Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

PubMed Central

Walsh, Derek; Mathews, Michael B.; Mohr, Ian

2013-01-01

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

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

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified amore » novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.« less

Optimal therapies of a virus replication model with pharmacological delays based on reverse transcriptase inhibitors and protease inhibitors

NASA Astrophysics Data System (ADS)

Pei, Yongzhen; Li, Changguo; Liang, Xiyin

2017-11-01

A short delay in the pharmacological effect on account of the time required for drug absorption, distribution, and penetration into target cells after application of any anti-viral drug, is defined by the pharmacological delay (Herz et al 1996 Proc. Natl Acad. Sci. USA 93 7247-51). In this paper, a virus replication model with Beddington-DeAngelis incidence rate and the pharmacological and intracellular delays is presented to describe the treatment to cure the virus infection. The optimal controls represent the efficiency of reverse transcriptase inhibitors and protease inhibitors in suppressing viral production and prohibiting new infections. Due to the fact that both the control and state variables contain delays, we derive a necessary conditions for our optimal problem. Based on these results, numerical simulations are implemented not only to show the optimal therapeutic schedules for different infection and release rates, but also to compare the effective of three treatment programs. Furthermore, comparison of therapeutic effects under different maximum tolerable dosages is shown. Our research indicates that (1) the proper and specific treatment program should be determined according to the infection rates of different virus particles; (2) the optimal combined drug treatment is the most efficient; (3) the appropriate proportion of medicament must be formulated during the therapy due to the non-monotonic relationship between maximum tolerable dosages and therapeutic effects; (4) the therapeutic effect is advantageous when the pharmacological delay is considered.

Manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development.

PubMed

Blakely, Pennelope K; Delekta, Phillip C; Miller, David J; Irani, David N

2015-02-01

While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.

Manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development

PubMed Central

Blakely, Pennelope K.; Delekta, Phillip C.; Miller, David J.; Irani, David N.

2014-01-01

While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular that age, gender and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery. PMID:25361697

Evasion of Early Antiviral Responses by Herpes Simplex Viruses

PubMed Central

Suazo, Paula A.; Ibañez, Francisco J.; Retamal-Díaz, Angello R.; Paz-Fiblas, Marysol V.; Bueno, Susan M.; Kalergis, Alexis M.; González, Pablo A.

2015-01-01

Besides overcoming physical constraints, such as extreme temperatures, reduced humidity, elevated pressure, and natural predators, human pathogens further need to overcome an arsenal of antimicrobial components evolved by the host to limit infection, replication and optimally, reinfection. Herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) infect humans at a high frequency and persist within the host for life by establishing latency in neurons. To gain access to these cells, herpes simplex viruses (HSVs) must replicate and block immediate host antiviral responses elicited by epithelial cells and innate immune components early after infection. During these processes, infected and noninfected neighboring cells, as well as tissue-resident and patrolling immune cells, will sense viral components and cell-associated danger signals and secrete soluble mediators. While type-I interferons aim at limiting virus spread, cytokines and chemokines will modulate resident and incoming immune cells. In this paper, we discuss recent findings relative to the early steps taking place during HSV infection and replication. Further, we discuss how HSVs evade detection by host cells and the molecular mechanisms evolved by these viruses to circumvent early antiviral mechanisms, ultimately leading to neuron infection and the establishment of latency. PMID:25918478

Selection of Optimal Polypurine Tract Region Sequences during Moloney Murine Leukemia Virus Replication

PubMed Central

Robson, Nicole D.; Telesnitsky, Alice

2000-01-01

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT. PMID:11044073

An integrated chemical biology approach reveals the mechanism of action of HIV replication inhibitors.

PubMed

Pagano, Nicholas; Teriete, Peter; Mattmann, Margrith E; Yang, Li; Snyder, Beth A; Cai, Zhaohui; Heil, Marintha L; Cosford, Nicholas D P

2017-12-01

Continuous flow (microfluidic) chemistry was employed to prepare a small focused library of dihydropyrimidinone (DHPM) derivatives. Compounds in this class have been reported to exhibit activity against the human immunodeficiency virus (HIV), but their molecular target had not been identified. We tested the initial set of DHPMs in phenotypic assays providing a hit (1i) that inhibited the replication of the human immunodeficiency virus HIV in cells. Flow chemistry-driven optimization of 1i led to the identification of HIV replication inhibitors such as 1l with cellular potency comparable with the clinical drug nevirapine (NVP). Mechanism of action (MOA) studies using cellular and biochemical assays coupled with 3D fingerprinting and in silico modeling demonstrated that these drug-like probe compounds exert their effects by inhibiting the viral reverse transcriptase polymerase (RT). This led to the design and synthesis of the novel DHPM 1at that inhibits the replication of drug resistant strains of HIV. Our work demonstrates that combining flow chemistry-driven analogue refinement with phenotypic assays, in silico modeling and MOA studies is a highly effective strategy for hit-to-lead optimization applicable to the discovery of future therapeutic agents. Copyright © 2017. Published by Elsevier Ltd.

Antibody-mediated targeting of replication-competent retroviral vectors.

PubMed

Tai, Chien-Kuo; Logg, Christopher R; Park, Jinha M; Anderson, W French; Press, Michael F; Kasahara, Noriyuki

2003-05-20

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

PubMed Central

Date, Tomoko; Akazawa, Daisuke; Tian, Xiao; Suzuki, Tetsuro; Kato, Takanobu; Tanaka, Yasuhito; Mizokami, Masashi; Wakita, Takaji; Toyoda, Tetsuya

2010-01-01

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells. PMID:20442786

The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

PubMed Central

Dhungel, Pragyesh; Cao, Shuai

2017-01-01

The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs. PMID:28854224

New frontiers in oncolytic viruses: optimizing and selecting for virus strains with improved efficacy

PubMed Central

2018-01-01

Oncolytic viruses have demonstrated selective replication and killing of tumor cells. Different types of oncolytic viruses – adenoviruses, alphaviruses, herpes simplex viruses, Newcastle disease viruses, rhabdoviruses, Coxsackie viruses, and vaccinia viruses – have been applied as either naturally occurring or engineered vectors. Numerous studies in animal-tumor models have demonstrated substantial tumor regression and prolonged survival rates. Moreover, clinical trials have confirmed good safety profiles and therapeutic efficacy for oncolytic viruses. Most encouragingly, the first cancer gene-therapy drug – Gendicine, based on oncolytic adenovirus type 5 – was approved in China. Likewise, a second-generation oncolytic herpes simplex virus-based drug for the treatment of melanoma has been registered in the US and Europe as talimogene laherparepvec. PMID:29445265

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

PubMed

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J; Lubick, Kirk J; Best, Sonja M; Stoyanov, Cristina T; Law, Lok Man J; Yuan, Zhenghong; Rice, Charles M; MacDonald, Margaret R

2011-01-13

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

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.

Generation and characterization of West Nile pseudo-infectious reporter virus for antiviral screening.

PubMed

Zhang, Hong-Lei; Ye, Han-Qing; Deng, Cheng-Lin; Liu, Si-Qing; Shi, Pei-Yong; Qin, Cheng-Feng; Yuan, Zhi-Ming; Zhang, Bo

2017-05-01

West Nile virus (WNV), a mosquito-borne flavivirus, is an important neurotropic human pathogen. As a biosafety level-3 (BSL-3) agent, WNV is strictly to BSL-3 laboratories for experimentations, thus greatly hindering the development of vaccine and antiviral drug. Here, we developed a novel pseudo-infectious WNV reporter virus expressing the Gaussia luciferase (Gluc). A stable 293T NS1 cell line expressing NS1 was selected for trans-supplying NS1 protein to support the replication of WNV-ΔNS1 virus and WNV-ΔNS1-Gluc reporter virus with large-fragment deletion of NS1. WNV-ΔNS1 virus and WNV-Gluc-ΔNS1 reporter virus were confined to complete their replication cycle in this 293T NS1 cell line, displaying nearly identical growth kinetics to WT WNV although the viral titers were lower than those of WT WNV. The reporter gene was stably maintained in virus genome at least within three rounds of passage in 293T NS1 cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo-infectious WNV-Gluc-ΔNS1 could be used for antiviral screening. Furthermore, a high-throughput screening (HTS) assay in a 96-well format was optimized and validated using several known WNV inhibitors, indicating that the optimized HTS assay was suitable for high-throughput screening WNV inhibitors. Our work provides a stable and safe tool to handle WNV outside of a BSL-3 facility and facilitates high throughput screening for anti-WNV drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo.

PubMed

Ma, Julia; Zhang, Xuexiang; Soloveva, Veronica; Warren, Travis; Guo, Fang; Wu, Shuo; Lu, Huagang; Guo, Jia; Su, Qing; Shen, Helen; Solon, Eric; Comunale, Mary Ann; Mehta, Anand; Guo, Ju-Tao; Bavari, Sina; Du, Yanming; Block, Timothy M; Chang, Jinhong

2018-02-01

Targeting host functions essential for viral replication has been considered as a broad spectrum and resistance-refractory antiviral approach. However, only a few host functions have, thus far, been validated as broad-spectrum antiviral targets in vivo. ER α-glucosidases I and II have been demonstrated to be essential for the morphogenesis of many enveloped viruses, including members from four families of viruses causing hemorrhagic fever. In vivo antiviral efficacy of various iminosugar-based ER α-glucosidase inhibitors has been reported in animals infected with Dengue, Japanese encephalitis, Ebola, Marburg and influenza viruses. Herein, we established Huh7.5-derived cell lines with ER α-glucosidase I or II knockout using CRISPR/Cas9 and demonstrated that the replication of Dengue, Yellow fever and Zika viruses was reduced by only 1-2 logs in the knockout cell lines. The results clearly indicate that only a partial suppression of viral replication can possibly be achieved with a complete inhibition of ER-α-glucosidases I or II by their inhibitors. We therefore explore to improve the antiviral efficacy of a lead iminosugar IHVR-19029 through combination with another broad-spectrum antiviral agent, favipiravir (T-705). Indeed, combination of IHVR-19029 and T-705 synergistically inhibited the replication of Yellow fever and Ebola viruses in cultured cells. Moreover, in a mouse model of Ebola virus infection, combination of sub-optimal doses of IHVR-19029 and T-705 significantly increased the survival rate of infected animals. We have thus proved the concept of combinational therapeutic strategy for the treatment of viral hemorrhagic fevers with broad spectrum host- and viral- targeting antiviral agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

PubMed Central

Ikizler, Mine R.; Kawaoka, Yoshihiro; Rudenko, Larisa G.; Treanor, John J.; Subbarao, Kanta; Wright, Peter F.

2012-01-01

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates. PMID:22915797

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.

Dengue virus induces and requires glycolysis for optimal replication.

PubMed

Fontaine, Krystal A; Sanchez, Erica L; Camarda, Roman; Lagunoff, Michael

2015-02-01

Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

PubMed

Azad, Kimi; Banerjee, Manidipa; Johnson, John E

2017-09-29

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

Vaccination with a codon-optimized A27L-containing plasmid decreases virus replication and dissemination after vaccinia virus challenge.

PubMed

Martínez, Osmarie; Bravo Cruz, Ariana; Santos, Saritza; Ramírez, Maite; Miranda, Eric; Shisler, Joanna; Otero, Miguel

2017-10-20

Smallpox is a disease caused by Variola virus (VARV). Although eradicated by WHO in 1980, the threat of using VARV on a bioterror attack has increased. The current smallpox vaccine ACAM2000, which consists of live vaccinia virus (VACV), causes complications in individuals with a compromised immune system or with previously reported skin diseases. Thus, a safer and efficacious vaccine needs to be developed. Previously, we reported that our virus-free DNA vaccine formulation, a pVAX1 plasmid encoding codon-optimized VACV A27L gene (pA27LOPT) with and without Imiquimod adjuvant, stimulates A27L-specific production of IFN-γ and increases humoral immunity 7days post-vaccination. Here, we investigated the immune response of our novel vaccine by measuring the frequency of splenocytes producing IFN-γ by ELISPOT, the TH1 and TH2 cytokine profiles, and humoral immune responses two weeks post-vaccination, when animals were challenged with VACV. In all assays, the A27-based DNA vaccine conferred protective immune responses. Specifically, two weeks after vaccination, mice were challenged intranasally with vaccinia virus, and viral titers in mouse lungs and ovaries were significantly lower in groups immunized with pA27LOPT and pA27LOPT+Imiquimod. These results demonstrate that our vaccine formulation decreases viral replication and dissemination in a virus-free DNA vaccine platform, and provides an alternative towards a safer an efficacious vaccine. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

USGS Publications Warehouse

Grady, C.A.; Gregg, J.L.; Wade, R.M.; Winton, J.R.; Hershberger, P.K.

2011-01-01

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between na??ve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >105 plaque-forming units (PFU) mL-1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory-reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among na??ve herring (1.2 ?? 103 PFU mL-1) than among groups that survived exposure to VHSV (1.0-2.9 ?? 102 PFU mL-1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39-47%) than among na??ve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring. ?? 2010 Blackwell Publishing Ltd.

