3 activation, which was detected even in the presence of either cytosine arabinoside or cycloheximide, indicating that viral DNA replication and protein synthesis were not required to activate the apoptotic process. The activation of caspase-3 was released from chloroquine inhibition 2 h after virus absorption, while the infection with UV-inactivated ASFV did not induce the activation of the caspase cascade. We conclude that ASFV induces apoptosis in the infected cell by an intracellular pathway probably triggered during the process of virus uncoating.

Are Ducks Contributing to the Endemicity of Highly Pathogenic H5N1 Influenza Virus in Asia?†

PubMed Central

Sturm-Ramirez, K. M.; Hulse-Post, D. J.; Govorkova, E. A.; Humberd, J.; Seiler, P.; Puthavathana, P.; Buranathai, C.; Nguyen, T. D.; Chaisingh, A.; Long, H. T.; Naipospos, T. S. P.; Chen, H.; Ellis, T. M.; Guan, Y.; Peiris, J. S. M.; Webster, R. G.

2005-01-01

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health. PMID:16103179

Studies of Genetic Variation in the AIDS Virus: Relevance to Disease Pathogenesis Anti-Viral Therapy, and Vaccine Development

DTIC Science & Technology

1990-06-30

lentiviral systems including equine infectious anemia virus (EIAV), visna virus, and simian immunodeficiency virus (SIV) (119,120,154). For EIAV, it is clear...a pig-tailed macaque that possesses altered biologic and antigenic properties leading to a broader host-range and a rapid, fatal immunodeficiency ...envelope/LTR region of a replication-defective variant of feline leukemia virus (FeLV), when introduced into a replication competent construct of FeLV, was

Cross-protection in nonhuman primates against Argentine hemorrhagic fever.

PubMed Central

Weissenbacher, M C; Coto, C E; Calello, M A; Rondinone, S N; Damonte, E B; Frigerio, M J

1982-01-01

The susceptibility of the marmoset Callithrix jacchus to Tacaribe virus infection was investigated to perform cross-protection studies between Junin and Tacaribe viruses. Five marmosets inoculated with Tacaribe virus failed to show any signs of disease, any alterations in erythrocyte, leukocyte, reticulocyte, and platelet counts or any changes in hematocrit or hemoglobin values. No Tacaribe virus could be recovered from blood at any time postinfection. Anti-Tacaribe neutralizing antibodies appeared 3 weeks postinfection. The five Tacaribe-infected marmosets and four noninfected controls were challenged with the pathogenic strain of Junin virus on day 60 post-Tacaribe infection. The former group showed no signs of disease, no viremia, and no challenge virus replication, whereas the control group exhibited the typical symptoms of Argentine hemorrhagic fever, high viremia, and viral titers in organs. Soon after challenge, the Tacaribe-protected marmosets synthesized neutralizing antibodies against Junin virus. These results indicate that the marmoset C. jacchus can be considered an experimental model for protection studies with arenaviruses and that the Tacaribe virus could be considered as a potential vaccine against Junin virus. PMID:6276301

Chimpanzee GB virus C and GB virus A E2 envelope glycoproteins contain a peptide motif that inhibits human immunodeficiency virus type 1 replication in human CD4+ T-cells

PubMed Central

McLinden, James H.; Stapleton, Jack T.; Klinzman, Donna; Murthy, Krishna K.; Chang, Qing; Kaufman, Thomas M.; Bhattarai, Nirjal

2013-01-01

GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4+ Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus–HIV interactions. PMID:23288422

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection

PubMed Central

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N.; Gestwicki, Jason E.; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-01-01

Summary Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. PMID:26582131

PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins.

PubMed

Li, Lili; Zhao, Hui; Liu, Ping; Li, Chunfeng; Quanquin, Natalie; Ji, Xue; Sun, Nina; Du, Peishuang; Qin, Cheng-Feng; Lu, Ning; Cheng, Genhong

2018-06-19

Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

PubMed Central

Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2015-01-01

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

A novel sheet-like virus particle array is a hallmark of Zika virus infection.

PubMed

Liu, Jun; Kline, Brandon A; Kenny, Tara A; Smith, Darci R; Soloveva, Veronica; Beitzel, Brett; Pang, Song; Lockett, Stephen; Hess, Harald F; Palacios, Gustavo; Kuhn, Jens H; Sun, Mei G; Zeng, Xiankun

2018-04-25

Zika virus (ZIKV) is an emerging flavivirus that caused thousands of human infections in recent years. Compared to other human flaviviruses, ZIKV replication is not well understood. Using fluorescent, transmission electron, and focused ion beam-scanning electron microscopy, we examined ZIKV replication dynamics in Vero 76 cells and in the brains of infected laboratory mice. We observed the progressive development of a perinuclear flaviviral replication factory both in vitro and in vivo. In vitro, we illustrated the ZIKV lifecycle from particle cell entry to egress. ZIKV particles assembled and aggregated in an induced convoluted membrane structure and ZIKV strain-specific membranous vesicles. While most mature virus particles egressed via membrane budding, some particles also likely trafficked through late endosomes and egressed through membrane abscission. Interestingly, we consistently observed a novel sheet-like virus particle array consisting of a single layer of ZIKV particles. Our study further defines ZIKV replication and identifies a novel hallmark of ZIKV infection.

Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine.

PubMed

Mancera Gracia, Jose Carlos; Van den Hoecke, Silvie; Saelens, Xavier; Van Reeth, Kristien

2017-01-01

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations.

Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine

PubMed Central

Van den Hoecke, Silvie; Saelens, Xavier; Van Reeth, Kristien

2017-01-01

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations. PMID:28384328

Timing Is Everything: Coordinated Control of Host Shutoff by Influenza A Virus NS1 and PA-X Proteins

PubMed Central

Khaperskyy, Denys A.

2015-01-01

Like all viruses, influenza viruses (IAVs) use host translation machinery to decode viral mRNAs. IAVs ensure efficient translation of viral mRNAs through host shutoff, a process whereby viral proteins limit the accumulation of host proteins through subversion of their biogenesis. Despite its small genome, the virus deploys multiple host shutoff mechanisms at different stages of infection, thereby ensuring successful replication while limiting the communication of host antiviral responses. In this Gem, we review recent data on IAV host shutoff proteins, frame the outstanding questions in the field, and propose a temporally coordinated model of IAV host shutoff. PMID:25878098

Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

PubMed Central

Crescenzo-Chaigne, Bernadette; Barbezange, Cyril; van der Werf, Sylvie

2008-01-01

Background The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the in vivo reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases. Results The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex. Conclusion In the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex. PMID:18973655

Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinto, Amelia K.; Williams, Graham D.; Szretter, Kristy J.

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5'-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infectedIfit1 -/-and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virusmore » [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack ofIfit1gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical betweenIfit1 -/-and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCENegative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis bothin vitroandin vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.« less

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

NASA Astrophysics Data System (ADS)

Mousnier, Aurélie; Bell, Andrew S.; Swieboda, Dawid P.; Morales-Sanfrutos, Julia; Pérez-Dorado, Inmaculada; Brannigan, James A.; Newman, Joseph; Ritzefeld, Markus; Hutton, Jennie A.; Guedán, Anabel; Asfor, Amin S.; Robinson, Sean W.; Hopkins-Navratilova, Iva; Wilkinson, Anthony J.; Johnston, Sebastian L.; Leatherbarrow, Robin J.; Tuthill, Tobias J.; Solari, Roberto; Tate, Edward W.