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.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

PubMed

Alberti, Michael O; Jones, Jennifer J; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K; Edmonds, Tara G; Kappes, John C; Ochsenbauer, Christina

2015-12-01

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

PubMed Central

Alberti, Michael O.; Jones, Jennifer J.; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K.; Edmonds, Tara G.; Kappes, John C.

2015-01-01

Abstract We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a “ribosome skipping” T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4+ T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, “6ATRi”] demonstrated Nef expression and function similar to parental “nonreporter” virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the “6ATRi” element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function. PMID:26101895

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

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.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. PMID:28515305

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

PubMed

Kjær, Jonas; Belsham, Graham J

2018-04-15

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV. Copyright © 2018 American Society for Microbiology.

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

Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

PubMed Central

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J.; Lubick, Kirk J.; Best, Sonja M.; Stoyanov, Cristina T.; Law, Lok Man J.; Yuan, Zhenghong; Rice, Charles M.; MacDonald, Margaret R.

2011-01-01

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule. PMID:21249176

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

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

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

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

Rouha, Harald; Hoenninger, Verena M.; Thurner, Caroline

2011-08-15

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, butmore » although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.« less

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug.

PubMed

Mastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M; Frick, David N; Bolognesi, Martino; Milani, Mario

2012-08-01

Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC₅₀ values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug

PubMed Central

Mastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M.; Frick, David N.; Bolognesi, Martino; Milani, Mario

2012-01-01

Objectives Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Methods Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Results Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. Conclusions The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV. PMID:22535622

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.

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

PubMed

Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2016-06-01

Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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.

Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8+ T-cell priming and viral control

PubMed Central

Duhan, Vikas; Khairnar, Vishal; Friedrich, Sarah-Kim; Zhou, Fan; Gassa, Asmae; Honke, Nadine; Shaabani, Namir; Gailus, Nicole; Botezatu, Lacramioara; Khandanpour, Cyrus; Dittmer, Ulf; Häussinger, Dieter; Recher, Mike; Hardt, Cornelia; Lang, Philipp A.; Lang, Karl S.

2016-01-01

Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8+ T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8+ T cells and for viral control. In contrast to specific antibodies, memory CD8+ T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus. PMID:26805453

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

Identification of differentially regulated maize proteins conditioning Sugarcane mosaic virus systemic infection.

PubMed

Chen, Hui; Cao, Yanyong; Li, Yiqing; Xia, Zihao; Xie, Jipeng; Carr, John P; Wu, Boming; Fan, Zaifeng; Zhou, Tao

2017-08-01

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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

DARPin-targeting of Measles Virus: Unique Bispecificity, Effective Oncolysis, and Enhanced Safety

PubMed Central

Friedrich, Katrin; Hanauer, Jan RH; Prüfer, Steffen; Münch, Robert C; Völker, Iris; Filippis, Christodoulos; Jost, Christian; Hanschmann, Kay-Martin; Cattaneo, Roberto; Peng, Kah-Whye; Plückthun, Andreas; Buchholz, Christian J; Cichutek, Klaus; Mühlebach, Michael D

2013-01-01

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics. PMID:23380817

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

PubMed

Watanabe, Tsunamasa; Hatakeyama, Hiroto; Matsuda-Yasui, Chiho; Sato, Yusuke; Sudoh, Masayuki; Takagi, Asako; Hirata, Yuichi; Ohtsuki, Takahiro; Arai, Masaaki; Inoue, Kazuaki; Harashima, Hideyoshi; Kohara, Michinori

2014-04-23

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

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.

Identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection.

PubMed

Simpson-Holley, Martha; Colgrove, Robert C; Nalepa, Grzegorz; Harper, J Wade; Knipe, David M

2005-10-01

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue.

PubMed

Behnam, Mira A M; Graf, Dominik; Bartenschlager, Ralf; Zlotos, Darius P; Klein, Christian D

2015-12-10

The dengue virus (DENV) and West Nile Virus (WNV) NS2B-NS3 proteases are attractive targets for the development of dual-acting therapeutics against these arboviral pathogens. We present the synthesis and extensive biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenylglycine as non-natural peptidic building blocks synthesized via a copper-complex intermediate. A three-step optimization strategy, beginning with fragment growth of the C-terminal 4-hydroxyphenylglycine to the benzyloxy ether, followed by C- and N-terminal optimization, and finally fragment merging generated compounds with in vitro affinities in the low nanomolar range. The most promising derivative reached Ki values of 12 nM at the DENV-2 and 39 nM at the WNV proteases. Several of the newly discovered protease inhibitors yielded a significant reduction of dengue and West Nile virus titers in cell-based assays of virus replication, with an EC50 value of 3.4 μM at DENV-2 and 15.5 μM at WNV for the most active analogue.

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.

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

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

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

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

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

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

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

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

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

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.

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

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.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

PubMed

Tsetsarkin, Konstantin A; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G

2016-09-13

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

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.

Molecular Studies of HTLV-1 Replication: An Update

PubMed Central

Martin, Jessica L.; Maldonado, José O.; Mueller, Joachim D.; Zhang, Wei; Mansky, Louis M.

2016-01-01

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. Studies on HTLV-1 have been instrumental for our understanding of the molecular pathology of virus-induced cancers. HTLV-1 is the etiological agent of an adult T-cell leukemia (ATL) and can lead to a variety of neurological pathologies, including HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP). The ability to treat the aggressive ATL subtypes remains inadequate. HTLV-1 replicates by (1) an infectious cycle involving virus budding and infection of new permissive target cells and (2) mitotic division of cells harboring an integrated provirus. Virus replication initiates host antiviral immunity and the checkpoint control of cell proliferation, but HTLV-1 has evolved elegant strategies to counteract these host defense mechanisms to allow for virus persistence. The study of the molecular biology of HTLV-1 replication has provided crucial information for understanding HTLV-1 replication as well as aspects of viral replication that are shared between HTLV-1 and human immunodeficiency virus type 1 (HIV-1). Here in this review, we discuss the various stages of the virus replication cycle—both foundational knowledge as well as current updates of ongoing research that is important for understanding HTLV-1 molecular pathogenesis as well as in developing novel therapeutic strategies. PMID:26828513

Construction and production of oncotropic vectors, derived from MVM(p), that share reduced sequence homology with helper plasmids.

PubMed

Clément, Nathalie; Velu, Thierry; Brandenburger, Annick

2002-09-01

The production of currently available vectors derived from autonomous parvoviruses requires the expression of capsid proteins in trans, from helper sequences. Cotransfection of a helper plasmid always generates significant amounts of replication-competent virus (RCV) that can be reduced by the integration of helper sequences into a packaging cell line. Although stocks of minute virus of mice (MVM)-based vectors with no detectable RCV could be produced by transfection into packaging cells; the latter appear after one or two rounds of replication, precluding further amplification of the vector stock. Indeed, once RCVs become detectable, they are efficiently amplified and rapidly take over the culture. Theoretically RCV-free vector stocks could be produced if all homology between vector and helper DNA is eliminated, thus preventing homologous recombination. We constructed new vectors based on the structure of spontaneously occurring defective particles of MVM. Based on published observations related to the size of vectors and the sequence of the viral origin of replication, these vectors were modified by the insertion of foreign DNA sequences downstream of the transgene and by the introduction of a consensus NS-1 nick site near the origin of replication to optimize their production. In one of the vectors the inserted fragment of mouse genomic DNA had a synergistic effect with the modified origin of replication in increasing vector production.

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.

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.

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

PubMed Central

Wernet, Mathias F.; Klovstad, Martha; Clandinin, Thomas R.

2014-01-01

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development. PMID:25386852

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.

A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection.

PubMed

Harmon, Brooke; Bird, Sara W; Schudel, Benjamin R; Hatch, Anson V; Rasley, Amy; Negrete, Oscar A

2016-08-15

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses. Copyright © 2016 Harmon et al.

A genome-wide RNA interference screen identifies a role for Wnt/β-catenin signaling during Rift Valley Fever Virus infection

DOE PAGES

Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.; ...

2016-05-25

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses Lamore » Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. Lastly, these studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.« less

A genome-wide RNA interference screen identifies a role for Wnt/β-catenin signaling during Rift Valley Fever Virus infection

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

Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses Lamore » Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. Lastly, these studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.« less

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

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

PubMed

Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Fanales-Belasio, Emanuele; Moretti, Sonia; Sernicola, Leonardo; Cara, Andrea; Negri, Donatella R M; Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Scoglio, Arianna; Caputo, Antonella; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Ciccozzi, Massimo; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

2004-09-03

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

PubMed Central

Lowry, Kym; Woodman, Andrew; Cook, Jonathan; Evans, David J.

2014-01-01

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses. PMID:24945141

Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

NASA Astrophysics Data System (ADS)

Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

1997-04-01

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

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

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.

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.

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.

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo.

PubMed

Sattler, Christine; Steer, Beatrix; Adler, Heiko

2016-03-01

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.

A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus.

PubMed

Cheng, Han; Koning, Katie; O'Hearn, Aileen; Wang, Minxiu; Rumschlag-Booms, Emily; Varhegyi, Elizabeth; Rong, Lijun

2015-11-24

Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

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

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.

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

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.

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety. PMID:26269179

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.

Suspension culture process for H9N2 avian influenza virus (strain Re-2).

PubMed

Wang, Honglin; Guo, Suying; Li, Zhenguang; Xu, Xiaoqin; Shao, Zexiang; Song, Guicai

2017-10-01

H9N2 avian influenza virus has caused huge economic loss for the Chinese poultry industry since it was first identified. Vaccination is frequently used as a control method for the disease. Meanwhile suspension culture has become an important tool for the development of influenza vaccines. To optimize the suspension culture conditions for the avian influenza H9N2 virus (Re-2 strain) in Madin-Darby Canine Kidney (MDCK) cells, we studied the culture conditions for cell growth and proliferation parameters for H9N2 virus replication. MDCK cells were successfully cultured in suspension, from a small scale to industrial levels of production, with passage time and initial cell density being optimized. The influence of pH on the culture process in the reactor has been discussed and the process parameters for industrial production were explored via amplification of the 650L reactor. Subsequently, we cultivated cells at high cell density and harvested high amounts of virus, reaching 10log2 (1:1024). Furthermore an animal experiment was conducted to detect antibody. Compared to the chicken embryo virus vaccine, virus cultured from MDCK suspension cells can produce a higher amount of antibodies. The suspension culture process is simple and cost efficient, thus providing a solid foundation for the realization of large-scale avian influenza vaccine production.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

PubMed

Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

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.

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.

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.

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.

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.

The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses

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

Terrier, Olivier; Moules, Vincent; Carron, Coralie

Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtypemore » origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.« less

siRNA for Influenza Therapy.

PubMed

Barik, Sailen

2010-07-01

Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world's population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here.

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

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.

Inactivated HSV-2 in MPL/alum adjuvant provides nearly complete protection against genital infection and shedding following long term challenge and rechallenge.

PubMed

Morello, Christopher S; Kraynyak, Kimberly A; Levinson, Michael S; Chen, Zhijiang; Lee, Kuo-Fen; Spector, Deborah H

2012-10-12

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals. Copyright © 2012 Elsevier Ltd. All rights reserved.

Feline immunodeficiency virus envelope glycoproteins antagonize tetherin through a distinctive mechanism that requires virion incorporation.

PubMed

Morrison, James H; Guevara, Rebekah B; Marcano, Adriana C; Saenz, Dyana T; Fadel, Hind J; Rogstad, Daniel K; Poeschla, Eric M

2014-03-01

BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env(-) particles do not. HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV tetherin antagonist is also its Env protein, but the mechanism is distinctive. Unlike other tetherin antagonists, FIV Env cannot act in trans to rescue vpu-deficient HIV-1. It must be incorporated specifically into FIV virions to be active. Also unlike other retroviral antagonists, but similar to Ebola virus Env, it does not act by downregulating or degrading tetherin. FIV Env might exclude tetherin locally or direct assembly to tetherin-negative membrane domains. Other distinctive features are apparent, including evidence that this virus evolved an equilibrium in which tetherin is both restriction factor and cofactor, as FIV requires tetherin for optimal particle release.

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 ...

Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

PubMed

Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2015-03-02

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba. Copyright © 2015 Elsevier B.V. All rights reserved.

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses.

PubMed

Izuogu, Adaeze O; McNally, Kristin L; Harris, Stephen E; Youseff, Brian H; Presloid, John B; Burlak, Christopher; Munshi-South, Jason; Best, Sonja M; Taylor, R Travis

2017-01-01

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1-30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.

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.

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

PubMed Central

Sattler, Christine; Steer, Beatrix; Adler, Heiko

2016-01-01

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues. PMID:27007137

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.

STAT2-dependent immune responses ensure host survival despite the presence of a potent viral antagonist.