2018-06-01

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

The Mechanism of Viral Replication. Structure of Replication Complexes of Encephalomyocarditis Virus

PubMed Central

Thach, Sigrid S.; Dobbertin, Darrell; Lawrence, Charles; Golini, Fred; Thach, Robert E.

1974-01-01

The structure of the purified replicative intermediate of encephalomyocarditis virus was determined by electron microscopy. Approximately 80% of the replicative intermediate complexes were characterized by a filament of double-stranded RNA of widely variable length, which had a “bush” of single-stranded RNA at one end. In many examples one or more additional single-stranded bushes were appended internally to the double-stranded RNA filament. These results support the view that before deproteinization, replicative intermediate contains little if any double-stranded RNA. Images PMID:4366773

Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes

PubMed Central

Le Coupanec, Alain; Tchankouo-Nguetcheu, Stéphane; Roux, Pascal; Khun, Huot; Huerre, Michel; Morales-Vargas, Ronald; Enguehard, Margot; Lavillette, Dimitri; Missé, Dorothée

2017-01-01

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells. PMID:28777313

Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes.

PubMed

Le Coupanec, Alain; Tchankouo-Nguetcheu, Stéphane; Roux, Pascal; Khun, Huot; Huerre, Michel; Morales-Vargas, Ronald; Enguehard, Margot; Lavillette, Dimitri; Missé, Dorothée; Choumet, Valérie

2017-08-04

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti . The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.

In vitro infection of salmonid epidermal tissues by infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus

USGS Publications Warehouse

Yamamoto, T.; Batts, W.N.; Winton, J.R.

1992-01-01

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.

The molecular biology of Bluetongue virus replication.

PubMed

Patel, Avnish; Roy, Polly

2014-03-01

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice.

PubMed

Belser, Jessica A; Gustin, Kortney M; Pearce, Melissa B; Maines, Taronna R; Zeng, Hui; Pappas, Claudia; Sun, Xiangjie; Carney, Paul J; Villanueva, Julie M; Stevens, James; Katz, Jacqueline M; Tumpey, Terrence M

2013-09-26

On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication.

PubMed

Schuchman, Ryan; Kilianski, Andy; Piper, Amanda; Vancini, Ricardo; Ribeiro, José M C; Sprague, Thomas R; Nasar, Farooq; Boyd, Gabrielle; Hernandez, Raquel; Glaros, Trevor

2018-05-09

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. IMPORTANCE Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are no vaccines or prophylactics for these agents leaving one third of the world population at risk of infection. Identifying effective antivirals has been a long term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses. Copyright © 2018 Schuchman et al.

Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells

PubMed Central

He, Ganlin; Cardona, Carol J.

2018-01-01

The PB1-F2 protein of influenza A virus has been considered a virulence factor, but its function in inducing apoptosis may be of disadvantage to viral replication. Host mechanisms to regulate PB1-F2-induced apoptosis remain unknown. We generated a PB1-F2-deficient avian influenza virus (AIV) H9N2 and found that the mutant virus replicated less efficiently in human lung epithelial cells. The PB1-F2-deficient virus produced less apoptotic cells, indicating that PB1-F2 of the H9N2 virus promotes apoptosis, occurring at the early stage of infection, in the lung epithelial cells. To understand how host cells regulate PB1-F2-induced apoptosis, we explored to identify cellular proteins interacting with PB1-F2 and found that HCLS1-associated protein X-1 (HAX-1), located mainly in the mitochondria as an apoptotic inhibitor, interacted with PB1-F2. Increased procaspase-9 activations, induced by PB1-F2, could be suppressed by HAX-1. In HAX-1 knockdown A549 cells, the replication of AIV H9N2 was suppressed in parallel to the activation of caspase-3 activation, which increased at the early stage of infection. We hypothesize that HAX-1 promotes AIV replication by interacting with PB1-F2, resulting in the suppression of apoptosis, prolonged cell survival, and enhancement of viral replication. Our data suggest that HAX-1 may be a promoting factor for AIV H9N2 replication through desensitizing PB1-F2 from its apoptotic induction in human lung epithelial cells. PMID:29576744

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses.

PubMed

Kandeil, Ahmed; Moatasim, Yassmin; Gomaa, Mokhtar R; Shehata, Mahmoud M; El-Shesheny, Rabeh; Barakat, Ahmed; Webby, Richard J; Ali, Mohamed A; Kayali, Ghazi

2016-01-04

Avian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells. To investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens. We observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding. Our findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells.

PubMed

Mänz, Benjamin; de Graaf, Miranda; Mögling, Ramona; Richard, Mathilde; Bestebroer, Theo M; Rimmelzwaan, Guus F; Fouchier, Ron A M

2016-07-01

A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts. Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions-most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2-have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Prereplicative events involving simian virus 40 DNA in permissive cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinaldy, A.; Feunteun, J.; Rosenberg, B.H.

1982-01-01

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA inmore » the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TaqI restriction enzyme was used to provide viral functions required for replication, and is a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.« less

Global genomics and proteomics approaches to identify host factors as targets to induce resistance against Tomato bushy stunt virus.

PubMed

Nagy, Peter D; Pogany, Judit

2010-01-01

The success of RNA viruses as pathogens of plants, animals, and humans depends on their ability to reprogram the host cell metabolism to support the viral infection cycle and to suppress host defense mechanisms. Plus-strand (+)RNA viruses have limited coding potential necessitating that they co-opt an unknown number of host factors to facilitate their replication in host cells. Global genomics and proteomics approaches performed with Tomato bushy stunt virus (TBSV) and yeast (Saccharomyces cerevisiae) as a model host have led to the identification of 250 host factors affecting TBSV RNA replication and recombination or bound to the viral replicase, replication proteins, or the viral RNA. The roles of a dozen host factors involved in various steps of the replication process have been validated in yeast as well as a plant host. Altogether, the large number of host factors identified and the great variety of cellular functions performed by these factors indicate the existence of a truly complex interaction between TBSV and the host cell. This review summarizes the advantages of using a simple plant virus and yeast as a model host to advance our understanding of virus-host interactions at the molecular and cellular levels. The knowledge of host factors gained can potentially be used to inhibit virus replication via gene silencing, expression of dominant negative mutants, or design of specific chemical inhibitors leading to novel specific or broad-range resistance and antiviral tools against (+)RNA plant viruses. Copyright © 2010 Elsevier Inc. All rights reserved.

Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross

PubMed Central

Ferris, Martin T.; Aylor, David L.; Bottomly, Daniel; Whitmore, Alan C.; Aicher, Lauri D.; Bell, Timothy A.; Bradel-Tretheway, Birgit; Bryan, Janine T.; Buus, Ryan J.; Gralinski, Lisa E.; Haagmans, Bart L.; McMillan, Leonard; Miller, Darla R.; Rosenzweig, Elizabeth; Valdar, William; Wang, Jeremy; Churchill, Gary A.; Threadgill, David W.; McWeeney, Shannon K.; Katze, Michael G.; Pardo-Manuel de Villena, Fernando; Baric, Ralph S.; Heise, Mark T.