PubMed

Le-Trilling, Vu Thuy Khanh; Wohlgemuth, Kerstin; Rückborn, Meike U; Jagnjic, Andreja; Maaßen, Fabienne; Timmer, Lejla; Katschinski, Benjamin; Trilling, Mirko

2018-05-09

Pathogen encounter induces interferons which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, host and pathogens are situated in a continuous arms race which shapes host evolution towards optimized immune responses and the pathogens towards enhanced immune evasive properties.Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2 which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo In contrast to wt-MCMV, ΔM27-MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g. liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection.Taken together, our study reveals the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary stand-off situation with fatal consequences when the equilibrium is disturbed. IMPORTANCE The host limits viral replication by interferons which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g. cytomegaloviruses, Zika virus, Dengue virus, and several paramyxoviruses). We analyzed infections of mouse CMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate their importance for host and virus in vitro and in vivo The inability to counteract STAT2 directly translates into exaggerated IFN susceptibility in vitro and pronounced attenuation in vivo Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus. Copyright © 2018 Le-Trilling et al.

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.

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

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…

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

Relative resistance of HIV-1 founder viruses to control by interferon-alpha

PubMed Central

2013-01-01

Background Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. Results The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNβ (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNβ, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. Conclusions The establishment of systemic HIV-1 infection by relatively IFNα-resistant founder viruses lends strong support to the hypothesis that IFNα plays an important role in the control of HIV-1 replication during the earliest stages of infection, prior to systemic viral spread. These findings suggest that it may be possible to harness the antiviral activity of type 1 IFNs in prophylactic and potentially also therapeutic strategies to combat HIV-1 infection. PMID:24299076

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.

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

Virus like particles as a platform for cancer vaccine development.

PubMed

Ong, Hui Kian; Tan, Wen Siang; Ho, Kok Lian

2017-01-01

Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.

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.

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

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.

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.

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.

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.

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

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.

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.

Enhanced replication of herpes simplex virus type 1 in human cells

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

Miller, C.S.; Smith, K.O.

1991-02-01

The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate (MMS), methyl methanethiosulfonate (MMTS), ultraviolet light (UV), or gamma radiation (GR)) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of themore » infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes.« less

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.

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.

Transcriptome analysis of rainbow trout infected with high and low virulence strains of Infectious hematopoietic necrosis virus

USGS Publications Warehouse

Purcell, Maureen K.; Marjara, Inderjit Singh; Batts, William; Kurath, Gael; Hansen, John D.

2010-01-01

There are three main genetic lineages or genogroups of Infectious hematopoietic necrosis virus (IHNV) in N. America. Strains representing the M genogroup are more virulent in rainbow trout relative to the U genogroup. In this study, we used microarray analysis to evaluate potential mechanisms responsible for host-specific virulence in rainbow trout that were given intraperitoneal injections of buffer or a representative M or U type virus strain. Reverse transcriptase quantitative PCR (RT-qPCR) was used to assess viral load and gene expression of select immune genes. Viral load was significantly higher in trout infected with the M virus starting at 24 h post-infection (p.i.) and continuing until 72 h p.i. Microarray analysis of the 48 h time point revealed 153 up-regulated and 248 down-regulated features in response to M virus infection but only 62 up-regulated and 49 down-regulated features following U virus infection. Translation and transcription features were among the most frequent down-regulated features in response to M virus infection and may be associated with the host cell shutoff phenomenon. A greater host cell shutoff response by the M virus may facilitate subversion of the host cell transcriptional machinery and enhance viral replication, suggesting the M virus may be better optimized to manipulate the rainbow trout transcriptional and translational machinery. Anti-viral associated features were the most commonly up-regulated features. A common set of features were up-regulated in both the M and U infection groups, but were induced to a higher magnitude in the M infection group. Gene expression of the anti-viral genes Mx-1 and Vig-1 was correlated but not entirely dependent on viral load in the anterior kidney. Slower replication of the U virus may allow the host more time to induce protective anti-viral immune mechanisms.

Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

PubMed

Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

2008-05-01

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

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

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

Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine.

PubMed

Ferrier-Rembert, Audrey; Drillien, Robert; Tournier, Jean-Nicolas; Garin, Daniel; Crance, Jean-Marc

2008-03-25

This study assessed three non-replicating smallpox vaccine candidates (modified vaccinia Ankara (MVA), NYVAC and HR) for their immunogenicity and ability to protect mice against an intranasal cowpox virus challenge and compared them with the traditional replicating vaccine. A single immunisation with the non-replicating vaccines induced a complete protection from death at short-term, but was not fully protective when mice were challenged 150 days post-vaccination with protection correlated with the specific neutralizing antibodies and CD4(+) T-cells responses. Prime-boost vaccination enabled effective long-term protection from death for mice vaccinated with MVA, but protection from disease and CD4(+) T-cell level were lower than the ones induced by the traditional vaccine over the long-term period. Further investigations are necessary with MVA to determine the optimal conditions of immunisation to induce at long-term immunogenicity and protection observed with the 1st generation smallpox vaccine.

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.

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

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.

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.

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.

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.

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

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

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

Purification and crystallization of dengue and West Nile virus NS2B-NS3 complexes.

PubMed

D'Arcy, Allan; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

2006-02-01

Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B-NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

[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

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.

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

Initial HIV-1 Antigen-Specific CD8+ T Cells in Acute HIV-1 Infection Inhibit Transmitted/Founder Virus Replication

PubMed Central

Freel, Stephanie A.; Picking, Ralph A.; Ferrari, Guido; Ding, Haitao; Ochsenbauer, Christina; Kappes, John C.; Kirchherr, Jennifer L.; Soderberg, Kelly A.; Weinhold, Kent J.; Cunningham, Coleen K.; Denny, Thomas N.; Crump, John A.; Cohen, Myron S.; McMichael, Andrew J.; Haynes, Barton F.

2012-01-01

CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8+ T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8+ responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8+ T cells during AHI. Autologous and heterologous CD8+ T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8+ T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8+ antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8+-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8+ T cell-mediated inhibition of virus replication. CD8+ T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies. PMID:22514337

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.

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.

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.

Feline Immunodeficiency Virus Envelope Glycoproteins Antagonize Tetherin through a Distinctive Mechanism That Requires Virion Incorporation

PubMed Central

Guevara, Rebekah B.; Marcano, Adriana C.; Saenz, Dyana T.; Fadel, Hind J.; Rogstad, Daniel K.

2014-01-01

ABSTRACT BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env− particles do not. IMPORTANCE HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV tetherin antagonist is also its Env protein, but the mechanism is distinctive. Unlike other tetherin antagonists, FIV Env cannot act in trans to rescue vpu-deficient HIV-1. It must be incorporated specifically into FIV virions to be active. Also unlike other retroviral antagonists, but similar to Ebola virus Env, it does not act by downregulating or degrading tetherin. FIV Env might exclude tetherin locally or direct assembly to tetherin-negative membrane domains. Other distinctive features are apparent, including evidence that this virus evolved an equilibrium in which tetherin is both restriction factor and cofactor, as FIV requires tetherin for optimal particle release. PMID:24390322

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

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus*

PubMed Central

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-01-01

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (NTAIL). XD binding induces NTAIL α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced NTAIL folding, XD-NTAIL binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced NTAIL α-helical folding were created within Box-2 of Edmonston MeV NTAIL. Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-NTAIL binding affinity or reduction/loss of XD-induced NTAIL alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity. PMID:22318731

Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.

PubMed

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-04-06

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.

The Contribution of Systemic and Pulmonary Immune Effectors to Vaccine-Induced Protection from H5N1 Influenza Virus Infection

PubMed Central

Lau, Yuk-Fai; Wright, Amber R.

2012-01-01

Live attenuated influenza vaccines (LAIVs) are effective in providing protection against influenza challenge in animal models and in preventing disease in humans. We previously showed that LAIVs elicit a range of immune effectors and that successful induction of pulmonary cellular and humoral immunity in mice requires pulmonary replication of the vaccine virus. An upper respiratory tract immunization (URTI) model was developed in mice to mimic the human situation, in which the vaccine virus does not replicate in the lower respiratory tract, allowing us to assess the protective efficacy of an H5N1 LAIV against highly pathogenic H5N1 virus challenge in the absence of significant pulmonary immunity. Our results show that, after one dose of an H5N1 LAIV, pulmonary influenza-specific lymphocytes are the main contributors to clearance of challenge virus from the lungs and that contributions of influenza-specific enzyme-linked immunosorbent assay (ELISA) antibodies in serum and splenic CD8+ T cells were negligible. Complete protection from H5N1 challenge was achieved after two doses of H5N1 LAIV and was associated with maturation of the antibody response. Although passive transfer of sera from mice that received two doses of vaccine prevented lethality in naive recipients following challenge, the mice showed significant weight loss, with high pulmonary titers of the H5N1 virus. These data highlight the importance of mucosal immunity in mediating optimal protection against H5N1 infection. Understanding the requirements for effective induction and establishment of these protective immune effectors in the respiratory tract paves the way for a more rational and effective vaccine approach in the future. PMID:22379093

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

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.

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

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

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.

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.

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

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

Increased Relative Risk of Tick-Borne Encephalitis in Warmer Weather.

PubMed

Daniel, Milan; Danielová, Vlasta; Fialová, Alena; Malý, Marek; Kříž, Bohumír; Nuttall, Patricia A

2018-01-01

Tick-borne encephalitis (TBE) is a serious acute neuroinfection of humans caused by a tick-borne flavivirus. The disease is typically seasonal, linked to the host-seeking activity of Ixodes ricinus (predominantly nymphs), the principal European tick vector species. To address the need for accurate risk predictions of contracting TBE, data on 4,044 TBE cases reported in the Czech Republic during 2001-2006 were compared with questing activity of I. ricinus nymphs monitored weekly at a defined location for the same 6-year period. A time shift of 21 days between infected tick bite and recorded disease onset provided the optimal model for comparing the number of cases of TBE with numbers of questing nymphs. Mean annual distribution of TBE cases and tick counts showed a similar bimodal distribution. Significantly, the ratio of TBE cases to questing nymphs was highest in the summer-autumn period even though the number of questing nymphs peaked in the spring-summer period. However, this pattern changed during a period of extreme meteorological events of flooding and abnormally high temperatures, indicating that changes in climate affect the incidence of TBE. Previous studies failed to link human behavior with changes in incidence of TBE but showed extrinsic temperature impacts arbovirus replication. Hence, we hypothesize the apparent discrepancy between peak nymphal tick activity and greatest risk of contracting TBE is due to the effect of temperature on virus replication in the tick vector. Relative proportions of questing nymphs and the numbers of weeks in which they were found were greater in summer-autumn compared with spring-summer at near-ground temperatures >5°C and at standard day and weekly average temperatures of >15°C. Thus, during the summer-autumn period, the virus dose in infected tick bites is likely greater owing to increased virus replication at higher microclimatic temperatures, consequently increasing the relative risk of contracting TBE per summer-autumn tick bite. The data support the use of weather-based forecasts of tick attack risk (based on daytime ambient temperature) supplemented with weekly average temperature (as a proxy for virus replication) to provide much-needed real-time forecasts of TBE risk.

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.

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...

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

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.

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.

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

The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1–3 synthesis

PubMed Central

Volkova, Eugenia; Gorchakov, Rodion; Frolov, Ilya

2008-01-01

Alphaviruses are regarded as attractive systems for expression of heterologous genes and development of recombinant vaccines. Venezuelan equine encephalitis virus (VEE)-based vectors are particularly promising because of their specificity to lymphoid tissues and strong resistance to interferon. To improve understanding of the VEE genome packaging and optimize application of this virus as a vector, we analyzed in more detail the mechanism of packaging of the VEE-specific RNAs. The presence of the RNAs in the VEE particles during serial passaging in tissue culture was found to depend not only on the presence of packaging signal(s), but also on the ability of these RNAs to express in cis nsP1, nsP2 and nsP3 in the form of a P123 precursor. Packaging of VEE genomes into infectious virions was also found to be more efficient compared to that of Sindbis virus, in spite of lower levels of RNA replication and structural protein production. PMID:16239019

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

PubMed

Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

2016-04-13

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qβ.

PubMed

Arribas, María; Cabanillas, Laura; Kubota, Kirina; Lázaro, Ester

2016-10-01

RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qβ, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

A doxycycline-dependent human immunodeficiency virus type 1 replicates in vivo without inducing CD4+ T-cell depletion

PubMed Central

Legrand, Nicolas; van der Velden, Gisela J.; Fang, Raphaël Ho Tsong; Douaisi, Marc; Weijer, Kees; Das, Atze T.; Blom, Bianca; Uittenbogaart, Christel H.; Berkhout, Ben

2012-01-01

A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4+ T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat–transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus–host interactions in vivo in a controlled fashion. PMID:22647372

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

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.

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

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

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

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

A Novel Expression Cassette of Lyssavirus Shows that the Distantly Related Mokola Virus Can Rescue a Defective Rabies Virus Genome

PubMed Central

Le Mercier, Philippe; Jacob, Yves; Tanner, Kyle; Tordo, Noël

2002-01-01

By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus. PMID:11799201

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

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.

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

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

PubMed

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

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

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.

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

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

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.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

PubMed

Beljanski, Vladimir; Chiang, Cindy; Kirchenbaum, Greg A; Olagnier, David; Bloom, Chalise E; Wong, Terianne; Haddad, Elias K; Trautmann, Lydie; Ross, Ted M; Hiscott, John

2015-10-01

The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

ONYX-015: mechanisms of action and clinical potential of a replication-selective adenovirus

PubMed Central

Ries, S; Korn, W M

2002-01-01

Accumulated knowledge in the molecular processes of tumour development combined with the availability of genetically modified viruses resemble the basis for new promising cancer therapeutics. The main advantages of employing replication-competent viruses are achievement of tumour selective killing and amplification of their oncolytic potential within the tumour mass. In this review, we describe the development of ONYX-015, one of the first and most advanced replication-competent viruses for cancer therapy. We discuss the molecular biology of this therapeutic approach and the interesting results obtained with this virus in clinical trials. British Journal of Cancer (2002) 86, 5–11. DOI: 10.1038/sj/bjc/6600006 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11857003

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

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

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

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

PubMed

Ilkow, Carolina S; Goping, Ing Swie; Hobman, Tom C

2011-02-01

Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

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.

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

Infection and Replication of Influenza Virus at the Ocular Surface.

PubMed

Creager, Hannah M; Kumar, Amrita; Zeng, Hui; Maines, Taronna R; Tumpey, Terrence M; Belser, Jessica A

2018-04-01

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film. IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin.

PubMed

Kumaki, Yohichi; Wandersee, Miles K; Smith, Aaron J; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M; Bailey, Kevin W; Vest, Zachary G; Li, Joseph K-K; Chan, Paul Kay-Sheung; Smee, Donald F; Barnard, Dale L

2011-04-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

PubMed Central

Kumaki, Yohichi; Wandersee, Miles K.; Smith, Aaron J.; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M.; Bailey, Kevin W.; Vest, Zachary G.; Li, Joseph K.-K.; Chan, Paul Kay-Sheung; Smee, Donald F.; Barnard, Dale L.

2011-01-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 hours before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p<0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. PMID:21338626

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

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

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

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirusmore » pathogenesis.« less

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

PubMed

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

2015-11-01

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirus pathogenesis.

Cycluridine: A novel antiviral effective against flaviviruses

PubMed Central

Galabov, Angel S; Mukova, Lucia; Abashev, Yuriy P; Wassilewa, Lilia; Tzvetkov, Petko; Minkov, Vassil; Barinskiy, Igor F; Rice, Charles M; Ouzounov, Sergey; Sidzhakova, Dorotea

2017-01-01

This review describes the contemporary state of research for antivirals effective against flaviviruses, especially focusing on inhibitors of the pestivirus causative agent of bovine viral diarrhoea virus. We highlight cycluridine, an originally synthesized Mannich’s base [a tetrahydro-2(1H)-pyrimidinones derivative], as a highly effective antiviral possessing a strong inhibitory effect on bovine viral diarrhoea virus replication. Cycluridine was active against replication of a wide variety of bovine viral diarrhoea virus strains in cell cultures. The drug-sensitive period in the bovine viral diarrhoea virus replication cycle included the latent period and the exponential phase; a 90-min delay in the peak of viral RNA synthesis was observed. Cycluridine administered orally manifested a pronounced protective effect in calves with natural mucosal disease/viral diarrhoea and calves experimentally infected with bovine viral diarrhoea virus. Its magnitude of activity and selectivity places cycluridine in the lead among all known substances with anti- bovine viral diarrhoea virus activity. Additionally, cycluridine applied subcutaneously showed anti-tick-born encephalitis virus activity, manifesting a marked protective effect in mice infected with tick-born encephalitis virus. Cycluridine could be a prospective antiviral in veterinary and medical practice for the treatment of bovine viral diarrhoea virus and other flavivirus infections. PMID:28768435

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro.

PubMed

Ocazionez, Raquel Elvira; Meneses, Rocio; Torres, Flor Angela; Stashenko, Elena

2010-05-01

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

PubMed Central

Schoelz, James E.; Harries, Phillip A.; Nelson, Richard S.

2011-01-01

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread. PMID:21896501

Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

PubMed Central

Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling

2015-01-01

ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health. PMID:26581996

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

PubMed Central

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R.; Kolb, Gaëlle; Wiley, Michael R.; Jozwick, Lucas; Kuhn, Jens H.; Palacios, Gustavo; Radoshitzky, Sheli R.; J. Le Grice, Stuart F.; Johnson, Reed F.

2016-01-01

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. PMID:27651462

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

PubMed

Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

2009-06-20

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Therapeutic potential of oncolytic Newcastle disease virus: a critical review.

PubMed

Tayeb, Shay; Zakay-Rones, Zichria; Panet, Amos

2015-01-01

Newcastle disease virus (NDV) features a natural preference for replication in many tumor cells compared with normal cells. The observed antitumor effect of NDV appears to be a result of both selective killing of tumor cells and induction of immune responses. Genetic manipulations to change viral tropism and arming the virus with genes encoding for cytokines improved the oncolytic capacity of NDV. Several intracellular proteins in tumor cells, including antiapoptotic proteins (Livin) and oncogenic proteins (H-Ras), are relevant for the oncolytic activity of NDV. Defects in the interferon system, found in some tumor cells, also contribute to the oncolytic selectivity of NDV. Notwithstanding, NDV displays effective oncolytic activity in many tumor types, despite having intact interferon signaling. Taken together, several cellular systems appear to dictate the selective oncolytic activity of NDV. Some barriers, such as neutralizing antibodies elicited during NDV treatment and the extracellular matrix in tumor tissue appear to interfere with spread of NDV and reduce oncolysis. To further understand the oncolytic activity of NDV, we compared two NDV strains, ie, an attenuated virus (NDV-HUJ) and a pathogenic virus (NDV-MTH-68/H). Significant differences in amino acid sequence were noted in several viral proteins, including the fusion precursor (F0) glycoprotein, an important determinant of replication and pathogenicity. However, no difference in the oncolytic activity of the two strains was noted using human tumor tissues maintained as organ cultures or in mouse tumor models. To optimize virotherapy in clinical trials, we describe here a unique organ culture methodology, using a biopsy taken from a patient's tumor before treatment for ex vivo infection with NDV to determine the oncolytic potential on an individual basis. In conclusion, oncolytic NDV is an excellent candidate for cancer therapy, but more knowledge is needed to ensure success in clinical trials.

Empirical fitness models for hepatitis C virus immunogen design

NASA Astrophysics Data System (ADS)

Hart, Gregory R.; Ferguson, Andrew L.

2015-12-01

Hepatitis C virus (HCV) afflicts 170 million people worldwide, 2%-3% of the global population, and kills 350 000 each year. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic in the developing world where expensive drug therapies are not available. Despite 20 years of research, the high mutability of the virus and lack of knowledge of what constitutes effective immune responses have impeded development of an effective vaccine. Coupling data mining of sequence databases with spin glass models from statistical physics, we have developed a computational approach to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence. These landscapes explicitly connect viral genotype to phenotypic fitness, and reveal vulnerable immunological targets within the viral proteome that can be exploited to rationally design vaccine immunogens. We have recovered the empirical fitness landscape for the HCV RNA-dependent RNA polymerase (protein NS5B) responsible for viral genome replication, and validated the predictions of our model by demonstrating excellent accord with experimental measurements and clinical observations. We have used our landscapes to perform exhaustive in silico screening of 16.8 million T-cell immunogen candidates to identify 86 optimal formulations. By reducing the search space of immunogen candidates by over five orders of magnitude, our approach can offer valuable savings in time, expense, and labor for experimental vaccine development and accelerate the search for a HCV vaccine. Abbreviations: HCV—hepatitis C virus, HLA—human leukocyte antigen, CTL—cytotoxic T lymphocyte, NS5B—nonstructural protein 5B, MSA—multiple sequence alignment, PEG-IFN—pegylated interferon.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

PubMed

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-09-30

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome

PubMed Central

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-01-01

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts’ fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates ‘mother bias’ (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. PMID:27492289

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.

Cellular Restriction Factors of Feline Immunodeficiency Virus

PubMed Central

Zielonka, Jörg; Münk, Carsten

2011-01-01

Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

Focal adhesion kinase (FAK) regulates polymerase activity of multiple influenza A virus subtypes.

PubMed

Elbahesh, Husni; Bergmann, Silke; Russell, Charles J

2016-12-01

Influenza A viruses (IAVs) cause numerous pandemics and yearly epidemics resulting in ~500,000 annual deaths globally. IAV modulates cellular signaling pathways at every step of the infection cycle. Focal adhesion kinase (FAK) has been shown to play a critical role in endosomal trafficking of influenza A viruses, yet it is unclear how FAK kinase activity regulates IAV replication. Using mini-genomes derived from H1N1, H5N1 and H7N9 viruses, we dissected RNA replication by IAVs independent of viral entry or release. Our results show FAK activity promotes efficient IAV polymerase activity and inhibiting FAK activity with a chemical inhibitor or a kinase-dead mutant significantly reduces IAV polymerase activity. Using co-immunoprecipitations and proximity ligation assays, we observed interactions between FAK and the viral nucleoprotein, supporting a direct role of FAK in IAV replication. Altogether, the data indicates that FAK kinase activity is important in promoting IAV replication by regulating its polymerase activity. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1991-01-01

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). Images PMID:2041773

Competitive replication kinetics and pathogenicity in pigs co-infected with historical and newly invading classical swine fever viruses.

PubMed

Huang, Yu-Liang; Deng, Ming-Chung; Tsai, Kuo-Jung; Liu, Hsin-Meng; Huang, Chin-Cheng; Wang, Fun-In; Chang, Chia-Yi

2017-01-15

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication and Adaptive Mutations of Low Pathogenic Avian Influenza Viruses in Tracheal Organ Cultures of Different Avian Species

PubMed Central

Petersen, Henning; Matrosovich, Mikhail; Pleschka, Stephan; Rautenschlein, Silke

2012-01-01

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants. PMID:22912693

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.

Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation-A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion.

PubMed

Goldaracena, N; Spetzler, V N; Echeverri, J; Kaths, J M; Cherepanov, V; Persson, R; Hodges, M R; Janssen, H L A; Selzner, N; Grant, D R; Feld, J J; Selzner, M

2017-04-01

Normothermic ex vivo liver perfusion (NEVLP) offers the potential to optimize graft function prior to liver transplantation (LT). Hepatitis C virus (HCV) is dependent on the presence of miRNA(microRNA)-122. Miravirsen, a locked-nucleic acid oligonucleotide, sequesters miR-122 and inhibits HCV replication. The aim of this study was to assess the efficacy of delivering miravirsen during NEVLP to inhibit miR-122 function in a pig LT model. Pig livers were treated with miravirsen during NEVLP or cold storage (CS). Miravirsen absorption, miR-122 sequestration, and miR-122 target gene derepression were determined before and after LT. The effect of miravirsen treatment on HCV infection of hepatoma cells was also assessed. NEVLP improved miravirsen uptake versus CS. Significant miR-122 sequestration and miR-122 target gene derepression were seen with NEVLP but not with CS. In vitro data confirmed miravirsen suppression of HCV replication after established infection and prevented HCV infection with pretreatment of cells, analogous to the pretreatment of grafts in the transplant setting. In conclusion, miravirsen delivery during NEVLP is a potential strategy to prevent HCV reinfection after LT. This is the first large-animal study to provide "proof of concept" for using NEVLP to modify and optimize liver grafts for transplantation. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Hepatitis B virus molecular biology and pathogenesis

PubMed Central

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. PMID:28042609

A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

PubMed

Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

2014-04-01

Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

Foot-and-mouth disease virus 5’-terminal S fragment is required for replication and modulation of the innate immune response in host cells

USDA-ARS?s Scientific Manuscript database

The foot-and-mouth disease virus (FMDV) contains a 5’ untranslated region (5’UTR) with multiple structural domains that regulate viral genome replication, translation, and virus-host interactions. At its 5’terminus, the S fragment of over 360 bp is predicted to form a stable stem-loop that is separ...

Replication Capacity of Avian Influenza A(H9N2) Virus in Pet Birds and Mammals, Bangladesh.

PubMed

Lenny, Brian J; Shanmuganatham, Karthik; Sonnberg, Stephanie; Feeroz, Mohammed M; Alam, S M Rabiul; Hasan, M Kamrul; Jones-Engel, Lisa; McKenzie, Pamela; Krauss, Scott; Webster, Robert G; Jones, Jeremy C

2015-12-01

Avian influenza A(H9N2) is an agricultural and public health threat. We characterized an H9N2 virus from a pet market in Bangladesh and demonstrated replication in samples from pet birds, swine tissues, human airway and ocular cells, and ferrets. Results implicated pet birds in the potential dissemination and zoonotic transmission of this virus.