2013-01-01

Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss. PMID:23468633

Evidence of Ebola Virus Replication and High Concentration in Semen of a Patient During Recovery.

PubMed

Barnes, Kayla G; Kindrachuk, Jason; Lin, Aaron E; Wohl, Shirlee; Qu, James; Tostenson, Samantha D; Dorman, William R; Busby, Michele; Siddle, Katherine J; Luo, Cynthia Y; Matranga, Christian B; Davey, Richard T; Sabeti, Pardis C; Chertow, Daniel S

2017-10-15

In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Hepatitis B virus molecular biology and pathogenesis.

PubMed

Lamontagne, R Jason; Bagga, Sumedha; Bouchard, Michael J

2016-01-01

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350-500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae . In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC.

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Characterization of the replication cycle of the Lymantria dispar nuclear polyhedrosis virus

Treesearch

Christopher I. Riegel; James M. Slavicek

1997-01-01

The life cycle of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) was characterized through analysis of budded virus (BV) release, the temporal formation of polyhedra, the temporal transcription pattern of representative early, late, and hyper-expressed late genes, and the onset of DNA replication in the Ld652Y cell line. Transcripts from...

Availability of activated CD4+ T cells dictates the level of viremia in naturally SIV-infected sooty mangabeys.

PubMed

Klatt, Nichole R; Villinger, Francois; Bostik, Pavel; Gordon, Shari N; Pereira, Lara; Engram, Jessica C; Mayne, Ann; Dunham, Richard M; Lawson, Benton; Ratcliffe, Sarah J; Sodora, Donald L; Else, James; Reimann, Keith; Staprans, Silvija I; Haase, Ashley T; Estes, Jacob D; Silvestri, Guido; Ansari, Aftab A

2008-06-01

Naturally SIV-infected sooty mangabeys (SMs) remain asymptomatic despite high virus replication. Elucidating the mechanisms underlying AIDS resistance of SIV-infected SMs may provide crucial information to better understand AIDS pathogenesis. In this study, we assessed the determinants of set-point viremia in naturally SIV-infected SMs, i.e., immune control of SIV replication versus target cell limitation. We depleted CD4+ T cells in 6 naturally SIV-infected SMs by treating with humanized anti-CD4 mAb (Cdr-OKT4A-huIgG1). CD4+ T cells were depleted almost completely in blood and BM and at variable levels in mucosal tissues and LNs. No marked depletion of CD14+ monocytes was observed. Importantly, CD4+ T cell depletion was associated with a rapid, significant decline in viral load, which returned to baseline level at day 30-45, coincident with an increased fraction of proliferating and activated CD4+ T cells. Throughout the study, virus replication correlated with the level of proliferating CD4+ T cells. CD4+ T cell depletion did not induce any changes in the fraction of Tregs or the level of SIV-specific CD8+ T cells. Our results suggest that the availability of activated CD4+ T cells, rather than immune control of SIV replication, is the main determinant of set-point viral load during natural SIV infection of SMs.

Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication

PubMed Central

Rahman, Masmudur M.; Liu, Jia; Chan, Winnie M.; Rothenburg, Stefan; McFadden, Grant

2013-01-01

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells. PMID:23853588

Immunization against Genital Herpes with a Vaccine Virus That has Defects in Productive and Latent Infection

NASA Astrophysics Data System (ADS)

da Costa, Xavier J.; Jones, Cheryl A.; Knipe, David M.

1999-06-01

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.

Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

PubMed Central

Mühlberger, Elke; Lötfering, Beate; Klenk, Hans-Dieter; Becker, Stephan

1998-01-01

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication. PMID:9765419

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

PubMed

Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

2015-03-01

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

PubMed Central

Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

1993-01-01

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells. Images PMID:7688453

Oxidative stress favours herpes virus infection in vertebrates: a meta-analysis.

PubMed

Sebastiano, Manrico; Chastel, Olivier; de Thoisy, Benoît; Eens, Marcel; Costantini, David

2016-08-01

Herpes viruses are responsible for a variety of pathological effects in humans and in both wild and domestic animals. One mechanism that has been proposed to facilitate replication and activity of herpes viruses is oxidative stress (OS). We used meta-analytical techniques to test the hypotheses that (1) herpes virus infection causes OS and (2) supplementation of antioxidants reduces virus load, indicating that replication is favoured by a state of OS. Results based on studies on mammals, including humans, and birds show that (1) OS is indeed increased by herpes virus infection across multiple tissues and species, (2) biomarkers of OS may change differently between tissues, and (3) the effect size does not differ among different virus strains. In addition, the increase of oxidative damage in blood (tissue commonly available in ecological studies) was similar to that in the tissues most sensitive to the herpes virus. Our results also show that administration of antioxidants reduces virus yield, indicating that a condition of OS is favorable for the viral replication. In addition, some antioxidants may be more efficient than others in reducing herpes virus yield. Our results point to a potential mechanism linking herpes virus infection to individual health status.

Evolutionary dynamics of giant viruses and their virophages.

PubMed

Wodarz, Dominik

2013-07-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested.

Evolutionary dynamics of giant viruses and their virophages

PubMed Central

Wodarz, Dominik

2013-01-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested. PMID:23919155

Mimicry of molecular pretenders: the terminal structures of satellites associated with plant RNA viruses.

PubMed

Huang, Ying-Wen; Hu, Chung-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu

2010-01-01

Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.

Vaccine-induced, simian immunodeficiency virus-specific CD8+ T cells reduce virus replication but do not protect from simian immunodeficiency virus disease progression.

PubMed

Engram, Jessica C; Dunham, Richard M; Makedonas, George; Vanderford, Thomas H; Sumpter, Beth; Klatt, Nichole R; Ratcliffe, Sarah J; Garg, Seema; Paiardini, Mirko; McQuoid, Monica; Altman, John D; Staprans, Silvija I; Betts, Michael R; Garber, David A; Feinberg, Mark B; Silvestri, Guido

2009-07-01

Our limited understanding of the interaction between primate lentiviruses and the host immune system complicates the design of an effective HIV/AIDS vaccine. To identify immunological correlates of protection from SIV disease progression, we immunized two groups of five rhesus macaques (RMs) with either modified vaccinia Ankara (MVA) or MVADeltaudg vectors that expressed SIVmac239 Gag and Tat. Both vectors raised a SIV-specific CD8(+) T cell response, with a magnitude that was greater in mucosal tissues than in peripheral blood. After challenge with SIVmac239, all vaccinated RMs showed mucosal and systemic CD8(+) T cell recall responses that appeared faster and were of greater magnitude than those in five unvaccinated control animals. All vaccinated RMs showed a approximately 1-log lower peak and early set-point SIV viral load than the unvaccinated animals, and then, by 8 wk postchallenge, exhibited levels of viremia similar to the controls. We observed a significant direct correlation between the magnitude of postchallenge SIV-specific CD8(+) T cell responses and SIV viral load. However, vaccinated RMs showed no protection from either systemic or mucosal CD4(+) T cell depletion and no improved survival. The observation that vaccine-induced, SIV-specific CD8(+) T cells that partially control SIVmac239 virus replication fail to protect from immunological or clinical progression of SIV infection underscores both the complexity of AIDS pathogenesis and the challenges of properly assessing the efficacy of candidate AIDS vaccines.