Evaluation of chimeric yellow fever 17D/dengue viral replication in ticks.

PubMed

Kazimírová, Mária; Mantel, Nathalie; Raynaud, Sandrine; Slovák, Mirko; Ustaniková, Katarína; Lang, Jean; Guy, Bruno; Barban, Veronique; Labuda, Milan

2012-11-01

Chimeric yellow fever 17D/DENV-1-4 viruses (CYD-1-4) have been developed as a tetravalent dengue vaccine candidate which is currently being evaluated in efficacy trials in Asia and America. While YF 17D and DENV are mosquito-borne flaviviruses, it has been shown that CYD-1-4 do not replicate after oral infection in mosquitoes and are not transmitted to new hosts. To further document the risk of environmental dissemination of these viruses, we evaluated the replication of CYD-1-4 in ticks, the vector of tick-borne encephalitis virus (TBEV), another member of the flavivirus family. Females of two hard tick species, Ixodes ricinus and Rhipicephalus appendiculatus, were inoculated intracoelomically with CYD-1-4 viruses and parent viruses (DENV-1-4 and YF 17D). Virus persistence and replication was assessed 2, 16, and 44 days post-inoculation by plaque titration and qRT-PCR. CYD-1-4 viruses were detected in I. ricinus ticks at early time points post-inoculation, but with infectious titers at least 100-fold lower than those observed in TBEV-infected ticks. Unlike TBEV, complete viral clearance occurred by day 44 in most ticks except for CYD-2, which had a tendency to decline. In addition, while about 70% of TBEV-infected I. ricinus nymphs acquired infection by co-feeding with infected tick females on non-viremic hosts, no co-feeding transmission of CYD-2 virus was detected. Based on these results, we conclude that the risk of dissemination of the candidate vaccine viruses by tick bite is highly unlikely.

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells.

PubMed

Massin, Pascale; Kuntz-Simon, Gaëlle; Barbezange, Cyril; Deblanc, Céline; Oger, Aurélie; Marquet-Blouin, Estelle; Bougeard, Stéphanie; van der Werf, Sylvie; Jestin, Véronique

2010-05-19

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication. Copyright 2009 Elsevier B.V. All rights reserved.

A Novel Self-Replicating Chimeric Lentivirus-Like Particle

PubMed Central

Young, Kelly R.; Madden, Victoria J.; Johnson, Philip R.; Johnston, Robert E.

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4+ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation. PMID:22013035

A novel self-replicating chimeric lentivirus-like particle.

PubMed

Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

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.

Replication, pathogenicity, shedding, and transmission of Zaire ebolavirus in pigs.

PubMed

Kobinger, Gary P; Leung, Anders; Neufeld, James; Richardson, Jason S; Falzarano, Darryl; Smith, Greg; Tierney, Kevin; Patel, Ami; Weingartl, Hana M

2011-07-15

(See the editorial commentary by Bausch, on pages 179-81.) Reston ebolavirus was recently detected in pigs in the Philippines. Specific antibodies were found in pig farmers, indicating exposure to the virus. This important observation raises the possibility that pigs may be susceptible to Ebola virus infection, including from other species, such as Zaire ebolavirus (ZEBOV), and can transmit to other susceptible hosts. This study investigated whether ZEBOV, a species commonly reemerging in central Africa, can replicate and induce disease in pigs and can be transmitted to naive animals. Domesticated Landrace pigs were challenged through mucosal exposure with a total of 1 ×10(6) plaque-forming units of ZEBOV and monitored for virus replication, shedding, and pathogenesis. Using similar conditions, virus transmission from infected to naive animals was evaluated in a second set of pigs. Following mucosal exposure, pigs replicated ZEBOV to high titers (reaching 10(7) median tissue culture infective doses/mL), mainly in the respiratory tract, and developed severe lung pathology. Shedding from the oronasal mucosa was detected for up to 14 days after infection, and transmission was confirmed in all naive pigs cohabiting with inoculated animals. These results shed light on the susceptibility of pigs to ZEBOV infection and identify an unexpected site of virus amplification and shedding linked to transmission of infectious virus.

Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.

PubMed

Amon, Wolfgang; White, Robert E; Farrell, Paul J

2006-05-01

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

PubMed Central

Townsend, R; Watts, J; Stanley, J

1986-01-01

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently. Images PMID:3951986

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

PubMed Central

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

2016-01-01

ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. PMID:27558420

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

PubMed

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

2016-11-01

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Transmitted virus fitness and host T cell responses collectively define divergent infection outcomes in two HIV-1 recipients

DOE PAGES

Yue, Ling; Pfafferott, Katja J.; Baalwa, Joshua; ...

2015-01-08

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/foundermore » (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.« less

Transmitted virus fitness and host T cell responses collectively define divergent infection outcomes in two HIV-1 recipients

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

Yue, Ling; Pfafferott, Katja J.; Baalwa, Joshua

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/foundermore » (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.« less

In vitro inhibition of human influenza A virus replication by chloroquine

PubMed Central

Ooi, Eng Eong; Chew, Janet Seok Wei; Loh, Jin Phang; Chua, Robert CS

2006-01-01

Chloroquine is a 9-aminoquinolone with well-known anti-malarial effects. It has biochemical properties that could be applied to inhibit viral replication. We report here that chloroquine is able to inhibit influenza A virus replication, in vitro, and the IC50s of chloroquine against influenza A viruses H1N1 and H3N2 are lower than the plasma concentrations reached during treatment of acute malaria. The potential of chloroquine to be added to the limited range of anti-influenza drugs should be explored further, particularly since antiviral drugs play a vital role in influenza pandemic preparedness. PMID:16729896

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

PubMed

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

2015-10-01

Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

PubMed Central

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

2015-01-01

ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. PMID:26246569

Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1

PubMed Central

Dziuba, Natallia; Ferguson, Monique R.; O'Brien, William A.; Sanchez, Anthony; Prussia, Andrew J.; McDonald, Natalie J.; Friedrich, Brian M.; Li, Guangyu; Shaw, Michael W.; Sheng, Jinsong; Hodge, Thomas W.; Rubin, Donald H.

2012-01-01

Abstract Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation. PMID:22404213

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

PubMed

Dziuba, Natallia; Ferguson, Monique R; O'Brien, William A; Sanchez, Anthony; Prussia, Andrew J; McDonald, Natalie J; Friedrich, Brian M; Li, Guangyu; Shaw, Michael W; Sheng, Jinsong; Hodge, Thomas W; Rubin, Donald H; Murray, James L

2012-10-01

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

PubMed

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

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

Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo

We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retardingmore » ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. - Highlights: •Hemin treatment protected monocyte-derived macrophages against Zika virus (ZIKV) infection. •Innate cellular protection against ZIKV infection correlated with Nrf2-dependent HO-1 expression. •Stimulation of innate cellular responses may provide a therapeutic strategy against ZIKV infection.« less

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells

PubMed Central

Qiao, Wenjie; Medina, Vicente; Falk, Bryce W.

2017-01-01

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses. PMID:29021801

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

A MicroRNA Screen Identifies the Wnt Signaling Pathway as a Regulator of the Interferon Response during Flavivirus Infection

PubMed Central

Smith, Jessica L.; Jeng, Sophia; McWeeney, Shannon K.

2017-01-01

ABSTRACT The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiflaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three flaviviruses, DENV, WNV, and Japanese encephalitis virus (JEV), using a high-content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/β-catenin signaling, demonstrated strong antiflaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate interferon regulatory factor 3 (IRF3) phosphorylation and translocation to the nucleus, the induction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3β (GSK3β)–TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating the replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research. IMPORTANCE MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through the posttranscriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high-content screen for miRNAs that inhibit the replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus 2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow-up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways but also provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions. PMID:28148804

A MicroRNA Screen Identifies the Wnt Signaling Pathway as a Regulator of the Interferon Response during Flavivirus Infection.

PubMed

Smith, Jessica L; Jeng, Sophia; McWeeney, Shannon K; Hirsch, Alec J

2017-04-15

The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiflaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three flaviviruses, DENV, WNV, and Japanese encephalitis virus (JEV), using a high-content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/β-catenin signaling, demonstrated strong antiflaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate interferon regulatory factor 3 (IRF3) phosphorylation and translocation to the nucleus, the induction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3β (GSK3β)-TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating the replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research. IMPORTANCE MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through the posttranscriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high-content screen for miRNAs that inhibit the replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus 2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow-up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways but also provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions. Copyright © 2017 American Society for Microbiology.

Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleoprotein during Replication in Chicken Brains ▿

PubMed Central

Tada, Tatsuya; Suzuki, Koutaro; Sakurai, Yu; Kubo, Masanori; Okada, Hironao; Itoh, Toshihiro; Tsukamoto, Kenji

2011-01-01

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP I109T). By analyzing viruses constructed by the reverse-genetic method, we established that the NP I109T substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP I109T substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP I109T substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP I109T mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains. PMID:21795332

Nonhuman Primate Models of Hepatitis A Virus and Hepatitis E Virus Infections.

PubMed

Lanford, Robert E; Walker, Christopher M; Lemon, Stanley M

2018-04-23

Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

PubMed

Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

2012-01-01

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

PubMed Central

Shuda, Masahiro; Feng, Huichen; Kwun, Hyun Jin; Rosen, Steven T.; Gjoerup, Ole; Moore, Patrick S.; Chang, Yuan

2008-01-01

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors. PMID:18812503

Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters.

PubMed

Orbanz, Jeannette; Finke, Stefan

2010-10-01

Bat lyssaviruses (Fam. Rhabdoviridae) represent a source for the infection of terrestial mammals and the development of rabies disease. Molecular differences in the replication of bat and non-bat lyssaviruses and their contribution to pathogenicity, however, are unknown. One reason for this is the lack of reverse genetics systems for bat-restricted lyssaviruses. To investigate bat lyssavirus replication and host adaptation, we developed a reverse genetics system for European bat lyssavirus type 1 (EBLV-1; genotype 5). This was achieved by co-transfection of HEK-293T cells with a full-length EBLV-1 genome cDNA and expression plasmids for EBLV-1 proteins, resulting in recombinant EBLV-1 (rEBLV-1). Replication of rEBLV-1 was comparable to that of parental virus, showing that rEBLV-1 is a valid tool to investigate EBLV-1 replication functions. In a first approach, we tested whether the terminal promoter sequences of EBLV-1 are genotype-specific. Although genotype 1 (rabies virus) minigenomes were successfully amplified by EBLV-1 helper virus, in the context of the complete virus, only the antigenome promoter (AGP) sequence of EBLV-1 was replaceable, as indicated by comparable replication of rEBLV-1 and the chimeric virus. These analyses demonstrate that the terminal AGPs of genotype 1 and genotype 5 lyssaviruses are compatible with those of the heterologous genotype.

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

Characterizing Bacteriophage PR772 as a Potential Surrogate for Adenovirus in Water Disinfection: A Comparative Analysis of Inactivation Kinetics and Replication Cycle Inhibition by Free Chlorine.

PubMed

Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

2016-03-01

Elucidating mechanisms by which pathogenic waterborne viruses become inactivated by drinking water disinfectants would facilitate the development of sensors to detect infectious viruses and novel disinfection strategies to provide safe water. Using bacteriophages as surrogates for human pathogenic viruses could assist in elucidating these mechanisms; however, an appropriate viral surrogate for human adenovirus (HAdV), a medium-sized virus with a double-stranded DNA genome, needs to be identified. Here, we characterized the inactivation kinetics of bacteriophage PR772, a member of the Tectiviridae family with many similarities in structure and replication to HAdV. The inactivation of PR772 and HAdV by free chlorine had similar kinetics that could be represented with a model previously developed for HAdV type 2 (HAdV-2). We developed and tested a quantitative assay to analyze several steps in the PR772 replication cycle to determine if both viruses being inactivated at similar rates resulted from similar replication cycle events being inhibited. Like HAdV-2, we observed that PR772 inactivated by free chlorine still attached to host cells, and viral DNA synthesis and early and late gene transcription were inhibited. Consequently, free chlorine exposure inhibited a replication cycle event that was post-binding but took place prior to early gene synthesis for both PR772 and HAdV-2.

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

PubMed Central

Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

2010-01-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

An Association between BK Virus Replication in Bone Marrow and Cytopenia in Kidney-Transplant Recipients

PubMed Central

Pambrun, Emilie; Mengelle, Catherine; Fillola, Geneviève; Laharrague, Patrick; Esposito, Laure; Cardeau-Desangles, Isabelle; Del Bello, Arnaud; Izopet, Jacques; Rostaing, Lionel; Kamar, Nassim

2014-01-01

The human polyomavirus BK (BKV) is associated with severe complications, such as ureteric stenosis and polyomavirus-associated nephropathy (PVAN), which often occur in kidney-transplant patients. However, it is unknown if BKV can replicate within bone marrow. The aim of this study was to search for BKV replication within the bone marrow of kidney-transplant patients presenting with a hematological disorder. Seventy-two kidney-transplant patients underwent bone-marrow aspiration for cytopenia. At least one virus was detected in the bone marrow of 25/72 patients (35%), that is, parvovirus B19 alone (n = 8), parvovirus plus Epstein-Barr virus (EBV) (n = 3), cytomegalovirus (n = 4), EBV (n = 2), BKV alone (n = 7), and BKV plus EBV (n = 1). Three of the eight patients who had BKV replication within the bone marrow had no detectable BKV replication in the blood. Neutropenia was observed in all patients with BKV replication in the bone marrow, and blockade of granulocyte maturation was observed. Hematological disorders disappeared in all patients after doses of immunosuppressants were reduced. In conclusion, an association between BKV replication in bone marrow and hematological disorders, especially neutropenia, was observed. Further studies are needed to confirm these findings. PMID:24868448

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.

PubMed

de Wilde, Adriaan H; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Chatterji, Udayan; de Jong, Danielle; Gallay, Philippe; Szuhai, Karoly; Posthuma, Clara C; Snijder, Eric J

2018-04-01

Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

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

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

Inside the lifestyle of the virophage.

PubMed

Desnues, C; Raoult, D

2010-01-01

We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in Acanthamoeba castellanii. A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by A. castellanii following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses. Copyright 2010 S. Karger AG, Basel.

Elimination of mitochondrial DNA is not required for herpes simplex virus 1 replication.

PubMed

Duguay, Brett A; Saffran, Holly A; Ponomarev, Alina; Duley, Shayla A; Eaton, Heather E; Smiley, James R

2014-03-01

Infection with herpes simplex virus type 1 (HSV-1) results in the rapid elimination of mitochondrial DNA (mtDNA) from host cells. It is known that a mitochondrial isoform of the viral alkaline nuclease (UL12) called UL12.5 triggers this process. However, very little is known about the impact of mtDNA depletion on viral replication or the biology of HSV-1 infections. These questions have been difficult to address because UL12.5 and UL12 are encoded by overlapping transcripts that share the same open reading frame. As a result, mutations that alter UL12.5 also affect UL12, and UL12 null mutations severely impair viral growth by interfering with the intranuclear processing of progeny viral genomes. Therefore, to specifically assess the impact of mtDNA depletion on viral replication, it is necessary to eliminate the activity of UL12.5 while preserving the nuclear functions of UL12. Previous work has shown that the human cytomegalovirus alkaline nuclease UL98 can functionally substitute for UL12 during HSV-1 replication. We found that UL98 is unable to deplete mtDNA in transfected cells and therefore generated an HSV-1 variant in which UL98 coding sequences replace the UL12/UL12.5 open reading frame. The resulting virus was severely impaired in its ability to trigger mtDNA loss but reached titers comparable to those of wild-type HSV-1 in one-step and multistep growth experiments. Together, these observations demonstrate that the elimination of mtDNA is not required for HSV-1 replication in cell culture. Herpes simplex virus types 1 and 2 destroy the DNA of host cell mitochondria, the powerhouses of cells. Epstein-Barr virus, a distantly related herpesvirus, has a similar effect, indicating that mitochondrial DNA destruction is under positive selection and thus confers a benefit to the virus. The present work shows that mitochondrial DNA destruction is not required for efficient replication of herpes simplex virus type 1 in cultured Vero kidney epithelial cells, suggesting that this activity likely benefits the virus in other cell types or in the intact human host.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

PubMed

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

Pharmacological inhibition of feline immunodeficiency virus (FIV).

PubMed

Mohammadi, Hakimeh; Bienzle, Dorothee

2012-05-01

Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses and causes an acquired immunodeficiency syndrome (AIDS) in domestic and non-domestic cats worldwide. Genome organization of FIV and clinical characteristics of the disease caused by the virus are similar to those of human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes and macrophages, and their replication cycle in infected cells is analogous. Due to marked similarity in genomic organization, virus structure, virus replication and disease pathogenesis of FIV and HIV, infection of cats with FIV is a useful tool to study and develop novel drugs and vaccines for HIV. Anti-retroviral drugs studied extensively in HIV infection have targeted different steps of the virus replication cycle: (1) inhibition of virus entry into susceptible cells at the level of attachment to host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus membrane with the cell membrane; (3) blockade of reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and viral DNA integration into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite much success of anti-retroviral therapy slowing disease progression in people, similar therapy has not been thoroughly investigated in cats. In this article we review current pharmacological approaches and novel targets for anti-lentiviral therapy, and critically assess potentially suitable applications against FIV infection in cats.

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

Vassilevska, Tanya

This is the first code, designed to run on a desktop, which models the intracellular replication and the cell-to-cell infection and demonstrates virus evolution at the molecular level. This code simulates the infection of a population of "idealized biological cells" (represented as objects that do not divide or have metabolism) with "virus" (represented by its genetic sequence), the replication and simultaneous mutation of the virus which leads to evolution of the population of genetically diverse viruses. The code is built to simulate single-stranded RNA viruses. The input for the code is 1. the number of biological cells in the culture,more » 2. the initial composition of the virus population, 3. the reference genome of the RNA virus, 4. the coordinates of the genome regions and their significance and, 5. parameters determining the dynamics of virus replication, such as the mutation rate. The simulation ends when all cells have been infected or when no more infections occurs after a given number of attempts. The code has the ability to simulate the evolution of the virus in serial passage of cell "cultures", i.e. after the end of a simulation, a new one is immediately scheduled with a new culture of infected cells. The code outputs characteristics of the resulting virus population dynamics and genetic composition of the virus population, such as the top dominant genomes, percentage of a genome with specific characteristics.« less

Ectromelia virus upregulates the expression of heat shock protein 70 to promote viral replication.

PubMed

Cheng, Wenyu; Jia, Huaijie; Wang, Xiaoxia; He, Xiaobing; Jin, Qiwang; Cao, Jingxin; Jing, Zhizhong

2018-08-01

The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein 70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcription‑quantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member 1B (Hspa1b), was highly upregulated during ECTV infection in vitro and in vivo. Subsequently, overexpression of Hspa1b protein and small interfering RNA‑mediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.

AR-12 suppresses dengue virus replication by down-regulation of PI3K/AKT and GRP78.

PubMed

Chen, Hsin-Hsin; Chen, Chien-Chin; Lin, Yee-Shin; Chang, Po-Chun; Lu, Zi-Yi; Lin, Chiou-Feng; Chen, Chia-Ling; Chang, Chih-Peng

2017-06-01

Dengue virus (DENV) infection has become a public health issue of worldwide concern and is a serious health problem in Taiwan, yet there are no approved effective antiviral drugs to treat DENV. The replication of DENV requires both viral and cellular factors. Targeting host factors may provide a potential antiviral strategy. It has been known that up-regulation of PI3K/AKT signaling and GRP78 by DENV infection supports its replication. AR-12, a celecoxib derivative with no inhibiting activity on cyclooxygenase, shows potent inhibitory activities on both PI3K/AKT signaling and GRP78 expression levels, and recently has been found to block the replication of several hemorrhagic fever viruses. However the efficacy of AR-12 in treating DENV infection is still unclear. Here, we provide evidence to show that AR-12 is able to suppress DENV replication before or after virus infection in cell culture and mice. The antiviral activities of AR-12 are positive against infection of the four different DENV serotypes. AR-12 significantly down-regulates the PI3K/AKT activity and GRP78 expression in DENV infected cells whereas AKT and GRP78 rescue are able to attenuate anti-DENV effect of AR-12. Using a DENV-infected suckling mice model, we further demonstrate that treatment of AR-12 before or after DENV infection reduces virus replication and mice mortality. In conclusion, we uncover the potential efficacy of AR-12 as a novel drug for treating dengue. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.; McGrath, M. S.; Hanks, D.; Erickson, S.; Pulliam, L.

1992-01-01

We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

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

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

PubMed

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

In this chapter, we describe the minigenome system for Ebola virus (EBOV), which reconstitutes EBOV polymerase activity in cells and can be used to model viral genome replication and transcription. This protocol comprises all steps including cell culture, plasmid preparation, transfection, and luciferase reporter assay readout.

Replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus and isolation of a td mutant from duck-adapted PR-RSV-C.

PubMed

Geryk, J; Mazo, A; Svoboda, J; Hlozánek, I

1980-01-01

The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.

The Cytoplasmic C-Tail of the Mouse Cytomegalovirus 7 Transmembrane Receptor Homologue, M78, Regulates Endocytosis of the Receptor and Modulates Virus Replication in Different Cell Types

PubMed Central

2016-01-01

Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes. PMID:27760189

Effect of sex steroid hormones on replication and transmission of major HIV subtypes.

PubMed

Ragupathy, Viswanath; Devadas, Krishnakumar; Tang, Shixing; Wood, Owen; Lee, Sherwin; Dastyer, Armeta; Wang, Xue; Dayton, Andrew; Hewlett, Indira

2013-11-01

The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV. Published by Elsevier Ltd.

Replication of 2 subtypes of low-pathogenicity avian influenza virus of duck and gull origins in experimentally infected Mallard ducks.

PubMed

Daoust, P-Y; van de Bildt, M; van Riel, D; van Amerongen, G; Bestebroer, T; Vanderstichel, R; Fouchier, R A M; Kuiken, T

2013-05-01

Many subtypes of low-pathogenicity avian influenza (LPAI) virus circulate in wild bird reservoirs, but their prevalence may vary among species. We aimed to compare by real-time reverse-transcriptase polymerase chain reaction, virus isolation, histology, and immunohistochemistry the distribution and pathogenicity of 2 such subtypes of markedly different origins in Mallard ducks (Anas platyrhynchos): H2N3 isolated from a Mallard duck and H13N6 isolated from a Ring-billed Gull (Larus delawarensis). Following intratracheal and intraesophageal inoculation, neither virus caused detectable clinical signs, although H2N3 virus infection was associated with a significantly decreased body weight gain during the period of virus shedding. Both viruses replicated in the lungs and air sacs until approximately day 3 after inoculation and were associated with a locally extensive interstitial, exudative, and proliferative pneumonia. Subtype H2N3, but not subtype H13N6, went on to infect the epithelia of the intestinal mucosa and cloacal bursa, where it replicated without causing lesions until approximately day 5 after inoculation. Larger quantities of subtype H2N3 virus were detected in cloacal swabs than in pharyngeal swabs. The possible clinical significance of LPAI virus-associated pulmonary lesions and intestinal tract infection in ducks deserves further evaluation.

42 CFR 73.3 - HHS select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-10-01

... virus Monkeypox virus Reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (Reconstructed 1918 Influenza virus...

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.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

PubMed

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

2016-11-16

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Rapid host immune response and viral dynamics in herpes simplex virus-2 infection

PubMed Central

Schiffer, Joshua T; Corey, Lawrence

2014-01-01

Herpes Simplex Virus-2 (HSV-2) is episodically shed throughout the human genital tract. While high viral load correlates with development of genital ulcers, shedding also commonly occurs even when ulcers are not present, allowing for silent transmission during coitus and contributing to high seroprevalence of HSV-2 worldwide. Frequent viral reactivation occurs despite diverse and complementary host and viral mechanisms within ganglionic tissue that predispose towards latency, suggesting that viral replication may be constantly occurring in a small minority of neurons within the ganglia. Within genital mucosa, the in vivo expansion and clearance rates of HSV-2 are extremely rapid. Resident dendritic cells and memory HSV-specific T cells persist at prior sites of genital tract reactivation, and in conjunction with prompt innate recognition of infected cells, lead to rapid containment of infected cells. Shedding episodes vary greatly in duration and severity within a single person over time: this heterogeneity appears best explained by variation in the densities of host immunity across the genital tract. The fact that immune responses usually control viral replication in genital skin prior to development of lesions provides optimism that enhancing such responses could lead to effective vaccines and immunotherapies. PMID:23467247

Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

PubMed

Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

2018-01-15

Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

USGS Publications Warehouse

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2017-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout.

PubMed

Wargo, Andrew R; Scott, Robert J; Kerr, Benjamin; Kurath, Gael

2017-01-02

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2days, defining a generally uniform early peak period of shedding from 1 to 4days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

PubMed Central

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2016-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority offish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. PMID:27771253

Autologous neutralizing antibody to human immunodeficiency virus-1 and replication-competent virus recovered from CD4+ T-cell reservoirs in pediatric HIV-1-infected patients on HAART.