Enhanced Control of Pathogenic Simian Immunodeficiency Virus SIVmac239 Replication in Macaques Immunized with an Interleukin-12 Plasmid and a DNA Prime-Viral Vector Boost Vaccine Regimen ▿ §

PubMed Central

Winstone, Nicola; Wilson, Aaron J.; Morrow, Gavin; Boggiano, Cesar; Chiuchiolo, Maria J.; Lopez, Mary; Kemelman, Marina; Ginsberg, Arielle A.; Mullen, Karl; Coleman, John W.; Wu, Chih-Da; Narpala, Sandeep; Ouellette, Ian; Dean, Hansi J.; Lin, Feng; Sardesai, Niranjan Y.; Cassamasa, Holly; McBride, Dawn; Felber, Barbara K.; Pavlakis, George N.; Schultz, Alan; Hudgens, Michael G.; King, C. Richter; Zamb, Timothy J.; Parks, Christopher L.; McDermott, Adrian B.

2011-01-01

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent “blips” in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication. PMID:21734035

The temperature-sensitive and attenuation phenotypes conferred by mutations in the influenza virus PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33 viruses.

PubMed

Broadbent, Andrew J; Santos, Celia P; Godbout, Rachel A; Subbarao, Kanta

2014-11-01

Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥ 100-fold) replication at 39 °C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391 E, 581 G, and 661 T), PB2 (265 S), and NP (34 G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04(ts sig) and CA07(ts sig) viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39 °C, but the replication of WSN(ts sig) was restricted ≥ 100-fold compared to that at 33 °C. Reassortant CA07(ts sig) viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥ 100-fold at 39 °C. In ferrets, the CA07(ts sig) virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07(ts sig) virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

PubMed Central

Broadbent, Andrew J.; Santos, Celia P.; Godbout, Rachel A.

2014-01-01

ABSTRACT Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥100-fold) replication at 39°C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04ts sig and CA07ts sig viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39°C, but the replication of WSNts sig was restricted ≥100-fold compared to that at 33°C. Reassortant CA07ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥100-fold at 39°C. In ferrets, the CA07ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCE Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses. PMID:25122786

Blocking Virus Replication during Acute Murine Cytomegalovirus Infection Paradoxically Prolongs Antigen Presentation and Increases the CD8+ T Cell Response by Preventing Type I IFN-Dependent Depletion of Dendritic Cells.

PubMed

Loo, Christopher P; Snyder, Christopher M; Hill, Ann B

2017-01-01

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8 + T cell response, which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication, we found that increased virus replication drove increased effector CD8 + T cell differentiation, as expected. Paradoxically, however, increased virus replication dramatically decreased the size of the CD8 + T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs, but they did not inhibit the response to "inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8 + T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

PubMed

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

PubMed

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

Sequential structures provide insights into the fidelity of RNA replication.

PubMed

Ferrer-Orta, Cristina; Arias, Armando; Pérez-Luque, Rosa; Escarmís, Cristina; Domingo, Esteban; Verdaguer, Nuria

2007-05-29

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication

PubMed Central

Zhang, Ran; Liu, Chuangang; Cao, Yunzi; Jamal, Muhammad; Chen, Xi; Zheng, Jinfang; Li, Liang; You, Jing; Zhu, Qi; Liu, Shiyong; Dai, Jinxia; Cui, Min; Fu, Zhen F.; Cao, Gang

2017-01-01

Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication. PMID:28388579

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

PubMed Central

Ando, Tomomi; Imamura, Hiromi; Suzuki, Ryosuke; Aizaki, Hideki; Watanabe, Toshiki; Wakita, Takaji; Suzuki, Tetsuro

2012-01-01

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome. PMID:22396648

Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts.

PubMed

Yuk, Seong-Su; Lee, Dong-Hun; Park, Jae-Keun; Tseren-Ochir, Erdene-Ochir; Kwon, Jung-Hoon; Noh, Jin-Yong; Lee, Joong-Bok; Park, Seung-Yong; Choi, In-Soo; Song, Chang-Seon

2016-04-27

Interferon gamma (IFN-γ), an immunoregulatory cytokine, is known to control many microbial infections. In a previous study, chicken interferon gamma (chIFN-γ) was found to be up-regulated following avian influenza virus (AIV) infection in specific pathogen-free chickens. We aimed to investigate whether the pre-immune state induced by chIFN-γ could generate an antiviral response against influenza virus. We generated a chIFN-γ-expressing plasmid and transfected it into chicken embryo fibroblasts (CEFs) and then infected the cells with human origin H1N1 or avian origin H9N2 influenza viruses. Viral titers of culture medium were evaluated in MDCK cell and the viral RNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase. To further evaluate the role of the antiviral effect of chIFN-γ by using a backward approach, synthetic small interfering RNAs (siRNA) targeting chIFN-γ were used to suppress chIFN-γ. The chIFN-γ-stimulated CEFs inhibited the replication of viral RNA (vRNA) and showed a mild decrease in the infectious virus load released in the culture medium. Compared to the mock-transfected control, the messenger RNA (mRNA) levels of type I IFNs and IFN-stimulated genes were up-regulated in the cells expressing chIFN-γ. After treatment with the siRNA, we detected a higher expression of viral genes than that observed in the mock-transfected control. Our results suggest that apart from the important role played by chIFN-γ in the antiviral state generated against influenza virus infection, the pre-immune state induced by chIFN-γ can be helpful in mitigating the propagation of influenza virus.

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

PubMed

Yang, Bo; Liu, Xi-Juan; Yao, Yongxuan; Jiang, Xuan; Wang, Xian-Zhang; Yang, Hong; Sun, Jin-Yan; Miao, Yun; Wang, Wei; Huang, Zhen-Li; Wang, Yanyi; Tang, Qiyi; Rayner, Simon; Britt, William J; McVoy, Michael A; Luo, Min-Hua; Zhao, Fei

2018-05-01

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target. IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy. Copyright © 2018 American Society for Microbiology.

Use of a highly sensitive strand-specific quantitative PCR to identify abortive replication in the mouse model of respiratory syncytial virus disease

PubMed Central

2010-01-01

Background The BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics. Results In the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells. Conclusions This is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions. PMID:20860795

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector

PubMed Central

Lichtenstein, DL; Spencer, JF; Doronin, K; Patra, D; Meyer, JM; Shashkova, EV; Kuppuswamy, M; Dhar, D; Thomas, MA; Tollefson, AE; Zumstein, LA; Wold, WSM; Toth, K

2012-01-01

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 × 1010 viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007. PMID:19197324

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.

PubMed

Lichtenstein, D L; Spencer, J F; Doronin, K; Patra, D; Meyer, J M; Shashkova, E V; Kuppuswamy, M; Dhar, D; Thomas, M A; Tollefson, A E; Zumstein, L A; Wold, W S M; Toth, K

2009-08-01

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

STAT3 Activation Promotes Oncolytic HSV1 Replication in Glioma Cells

PubMed Central

Okemoto, Kazuo; Wagner, Benjamin; Meisen, Hans; Haseley, Amy; Kaur, Balveen; Chiocca, Ennio Antonio

2013-01-01

Recent studies report that STAT3 signaling is a master regulator of mesenchymal transformation of gliomas and that STAT3 modulated genes are highly expressed in the mesenchymal transcriptome of gliomas. A currently studied experimental treatment for gliomas consists of intratumoral injection of oncolytic viruses (OV), such as oncolytic herpes simplex virus type 1 (oHSV). We have described one particular oHSV (rQNestin34.5) that exhibits potent anti-glioma activity in animal models. Here, we hypothesized that alterations in STAT3 signaling in glioma cells may affect the replicative ability of rQNestin34.5. In fact, human U251 glioma cells engineered to either over-express STAT3 or with genetic down-regulation of STAT3 supported oHSV replication to a significantly higher or lesser degree, respectively, when compared to controls. Administration of pharmacologic agents that increase STAT3 phosphorylation/activation (Valproic Acid) or increase STAT3 levels (Interleukin 6) also significantly enhanced oHSV replication. Instead, administration of inhibitors of STAT3 phosphorylation/activation (LLL12) significantly reduced oHSV replication. STAT3 led to a reduction in interferon signaling in oHSV infected cells and inhibition of interferon signaling abolished the effect of STAT3 on oHSV replication. These data thus indicate that STAT3 signaling in malignant gliomas enhances oHSV replication, likely by inhibiting the interferon response in infected glioma cells, thus suggesting avenues for possible potentiation of oncolytic virotherapy. PMID:23936533

Enhanced Replication of Virulent Newcastle Disease Virus in Chicken Macrophages Is due to Polarized Activation of Cells by Inhibition of TLR7.

PubMed

Zhang, Pingze; Ding, Zhuang; Liu, Xinxin; Chen, Yanyu; Li, Junjiao; Tao, Zhi; Fei, Yidong; Xue, Cong; Qian, Jing; Wang, Xueli; Li, Qingmei; Stoeger, Tobias; Chen, Jianjun; Bi, Yuhai; Yin, Renfu

2018-01-01

Newcastle disease (ND), caused by infections with virulent strains of Newcastle disease virus (NDV), is one of the most important infectious disease affecting wild, peridomestic, and domestic birds worldwide. Vaccines constructed from live, low-virulence (lentogenic) viruses are the most accepted prevention and control strategies for combating ND in poultry across the globe. Avian macrophages are one of the first cell lines of defense against microbial infection, responding to signals in the microenvironment. Although macrophages are considered to be one of the main target cells for NDV infection in vivo , very little is known about the ability of NDV to infect chicken macrophages, and virulence mechanisms of NDV as well as the polarized activation patterns of macrophages and correlation with viral infection and replication. In the present study, a cell culture model (chicken bone marrow macrophage cell line HD11) and three different virulence and genotypes of NDV (including class II virulent NA-1, class II lentogenic LaSota, and class I lentogenic F55) were used to solve the above underlying questions. Our data indicated that all three NDV strains had similar replication rates during the early stages of infection. Virulent NDV titers were shown to increase compared to the other lentogenic strains, and this growth was associated with a strong upregulation of both pro-inflammatory M1-like markers/cytokines and anti-inflammatory M2-like markers/cytokines in chicken macrophages. Virulent NDV was found to block toll-like receptor (TLR) 7 expression, inducing higher expression of type I interferons in chicken macrophages at the late stage of viral infection. Only virulent NDV replication can be inhibited by pretreatment with TLR7 ligand. Overall, this study demonstrated that virulent NDV activates a M1-/M2-like mixed polarized activation of chicken macrophages by inhibition of TLR7, resulting in enhanced replication compared to lentogenic viruses.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

PubMed

Organ, Edward L; Nalbantyan, Christopher D; Nanney, Lillian B; Woodward, Stephen C; Sheng, Jinsong; Dubois, Raymond N; Price, James; Sutcliffe, Marilyn; Coffey, Robert J; Rubin, Donald H

2004-07-01

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

PubMed Central

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

PubMed

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mourez, Thomas; APHP, GH Saint-Louis-Lariboisiere, Laboratoire de Bacteriologie-Virologie, F-75010 Paris; Universite Paris 7 Denis Diderot, F-75010 Paris

2011-10-25

We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimericmore » particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.« less

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Control of Ebola hemorrhagic fever: vaccine development and our Ebola project in Sierra Leone.

PubMed

Watanabe, Tokiko; Kawaoka, Yoshihiro

2016-01-01

Since December 2013, West Africa has experienced the worst Ebola virus outbreak in recorded history. Of the 28,639 cases reported to the World Health Organization as of March 2016, nearly half (14,124) occurred in Sierra Leone. With a case fatality rate of approximately 40%, this outbreak has claimed the lives of 11,316 individuals. No FDA-approved vaccines or drugs are available to prevent or treat Ebola virus infection. Experimental vaccines and therapies are being developed; however, their safety and efficacy are still being evaluated. Therefore, there is an urgent need to develop control measures to prevent or limit future Ebola virus outbreaks.Previously, we developed a replication-defective Ebola virus that lacks the coding region for the essential viral transcription activator VP30 (Ebola ΔVP30 virus). Here, we evaluated the vaccine efficacy of Ebola ΔVP30 virus in a non-human primate model and describe our collaborative Ebola project in Sierra Leone.

The C-Terminal Tail of TRIM56 Dictates Antiviral Restriction of Influenza A and B Viruses by Impeding Viral RNA Synthesis

PubMed Central

Liu, Baoming; Li, Nan L.; Shen, Yang; Bao, Xiaoyong; Elbahesh, Husni; Webby, Richard J.

2016-01-01

ABSTRACT Accumulating data suggest that tripartite-motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus, and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Here, we show that TRIM56 puts a check on replication of influenza A and B viruses in cell culture but does not inhibit Sendai virus or human metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza virus activity was independent of the E3 ligase activity, B-box, or coiled-coil domain. Rather, deletion of a 63-residue-long C-terminal-tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Together, these data reveal a novel antiviral activity of TRIM56 against influenza A and B viruses and provide insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. IMPORTANCE Options to treat influenza are limited, and drug-resistant influenza virus strains can emerge through minor genetic changes. Understanding novel virus-host interactions that alter influenza virus fitness may reveal new targets/approaches for therapeutic interventions. We show here that TRIM56, a tripartite-motif protein, is an intrinsic host restriction factor of influenza A and B viruses. Unlike its antiviral actions against positive-strand RNA viruses, the anti-influenza virus activity of TRIM56 was independent of the E3 ligase activity. Rather, expression of a short segment within the very C-terminal tail of TRIM56 inhibited the replication of influenza viruses as effectively as that of full-length TRIM56 by specifically targeting viral RNA synthesis. These data reveal the remarkable multifaceted activity of TRIM56, which has developed multiple domains to inhibit multiple viral families. They also raise the possibility of developing a broad-spectrum, TRIM56-based antiviral approach for addition to influenza prophylaxis and/or control strategies. PMID:26889027

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

PubMed

Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication. Copyright © 2017 American Society for Microbiology.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral position is available in the Viral Recombination Section (VRS), HIV Dynamics and Replication Program, CCR.  The VRS studies retroviral replication using human immunodeficiency viruses and other retroviruses, with a particular emphasis on the mechanisms of viral RNA biology, specific RNA packaging, virus assembly, and HIV replication.  Molecular tools and advanced imaging approaches are used to dissect various aspects of viral replication mechanisms.  A more complete description of the projects can be found at http://home.ncifcrf.gov/hivdrp/Hu_res.html.

Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.

PubMed

Santhakumar, Diwakar; Rohaim, Mohammed Abdel Mohsen Shahaat; Hussein, Hussein A; Hawes, Pippa; Ferreira, Helena Lage; Behboudi, Shahriar; Iqbal, Munir; Nair, Venugopal; Arns, Clarice W; Munir, Muhammad

2018-05-01

The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

PubMed Central

Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

2016-01-01

Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology. PMID:27812087

Primary simian immunodeficiency virus SIVmnd-2 infection in mandrills (Mandrillus sphinx).

PubMed

Onanga, Richard; Souquière, Sandrine; Makuwa, Maria; Mouinga-Ondeme, Augustin; Simon, François; Apetrei, Cristian; Roques, Pierre

2006-04-01

Mandrills are the only nonhuman primate (NHP) naturally infected by two types of simian immunodeficiency virus (SIV): SIVmnd-1 and SIVmnd-2. We have already reported that the high SIVmnd-1 replication during primary infection contrasts with only transient changes in CD4+ and CD8+ cell counts. Since early virus-host interactions predict viral control and disease progression in human immunodeficiency virus-infected patients, we investigated the dynamics of SIVmnd-2 primary infection in mandrills to examine the impact on immune effectors in blood and lymph nodes (LNs). To avoid in vitro strain selection, all mandrills in this study received plasma from SIVmnd-2-infected mandrills. SIVmnd-2 plasma viremia peaked at 10(7) to 10(8) RNA copies/ml between days 7 and 10. This peak was followed in all four monkeys by a decline in virus replication, with a set point level of 10(5) to 10(6) RNA copies/ml at day 42 postinfection (p.i.). Viral DNA load in PBMC and LNs also peaked between days 7 and 10 (10(5) to 10(6) DNA copies/10(6) cells) and stabilized at 10(3) to 10(4) DNA copies/10(6) cells during the chronic phase. Anti-SIVmnd-2 antibodies were detected starting from days 28 to 32. A transitory decline of CD3+ CD4+ cells in the LNs occurred in animals with high peak VLs. CD4+ and CD8+ T-cell activation in blood and LNs was noted between days 5 and 17 p.i., surrounding the peak of viral replication. This was most significant in the LNs. Activation markers then returned to preinfection values despite continuous and active viral replication during the chronic infection. The dynamics of SIVmnd-2 infection in mandrills showed a pattern similar to that of SIVmnd-1 infection. This might be a general feature of nonpathogenic SIV natural African NHP models.

Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

PubMed

Kirscher, Lorenz; Deán-Ben, Xosé Luis; Scadeng, Miriam; Zaremba, Angelika; Zhang, Qian; Kober, Christina; Fehm, Thomas Felix; Razansky, Daniel; Ntziachristos, Vasilis; Stritzker, Jochen; Szalay, Aladar A

2015-01-01

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

PubMed Central

Moisy, Dorothée; Avilov, Sergiy V.; Jacob, Yves; Laoide, Brid M.; Ge, Xingyi; Baudin, Florence; Jestin, Jean-Luc

2012-01-01

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses. PMID:22696656

Macrophages in Progressive Human Immunodeficiency Virus/Simian Immunodeficiency Virus Infections

PubMed Central

DiNapoli, Sarah R.; Hirsch, Vanessa M.

2016-01-01

The cells that are targeted by primate lentiviruses (HIV and simian immunodeficiency virus [SIV]) are of intense interest given the renewed effort to identify potential cures for HIV. These viruses have been reported to infect multiple cell lineages of hematopoietic origin, including all phenotypic and functional CD4 T cell subsets. The two most commonly reported cell types that become infected in vivo are memory CD4 T cells and tissue-resident macrophages. Though viral infection of CD4 T cells is routinely detected in both HIV-infected humans and SIV-infected Asian macaques, significant viral infection of macrophages is only routinely observed in animal models wherein CD4 T cells are almost entirely depleted. Here we review the roles of macrophages in lentiviral disease progression, the evidence that macrophages support viral replication in vivo, the animal models where macrophage-mediated replication of SIV is thought to occur, how the virus can interact with macrophages in vivo, pathologies thought to be attributed to viral replication within macrophages, how viral replication in macrophages might contribute to the asymptomatic phase of HIV/SIV infection, and whether macrophages represent a long-lived reservoir for the virus. PMID:27307568

Efficacy of Influenza Vaccination and Tamiflu® Treatment – Comparative Studies with Eurasian Swine Influenza Viruses in Pigs

PubMed Central

Duerrwald, Ralf; Schlegel, Michael; Bauer, Katja; Vissiennon, Théophile; Wutzler, Peter; Schmidtke, Michaela

2013-01-01

Recent epidemiological developments demonstrated that gene segments of swine influenza A viruses can account for antigenic changes as well as reduced drug susceptibility of pandemic influenza A viruses. This raises questions about the efficacy of preventive measures against swine influenza A viruses. Here, the protective effect of vaccination was compared with that of prophylactic Tamiflu® treatment against two Eurasian swine influenza A viruses. 11-week-old pigs were infected by aerosol nebulisation with high doses of influenza virus A/swine/Potsdam/15/1981 (H1N1/1981, heterologous challenge to H1N1 vaccine strain) and A/swine/Bakum/1832/2000 (H1N2/2000, homologous challenge to H1N2 vaccine strain) in two independent trials. In each trial (i) 10 pigs were vaccinated twice with a trivalent vaccine (RESPIPORC® FLU3; 28 and 7 days before infection), (ii) another 10 pigs received 150 mg/day of Tamiflu® for 5 days starting 12 h before infection, and (iii) 12 virus-infected pigs were left unvaccinated and untreated and served as controls. Both viruses replicated efficiently in porcine respiratory organs causing influenza with fever, dyspnoea, and pneumonia. Tamiflu® treatment as well as vaccination prevented clinical signs and significantly reduced virus shedding. Whereas after homologous challenge with H1N2/2000 no infectious virus in lung and hardly any lung inflammation were detected, the virus titre was not and the lung pathology was only partially reduced in H1N1/1981, heterologous challenged pigs. Tamiflu® application did not affect these study parameters. In conclusion, all tested preventive measures provided protection against disease. Vaccination additionally prevented virus replication and histopathological changes in the lung of homologous challenged pigs. PMID:23630601

Efficacy of influenza vaccination and tamiflu® treatment--comparative studies with Eurasian Swine influenza viruses in pigs.