PubMed

Ching, Natascha; Nielsen-Saines, Karin A; Deville, Jaime G; Wei, Lian S; Garratty, Eileen; Bryson, Yvonne J

2010-05-01

A patient's ability to produce autologous neutralizing antibody (ANAB) to current and past HIV isolates correlates with reduced disease progression and protects against maternal-fetal transmission. Little is known about the effects of prolonged viral suppression on the ANAB response in pediatric HIV-infected patients receiving HAART because the virus is hard to isolate, except by special methods. We therefore assessed ANAB to pre-HAART PBMC virus isolates and post-HAART replication-competent virus (RCV) isolates recovered from latent CD4(+) T-cell reservoirs in perinatally HIV-infected children by using a PBMC-based assay and 90% neutralization titers. We studied two infants and three children before and after HAART. At the time of RCV isolation (n = 4), plasma HIV RNA was <50 copies/ml. At baseline, four of five children had detectable ANAB titers to concurrent pre-HAART virus isolates. Although ANAB was detected in all subjects at several time points despite prolonged HAART and undetectable viremia, the response was variable. ANAB titers to concurrent post-HAART RCV and earlier pre-HAART plasma were present in 3 children suggesting prior exposure to this virus. Post-HAART RCV isolates had reduced replication kinetics in vitro compared to pre-HAART viruses. The presence of ANAB over time suggests that low levels of viral replication may still be ongoing despite HAART. The observation of baseline ANAB activity with earlier plasma against a later RCV suggests that the "latent" reservoir may be established early in life before HAART.

Short-lived infected cells support virus replication in sooty mangabeys naturally infected with simian immunodeficiency virus: implications for AIDS pathogenesis.

PubMed

Gordon, Shari N; Dunham, Richard M; Engram, Jessica C; Estes, Jacob; Wang, Zichun; Klatt, Nichole R; Paiardini, Mirko; Pandrea, Ivona V; Apetrei, Cristian; Sodora, Donald L; Lee, Ha Youn; Haase, Ashley T; Miller, Michael D; Kaur, Amitinder; Staprans, Silvija I; Perelson, Alan S; Feinberg, Mark B; Silvestri, Guido

2008-04-01

Sooty mangabeys (SMs) naturally infected with simian immunodeficiency virus (SIV) do not develop AIDS despite high levels of virus replication. At present, the mechanisms underlying this disease resistance are poorly understood. Here we tested the hypothesis that SIV-infected SMs avoid immunodeficiency as a result of virus replication occurring in infected cells that live significantly longer than human immunodeficiency virus (HIV)-infected human cells. To this end, we treated six SIV-infected SMs with potent antiretroviral therapy (ART) and longitudinally measured the decline in plasma viremia. We applied the same mathematical models used in HIV-infected individuals and observed that SMs naturally infected with SIV also present a two-phase decay of viremia following ART, with the bulk (92 to 99%) of virus replication sustained by short-lived cells (average life span, 1.06 days), and only 1 to 8% occurring in longer-lived cells. In addition, we observed that ART had a limited impact on CD4(+) T cells and the prevailing level of T-cell activation and proliferation in SIV-infected SMs. Collectively, these results suggest that in SIV-infected SMs, similar to HIV type 1-infected humans, short-lived activated CD4(+) T cells, rather than macrophages, are the main source of virus production. These findings indicate that a short in vivo life span of infected cells is a common feature of both pathogenic and nonpathogenic primate lentivirus infections and support a model for AIDS pathogenesis whereby the direct killing of infected cells by HIV is not the main determinant of disease progression.

Short-Lived Infected Cells Support Virus Replication in Sooty Mangabeys Naturally Infected with Simian Immunodeficiency Virus: Implications for AIDS Pathogenesis▿

PubMed Central

Gordon, Shari N.; Dunham, Richard M.; Engram, Jessica C.; Estes, Jacob; Wang, Zichun; Klatt, Nichole R.; Paiardini, Mirko; Pandrea, Ivona V.; Apetrei, Cristian; Sodora, Donald L.; Lee, Ha Youn; Haase, Ashley T.; Miller, Michael D.; Kaur, Amitinder; Staprans, Silvija I.; Perelson, Alan S.; Feinberg, Mark B.; Silvestri, Guido

2008-01-01

Sooty mangabeys (SMs) naturally infected with simian immunodeficiency virus (SIV) do not develop AIDS despite high levels of virus replication. At present, the mechanisms underlying this disease resistance are poorly understood. Here we tested the hypothesis that SIV-infected SMs avoid immunodeficiency as a result of virus replication occurring in infected cells that live significantly longer than human immunodeficiency virus (HIV)-infected human cells. To this end, we treated six SIV-infected SMs with potent antiretroviral therapy (ART) and longitudinally measured the decline in plasma viremia. We applied the same mathematical models used in HIV-infected individuals and observed that SMs naturally infected with SIV also present a two-phase decay of viremia following ART, with the bulk (92 to 99%) of virus replication sustained by short-lived cells (average life span, 1.06 days), and only 1 to 8% occurring in longer-lived cells. In addition, we observed that ART had a limited impact on CD4+ T cells and the prevailing level of T-cell activation and proliferation in SIV-infected SMs. Collectively, these results suggest that in SIV-infected SMs, similar to HIV type 1-infected humans, short-lived activated CD4+ T cells, rather than macrophages, are the main source of virus production. These findings indicate that a short in vivo life span of infected cells is a common feature of both pathogenic and nonpathogenic primate lentivirus infections and support a model for AIDS pathogenesis whereby the direct killing of infected cells by HIV is not the main determinant of disease progression. PMID:18216113

Hepatitis E virus persists in the presence of a type III interferon response.

PubMed

Yin, Xin; Li, Xinlei; Ambardekar, Charuta; Hu, Zhimin; Lhomme, Sébastien; Feng, Zongdi

2017-05-01

The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

PubMed

Cai, Jing; Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Tan, Yaqian; Cavenee, Webster K; Yan, Guangmei

2017-06-27

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses

NASA Astrophysics Data System (ADS)

Simon, Philippe F.; de La Vega, Marc-Antoine; Paradis, Éric; Mendoza, Emelissa; Coombs, Kevin M.; Kobasa, Darwyn; Beauchemin, Catherine A. A.

2016-04-01

Avian influenza viruses present an emerging epidemiological concern as some strains of H5N1 avian influenza can cause severe infections in humans with lethality rates of up to 60%. These have been in circulation since 1997 and recently a novel H7N9-subtyped virus has been causing epizootics in China with lethality rates around 20%. To better understand the replication kinetics of these viruses, we combined several extensive viral kinetics experiments with mathematical modelling of in vitro infections in human A549 cells. We extracted fundamental replication parameters revealing that, while both the H5N1 and H7N9 viruses replicate faster and to higher titers than two low-pathogenicity H1N1 strains, they accomplish this via different mechanisms. While the H7N9 virions exhibit a faster rate of infection, the H5N1 virions are produced at a higher rate. Of the two H1N1 strains studied, the 2009 pandemic H1N1 strain exhibits the longest eclipse phase, possibly indicative of a less effective neuraminidase activity, but causes infection more rapidly than the seasonal strain. This explains, in part, the pandemic strain’s generally slower growth kinetics and permissiveness to accept mutations causing neuraminidase inhibitor resistance without significant loss in fitness. Our results highlight differential growth properties of H1N1, H5N1 and H7N9 influenza viruses.

Susceptibility of chickens, quail, and pigeons to an H7N9 human influenza virus and subsequent egg-passaged strains.

PubMed

Uchida, Yuko; Kanehira, Katsushi; Takemae, Nobuhiro; Hikono, Hirokazu; Saito, Takehiko

2017-01-01

H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.

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.

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus.

PubMed

Nogales, Aitor; Rodriguez, Laura; DeDiego, Marta L; Topham, David J; Martínez-Sobrido, Luis

2017-09-01

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of the PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein had this inhibiting capability, while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PA WT + /NS1 MUT + ) or do not have (PA MUT - /NS1 WT - ) the ability to block host gene expression showed reduced pathogenicity in vivo However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PA MUT - /NS1 MUT + ) presented pathogenicity similar to that of a virus containing both wild-type proteins (PA WT + /NS1 WT - ). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV. IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition mechanisms, including prevention of host gene expression mediated by the viral PA-X and NS1 proteins. Here, we provide evidence demonstrating that optimal control of host protein synthesis by IAV PA-X and/or NS1 proteins is required for efficient IAV replication in the host. Moreover, we demonstrate the feasibility of genetically controlling the ability of IAV PA-X and NS1 proteins to inhibit host immune responses, providing an approach to develop more effective vaccines to combat disease caused by this important respiratory pathogen. Copyright © 2017 American Society for Microbiology.

Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants.

PubMed

Khandelwal, Nitin; Chander, Yogesh; Rawat, Krishan Dutt; Riyesh, Thachamvally; Nishanth, Chikkahonnaiah; Sharma, Shalini; Jindal, Naresh; Tripathi, Bhupendra N; Barua, Sanjay; Kumar, Naveen

2017-08-01

At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD 50 ) of 126.49 ng/egg and at an effective concentration 50 (EC 50 ) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts

PubMed Central

Rosato, Pamela C.

2014-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β, and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores, blindness, and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study, we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice, we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence or absence of innate immunity. In contrast, neurons were able to mount a robust antiviral response when provided with beta interferon, a molecule that strongly stimulates innate immunity, and that HSV-1 can combat this response through the γ34.5 viral gene. Our results have important implications for understanding how the nervous system defends itself against virus infections. PMID:24942587

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.

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

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

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2014-01-01

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. PMID:25054883

Protection From Varicella Zoster in Solid Organ Transplant Recipients Carrying Killer Cell Immunoglobulin-Like Receptor B Haplotypes.

PubMed

Schmied, Laurent; Terszowski, Grzegorz; Gonzalez, Asensio; Schmitter, Karin; Hirsch, Hans H; Garzoni, Christian; van Delden, Christian; Boggian, Katia; Mueller, Nicolas J; Berger, Christoph; Villard, Jean; Manuel, Oriol; Meylan, Pascal; Hess, Christoph; Stern, Martin

2015-12-01

Natural killer cell function is regulated by inhibitory and activating killer cell immunoglobulin-like receptors (KIR). Previous studies have documented associations of KIR genotype with the risk of cytomegalovirus (CMV) replication after solid organ transplantation. In this study of 649 solid organ transplant recipients, followed prospectively for infectious disease events within the Swiss Transplant Cohort Study, we were interested to see if KIR genotype associated with virus infections other than CMV. We found that KIR B haplotypes (which have previously been linked to protection from CMV replication) were associated with protection from varicella zoster virus infection (hazard ratio, 0.43; 95% confidence interval, 0.21-0.91; P = 0.03). No significant associations were detected regarding the risk of herpes simplex, Epstein-Barr virus or BK polyomavirus infections. In conclusion, these data provide evidence that the relative protection of KIR haplotype B from viral replication after solid organ transplantation may extend beyond CMV to other herpes viruses, such as varicella zoster virus and possibly Epstein-Barr virus.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

PubMed

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

PubMed Central

Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Suárez, Cristina; Salas, José

2013-01-01

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information. PMID:23824796

Differential replication of Foot-and-mouth disease viruses in mice determine lethality.

PubMed

Cacciabue, Marco; García-Núñez, María Soledad; Delgado, Fernando; Currá, Anabella; Marrero, Rubén; Molinari, Paula; Rieder, Elizabeth; Carrillo, Elisa; Gismondi, María Inés

2017-09-01

Adult C57BL/6J mice have been used to study Foot-and-mouth disease virus (FMDV) biology. In this work, two variants of an FMDV A/Arg/01 strain exhibiting differential pathogenicity in adult mice were identified and characterized: a non-lethal virus (A01NL) caused mild signs of disease, whereas a lethal virus (A01L) caused death within 24-48h independently of the dose used. Both viruses caused a systemic infection with pathological changes in the exocrine pancreas. Virus A01L reached higher viral loads in plasma and organs of inoculated mice as well as increased replication in an ovine kidney cell line. Complete consensus sequences revealed 6 non-synonymous changes between A01L and A10NL genomes that might be linked to replication differences, as suggested by in silico prediction studies. Our results highlight the biological significance of discrete genomic variations and reinforce the usefulness of this animal model to study viral determinants of lethality. Copyright © 2017 Elsevier Inc. All rights reserved.

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

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

Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.

We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain bymore » quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.« less

Archaeal Viruses: Diversity, Replication, and Structure.

PubMed

Dellas, Nikki; Snyder, Jamie C; Bolduc, Benjamin; Young, Mark J

2014-11-01

The Archaea-and their viruses-remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.

Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

PubMed

Zhou, Bin; Ma, Jingjiao; Liu, Qinfang; Bawa, Bhupinder; Wang, Wei; Shabman, Reed S; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B; Richt, Juergen A; Wentworth, David E; Ma, Wenjun

2014-10-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

PubMed Central

Bawa, Bhupinder; Wang, Wei; Shabman, Reed S.; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B.; Richt, Juergen A.; Wentworth, David E.; Ma, Wenjun

2014-01-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. PMID:25275541

Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets

PubMed Central

Belser, Jessica A.; Gustin, Kortney M.; Maines, Taronna R.; Pantin-Jackwood, Mary J.; Katz, Jacqueline M.; Tumpey, Terrence M.