PubMed

Duerrwald, Ralf; Schlegel, Michael; Bauer, Katja; Vissiennon, Théophile; Wutzler, Peter; Schmidtke, Michaela

2013-01-01

Recent epidemiological developments demonstrated that gene segments of swine influenza A viruses can account for antigenic changes as well as reduced drug susceptibility of pandemic influenza A viruses. This raises questions about the efficacy of preventive measures against swine influenza A viruses. Here, the protective effect of vaccination was compared with that of prophylactic Tamiflu® treatment against two Eurasian swine influenza A viruses. 11-week-old pigs were infected by aerosol nebulisation with high doses of influenza virus A/swine/Potsdam/15/1981 (H1N1/1981, heterologous challenge to H1N1 vaccine strain) and A/swine/Bakum/1832/2000 (H1N2/2000, homologous challenge to H1N2 vaccine strain) in two independent trials. In each trial (i) 10 pigs were vaccinated twice with a trivalent vaccine (RESPIPORC® FLU3; 28 and 7 days before infection), (ii) another 10 pigs received 150 mg/day of Tamiflu® for 5 days starting 12 h before infection, and (iii) 12 virus-infected pigs were left unvaccinated and untreated and served as controls. Both viruses replicated efficiently in porcine respiratory organs causing influenza with fever, dyspnoea, and pneumonia. Tamiflu® treatment as well as vaccination prevented clinical signs and significantly reduced virus shedding. Whereas after homologous challenge with H1N2/2000 no infectious virus in lung and hardly any lung inflammation were detected, the virus titre was not and the lung pathology was only partially reduced in H1N1/1981, heterologous challenged pigs. Tamiflu® application did not affect these study parameters. In conclusion, all tested preventive measures provided protection against disease. Vaccination additionally prevented virus replication and histopathological changes in the lung of homologous challenged pigs.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses. Copyright © 2017 American Society for Microbiology.

Rituximab-based treatment, HCV replication, and hepatic flares.

PubMed

Sagnelli, Evangelista; Pisaturo, Mariantonietta; Sagnelli, Caterina; Coppola, Nicola

2012-01-01

Rituximab, a chimeric mouse-human monoclonal antibody directed to the CD20 antigen expressed on pre-B lymphocytes and mature lymphocytes, causes a profound B-cell depletion. Due to its peculiar characteristics, this drug has been used to treat oncohaematological diseases, B cell-related autoimmune diseases, rheumatoid arthritis, and, more recently, HCV-associated mixed cryoglobulinaemic vasculitis. Rituximab-based treatment, however, may induce an increased replication of several viruses such as hepatitis B virus, cytomegalovirus, varicella-zoster virus, echovirus, and parvovirus B19. Recent data suggest that rituximab-based chemotherapy induces an increase in HCV expression in hepatic cells, which may become a target for a cell-mediated immune reaction after the withdrawal of treatment and the restoration of the immune control. Only a few small studies have investigated the occurrence of HCV reactivation and an associated hepatic flare in patients with oncohaematological diseases receiving R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). These studies suggest that the hepatic flares are frequently asymptomatic, but life-threatening liver failure occurs in nearly 10% of cases.

Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells.

PubMed

Novella, Isabel S; Ebendick-Corpus, Bonnie E; Zárate, Selene; Miller, Eric L

2007-06-01

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview.

PubMed

Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

2017-07-05

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV-host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus-host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding.

The chaperonin CCTα is required for efficient transcription and replication of rabies virus.

PubMed

Zhang, Jinyang; Ye, Chengjin; Ruan, Xizhen; Zan, Jie; Xu, Yunbin; Liao, Min; Zhou, Jiyong

2014-10-01

Negri bodies (NBs) are formed in the cytoplasm of rabies virus (RABV)-infected cells and are accompanied by a number of host factors to NBs, in which replication and transcription occur. Here, it was found that chaperonin containing TCP-1 subunit alpha (CCTα) relocalizes to NBs in RABV-infected cells, and that cotransfection of nucleo- and phospho-proteins of RABV is sufficient to recruit CCTα to the NBs' structure. Inhibition of CCTα expression by specific short hairpin RNA knockdown inhibited the replication and transcription of RABV. Therefore, this study showed that the host factor CCTα is associated with RABV infection and is very likely required for efficient virus transcription and replication. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

NASA Astrophysics Data System (ADS)

Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

1988-08-01

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.

PubMed

Brass, Abraham L; Huang, I-Chueh; Benita, Yair; John, Sinu P; Krishnan, Manoj N; Feeley, Eric M; Ryan, Bethany J; Weyer, Jessica L; van der Weyden, Louise; Fikrig, Erol; Adams, David J; Xavier, Ramnik J; Farzan, Michael; Elledge, Stephen J

2009-12-24

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens. Copyright 2009 Elsevier Inc. All rights reserved.

Pathogenesis, Transmissibility, and Ocular Tropism of a Highly Pathogenic Avian Influenza A (H7N3) Virus Associated with Human Conjunctivitis

PubMed Central

Belser, Jessica A.; Davis, C. Todd; Balish, Amanda; Edwards, Lindsay E.; Zeng, Hui; Maines, Taronna R.; Gustin, Kortney M.; Martínez, Irma López; Fasce, Rodrigo; Cox, Nancy J.; Katz, Jacqueline M.

2013-01-01

H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus (A/Mexico/InDRE7218/2012 [MX/7218]) and related avian H7 subtype viruses in mouse and ferret models. Several high- and low-pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tracts of ferrets; however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naive ferrets in a direct-contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet it downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naive-contact ferrets, while concurrently retaining the ability to replicate within ocular tissue and allowing the eye to serve as a portal of entry. PMID:23487452

Isolation of midgut escape mutants of two American genotype dengue 2 viruses from Aedes aegypti

PubMed Central

2013-01-01

Background Several studies have shown that American genotype dengue 2 viruses (DENV2) have reduced viral fitness in the mosquito vector, Aedes aegypti, compared to other DENV2 genotypes. Diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito. Results We analyzed the vector competence of Ae. aegypti for two American DENV2 strains, QR94 and PR159 originating from Mexico and Puerto-Rico, respectively. Both strains infected mosquito midguts following acquisition of infectious bloodmeals. However, DENV2-QR94 and DENV2-PR159 poorly disseminated from the midgut at 7 or 14 days post-bloodmeal (pbm). We detected one virus isolate, EM33, among 31 DENV2-QR94 infected mosquitoes, and one isolate, EM41, among 121 DENV2-PR159 infected mosquitoes, generating high virus titers in mosquito carcasses at 7 days pbm. In oral challenge experiments, EM33 and EM41 showed midgut dissemination rates of 40-50%. Replication efficiency of EM41 in secondary mosquito tissue was similar to that of a dissemination-competent control strain, whereas the replication efficiency of EM33 was significantly lower than that of the control virus. The genome sequence of DENV2-QR94 encoded seven unique amino acids (aa), which were not found in 100 of the most closely related DENV2 strains. EM33 had one additional aa change, E202K, in the E protein. DENV2-PR159 encoded four unique aa residues, one of them E202K, whereas EM41 had two additional aa substitutions, Q77E in the E protein and E93D in NS3. Conclusions Our results indicate that the midgut of Ae. aegypti acts as a selective sieve for DENV2 in which genetically distinct, dissemination-competent virus variants are rapidly selected from the viral quasispecies to be transmitted to vertebrates. PMID:23937713

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro

PubMed Central

JIA, HONGYU; LIU, CHANGHONG; YANG, YING; ZHU, HAIHONG; CHEN, FENG; LIU, JIHONG; ZHOU, LINFU

2015-01-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro. PMID:26640539

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

PubMed

Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

2015-11-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

PubMed

Martins, Mauricio A; Wilson, Nancy A; Piaskowski, Shari M; Weisgrau, Kim L; Furlott, Jessica R; Bonaldo, Myrna C; Veloso de Santana, Marlon G; Rudersdorf, Richard A; Rakasz, Eva G; Keating, Karen D; Chiuchiolo, Maria J; Piatak, Michael; Allison, David B; Parks, Christopher L; Galler, Ricardo; Lifson, Jeffrey D; Watkins, David I

2014-07-01

Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chauhan, Vinita S.; Furr, Samantha R.; Sterka, David G.

2010-05-10

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we havemore » confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.« less

Ostreid herpesvirus type 1 replication and host response in adult Pacific oysters, Crassostrea gigas.

PubMed

Segarra, Amélie; Baillon, Laury; Tourbiez, Delphine; Benabdelmouna, Abdellah; Faury, Nicole; Bourgougnon, Nathalie; Renault, Tristan

2014-10-08

Since 2008, massive mortality outbreaks associated with OsHV-1 detection have been reported in Crassostrea gigas spat and juveniles in several countries. Nevertheless, adult oysters do not demonstrate mortality in the field related to OsHV-1 detection and were thus assumed to be more resistant to viral infection. Determining how virus and adult oyster interact is a major goal in understanding why mortality events are not reported among adult Pacific oysters. Dual transcriptomics of virus-host interactions were explored by real-time PCR in adult oysters after a virus injection. Thirty-nine viral genes and five host genes including MyD88, IFI44, IkB2, IAP and Gly were measured at 0.5, 10, 26, 72 and 144 hours post infection (hpi). No viral RNA among the 39 genes was detected at 144 hpi suggesting the adult oysters are able to inhibit viral replication. Moreover, the IAP gene (oyster gene) shows significant up-regulation in infected adults compared to control adults. This result suggests that over-expression of IAP could be a reaction to OsHV-1 infection, which may induce the apoptotic process. Apoptosis could be a main mechanism involved in disease resistance in adults. Antiviral activity of haemolymph against herpes simplex virus (HSV-1) was not significantly different between infected adults versus control.

The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

PubMed Central

Zheng, Jian; Liu, Yinping; Sia, Sin Fun; Liu, Ming; Qin, Gang; Xiang, Zheng; Lam, Kwok-Tai

2011-01-01

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus–infected cells and inhibit viral replication in vitro. In Rag2−/−γc−/− immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus–infected Rag2−/−γc−/− mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget’s disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza. PMID:21708931

Correlation between Marek's disease virus pathotype and replication.

PubMed

Dunn, John R; Auten, Kiva; Heidari, Mohammad; Buscaglia, Celina

2014-06-01

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes MD, a lymphoproliferative disease in chickens. Pathotyping has become an increasingly important assay for monitoring shifts in virulence of field strains; however, it is time-consuming and expensive, and alternatives are needed to provide fast answers in the face of current outbreaks. The purpose of this study was to determine whether differences in virus replication between pathotypes that have been reported using a small number of virulent (v) and very virulent plus (vv+) MDV strains could be confirmed with a large collection of MD viruses. Based on pilot study data, bursa, brain, and lung samples were collected at 9 and 11 days postinoculation (dpi) from birds challenged with 1 of 15 MDV strains. The correlation between virus replication and virulence was confirmed between vMDV strains and higher virulent strains, but in most cases, there was no significant difference between very virulent (vv) and vv+MDV groups. At both 9 and 11 dpi, chickens infected with vv and vv+MDV had significantly lower body weights and relative thymus and bursa weights compared with chickens challenged with vMDV. However, similar to virus quantity, there was no significant difference between weights in birds challenged with vv or vv+MDV. The significant differences observed in maternal antibody negative (ab-) chickens were not significant in maternal antibody positive (ab+) chickens, demonstrating the requirement of ab- birds for this type of comparison. These data do not support the use of virus replication or organ weights as an alternative to pathotyping for discrimination between all three virulent MDV pathotypes but may be useful for determining a virus replication threshold to choose which field strains meet a minimum virulence to be pathotyped by traditional methods.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity*

PubMed Central

Manzoor, Rashid; Kuroda, Kazumichi; Yoshida, Reiko; Tsuda, Yoshimi; Fujikura, Daisuke; Miyamoto, Hiroko; Kajihara, Masahiro; Kida, Hiroshi; Takada, Ayato

2014-01-01

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments. PMID:24474693

Diverse Effects of Cyclosporine on Hepatitis C Virus Strain Replication

PubMed Central

Ishii, Naoto; Watashi, Koichi; Hishiki, Takayuki; Goto, Kaku; Inoue, Daisuke; Hijikata, Makoto; Wakita, Takaji; Kato, Nobuyuki; Shimotohno, Kunitada

2006-01-01

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents. PMID:16611911

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

PubMed

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N; Gestwicki, Jason E; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-11-19

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. Copyright © 2015 Elsevier Inc. All rights reserved.

Hepatitis C virus replicons: dinosaurs still in business?

PubMed Central

Woerz, I; Lohmann, V; Bartenschlager, R

2009-01-01

Since the molecular cloning of the hepatitis C virus (HCV) genome for the first time in 1989, there has been tremendous progress in our understanding of the multiple facets of the replication cycle of this virus. Key to this progress has been the development of systems to propagate the virus in cell culture, which turned out to be a notoriously difficult task. A major breakthrough has been the construction of subgenomic replicons that self-amplify in cultured human hepatoma cells. These RNAs recapitulate the intracellular steps of the HCV replication cycle and have been instrumental to decipher details of the RNA amplification steps including the identification of key host cell factors. However, reproduction of the complete viral replication cycle only became possible with the advent of a particular molecular HCV clone designated JFH-1 that replicates to very high levels and supports the production of infectious virus particles. The availability of this new culture system raises the question, whether the use of replicons is still justified. In this review, we will discuss the pros and cons of both systems.

Disruption of a stem-loop structure located upstream of pseudoknot domain in Tobacco mosaic virus enhanced its infectivity and viral RNA accumulation.

PubMed

Guo, Song; Wong, Sek-Man

2018-06-01

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication. Copyright © 2018 Elsevier Inc. All rights reserved.

cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras.

PubMed Central

Frolov, I; McBride, M S; Rice, C M

1998-01-01

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals. PMID:9814762

Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yakobson, B.; Hrynko, T.A.; Peak, M.J.

1989-03-01

Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting amore » requirement for de novo protein synthesis.« less