2012-01-01

While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes. PMID:22396651

Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis.

PubMed

Mandl, Christian W

2005-08-01

Tick-borne encephalitis virus (TBEV) is an important human pathogen that causes severe neurological illness in large areas of Europe and Asia. The neuropathogenesis of this disease agent is determined by its capacity to enter the central nervous system (CNS) after peripheral inoculation ("neuroinvasiveness") and its ability to replicate and cause damage within the CNS ("neurovirulence"). TBEV is a small, enveloped flavivirus with an unsegmented, positive-stranded RNA genome. Mutations affecting various steps of its natural replication cycle were shown to influence its neuropathogenic properties. This review describes experimental approaches and summarizes results on molecular determinants of neurovirulence and neuroinvasiveness that have been identified for this virus. It focuses on molecular mechanisms of three particular steps of the viral life cycle that have been studied in some detail for TBEV and two closely related tick-borne flaviviruses (Louping ill virus (LIV) and Langat virus (LGTV)), namely (i) the envelope protein E and its role in viral attachment to the cell surface, (ii) the 3'-noncoding region of the genome and its importance for viral RNA replication, and (iii) the capsid protein C and its role in the assembly process of infectious virus particles. Mutations affecting each of these three molecular targets significantly influence neuropathogenesis of TBEV, particularly its neuroinvasiveness. The understanding of molecular determinants of TBEV neuropathogenesis is relevant for vaccine development, also against other flaviviruses.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

PubMed Central

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

2011-01-01

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. PMID:21622960

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

PubMed Central

Meliopoulos, Victoria A.; Andersen, Lauren E.; Birrer, Katherine F.; Simpson, Kaylene J.; Lowenthal, John W.; Bean, Andrew G. D.; Stambas, John; Stewart, Cameron R.; Tompkins, S. Mark; van Beusechem, Victor W.; Fraser, Iain; Mhlanga, Musa; Barichievy, Samantha; Smith, Queta; Leake, Devin; Karpilow, Jon; Buck, Amy; Jona, Ghil; Tripp, Ralph A.

2012-01-01

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. PMID:22247330

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

PubMed Central

Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

2015-01-01

ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

NASA Astrophysics Data System (ADS)

Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

2014-04-01

Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

Oncolytic Herpes Simplex Viral Therapy: A Stride toward Selective Targeting of Cancer Cells.

PubMed

Sanchala, Dhaval S; Bhatt, Lokesh K; Prabhavalkar, Kedar S

2017-01-01

Oncolytic viral therapy, which makes use of replication-competent lytic viruses, has emerged as a promising modality to treat malignancies. It has shown meaningful outcomes in both solid tumor and hematologic malignancies. Advancements during the last decade, mainly genetic engineering of oncolytic viruses have resulted in improved specificity and efficacy of oncolytic viruses in cancer therapeutics. Oncolytic viral therapy for treating cancer with herpes simplex virus-1 has been of particular interest owing to its range of benefits like: (a) large genome and power to infiltrate in the tumor, (b) easy access to manipulation with the flexibility to insert multiple transgenes, (c) infecting majority of the malignant cell types with quick replication in the infected cells and (d) as Anti-HSV agent to terminate HSV replication. This review provides an exhaustive list of oncolytic herpes simplex virus-1 along with their genetic alterations. It also encompasses the major developments in oncolytic herpes simplex-1 viral therapy and outlines the limitations and drawbacks of oncolytic herpes simplex viral therapy.

Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection.

PubMed

Maeda, A; Wakiguchi, H; Yokoyama, W; Hisakawa, H; Tomoda, T; Kurashige, T

1999-04-01

Chronic active Epstein-Barr virus infection (CAEBV) is a severe illness with unusual EBV activation that persists for years, and its pathogenesis is largely unknown. After the creation of an accurate and reproducible polymerase chain reaction system to quantify EBV DNA, virus loads in peripheral blood lymphocytes (PBL) were determined in 54 children: 15 with CAEBV, 16 with infectious mononucleosis (IM), and 23 healthy children. Children with CAEBV and those with IM had high virus loads. Lower loads were detected in 47% of seropositive healthy donors. There were two distinct differences between children with CAEBV and those with IM: The former had greater viral replication (10(3)-10(7) copies/2.5x10(5) PBL) than those with IM, and viral replication declined in children with IM whereas active replication persisted for years in subjects with CAEBV. Persisting high virus loads are a possible diagnostic criterion for CAEBV. EBV loads may enable classification and prognosis of EBV infections.

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

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.

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: Potential as stable genetic markers

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

Reddy, S.T.; Stoker, A.W.; Bissell, M.J.

1991-12-01

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, the authors further show that blastoderms can be infected in culture and inmore » ovo. In ovo, lacZ expression is seen within 24 hours of virus inoculation, and by 96 hours stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.« less

VIRUS: first deployment of the massively replicated fiber integral field spectrograph for the upgraded Hobby-Eberly Telescope

NASA Astrophysics Data System (ADS)

Hill, Gary J.; Tuttle, Sarah E.; Vattiat, Brian L.; Lee, Hanshin; Drory, Niv; Kelz, Andreas; Ramsey, Jason; Peterson, Trent W.; DePoy, D. L.; Marshall, J. L.; Gebhardt, Karl; Chonis, Taylor; Dalton, Gavin; Farrow, Daniel; Good, John M.; Haynes, Dionne M.; Indahl, Briana L.; Jahn, Thomas; Kriel, Hermanus; Montesano, Francesco; Nicklas, Harald; Noyola, Eva; Prochaska, Travis; Allen, Richard D.; Bender, Ralf; Blanc, Guillermo; Fabricius, Maximilian H.; Finkelstein, Steve; Landriau, Martin; MacQueen, Phillip J.; Roth, M. M.; Savage, R. D.; Snigula, Jan M.; Anwad, Heiko

2016-08-01

The Visible Integral-field Replicable Unit Spectrograph (VIRUS) consists of 156 identical spectrographs (arrayed as 78 pairs) fed by 35,000 fibers, each 1.5 arcsec diameter, at the focus of the upgraded 10 m Hobby-Eberly Telescope (HET). VIRUS has a fixed bandpass of 350-550 nm and resolving power R 700. VIRUS is the first example of industrial-scale replication applied to optical astronomy and is capable of surveying large areas of sky, spectrally. The VIRUS concept offers significant savings of engineering effort, cost, and schedule when compared to traditional instruments. The main motivator for VIRUS is to map the evolution of dark energy for the Hobby-Eberly Telescope Dark Energy Experiment (HETDEX‡), using 0.8M Lyman-alpha emitting galaxies as tracers. The VIRUS array is undergoing staged deployment during 2016 and 2017. It will provide a powerful new facility instrument for the HET, well suited to the survey niche of the telescope, and will open up large spectroscopic surveys of the emission line universe for the first time. We will review the production, lessons learned in reaching volume production, characterization, and first deployment of this massive instrument.

A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics

PubMed Central

Pieknik, Julianna R.; Tang, Shuang

2018-01-01

Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection. PMID:29738431

[Comparative evaluation of Leningrad-3 mumps vaccine virus neurovirulence in a neonatal rat model].

PubMed

Ignat'ev, G M; Otrashevskaia, E V; Rubin, S A

2011-01-01

The neurovirulence and replication potential of several mumps virus strains, including Leningrad-3 mumps vaccine virus (FSUE SIC "Microgen", Russia) and wild type strains isolated in the Novosibirsk Region (Russia), were assessed in rat tests. The mean neurovirulence scores of the Leningrad-3 virus (< 4.0) were significantly lower than those of wild type strains (ranging from 6.1 to 15.2) and were in accordance with the scores determined for other attenuated mumps vaccine strains (usually ranging from 0 to 5). In general, the relative ability of the viruses to replicate in the rat brain tracked with their neurovirulence scores. These results indicate a low neurovirulence potential of the Leningrad-3 mumps vaccine virus for humans.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Porcine reproductive and respiratory syndrome virus infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff.

PubMed

Li, Yang; Fang, Liurong; Zhou, Yanrong; Tao, Ran; Wang, Dang; Xiao, Shaobo

2018-06-13

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest. IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2α (eIF2α). However, PRRSV nsp2 does not induce eIF2α phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by dephosphorylating eIF2α or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff. Copyright © 2018 American Society for Microbiology.

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

PubMed Central

Takeda, Makoto; Pekosz, Andrew; Shuck, Kevin; Pinto, Lawrence H.; Lamb, Robert A.

2002-01-01

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31 virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31 virus. We found that the M2-del29–31 virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31 virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31 virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle. PMID:11773413

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.

The Effect of Antiretroviral Combination Treatment on Epstein-Barr Virus (EBV) Genome Load in HIV-Infected Patients

PubMed Central

Friis, Anna M. C.; Gyllensten, Katarina; Aleman, Anna; Ernberg, Ingemar; Åkerlund, Börje

2010-01-01

We evaluated the effect of combination anti-retroviral treatment (cART) on the host control of EBV infection in moderately immunosuppressed HIV-1 patients. Twenty HIV-1 infected individuals were followed for five years with repeated measurements of EBV DNA load in peripheral blood lymphocytes in relation to HIV-RNA titers and CD4+ cell counts. Individuals with optimal response, i.e. durable non-detectable HIV-RNA, showed a decline of EBV load to the level of healthy controls. Individuals with non-optimal HIV-1 control did not restore their EBV control. Long-lasting suppression of HIV-replication after early initiation of cART is a prerequisite for re-establishing the immune control of EBV. PMID:21994658

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion.

PubMed

Wu, Ren-Huang; Tsai, Ming-Han; Chao, Day-Yu; Yueh, Andrew

2015-04-01

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

PubMed Central

Johnson, Stephen M.; Eltahla, Auda A.; Aloi, Maria; Aloia, Amanda L.; McDevitt, Christopher A.; Bull, Rowena A.

2017-01-01

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools. PMID:28956770

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice

PubMed Central

Matundan, Harry H.; Mott, Kevin R.; Allen, Sariah J.; Wang, Shaohui; Bresee, Catherine J.; Ghiasi, Yasamin N.; Town, Terrence

2016-01-01

ABSTRACT We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT(−) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation. PMID:27512072

Herpesvirus papio contains a plasmid origin of replication that acts in cis interspecies with an Epstein-Barr virus trans-acting function.

PubMed Central

Pesano, R L; Pagano, J S

1986-01-01

Herpesvirus papio (HVP) and Epstein-Barr virus (EBV) are closely related biologically and biochemically; lymphoblastoid cells infected with either virus contain episomal viral DNA. The putative origin of replication for EBV plasmids (oriP) has been assigned to a 1,790-base-pair fragment (cis) in the short unique region of the genome which requires a viral function supplied in trans from elsewhere in the genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We report here the identification of the putative origin of replication (cis) in HVP; we assigned it to the HVP EcoRI K fragment. The results indicate that the HVP replication process requires both a cis and a trans-acting function, analogous to that found in EBV. Images PMID:3023667

Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells

PubMed Central

Powell, Sharon K.; Artlip, Moria; Kaloss, Michele; Brazinski, Scott; Lyons, Russette; McGarrity, Gerard J.; Otto, Edward

1999-01-01

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone. PMID:10482636

Ebolaviruses: New roles for old proteins.

PubMed

Cantoni, Diego; Rossman, Jeremy S

2018-05-01

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein's primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity.

Ebolaviruses: New roles for old proteins

PubMed Central

2018-01-01

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein’s primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity. PMID:29723187

Immune response of mice to non-adapted avian influenza A virus.

PubMed

Stropkovská, A; Mikušková, T; Bobišová, Z; Košík, I; Mucha, V; Kostolanský, F; Varečková, E

2015-12-01

Human infections with avian influenza A viruses (IAVs) without or with clinical symptoms of disease were recently reported from several continents, mainly in high risk groups of people, who came into the contact with infected domestic birds or poultry. It was shown that avian IAVs are able to infect humans directly without previous adaptation, however, their ability to replicate and to cause a disease in this new host can differ. No spread of these avian IAVs among humans has been documented until now, except for one case described in Netherlands in the February of 2003 in people directly involved in handling IAV (H7N7)-infected poultry. The aim of our work was to examine whether a low pathogenic avian IAV can induce a virus-specific immune response of biological relevancy, in spite of its restricted replication in mammals. As a model we used a low pathogenic virus A/Duck/Czechoslovakia/1956 (H4N6) (A/Duck), which replicated well in MDCK cells and produced plaques on cell monolayers, but was unable to replicate productively in mouse lungs. We examined how the immune system of mice responds to the intranasal application of this non-adapted avian virus. Though we did not prove the infectious virus in lungs of mice following A/Duck application even after its multiple passaging in mice, we detected virus-specific vRNA till day 8 post infection. Moreover, we detected virus-specific mRNA and de novo synthesized viral nucleoprotein (NP) and membrane protein (M1) in lungs of mice on day 2 and 4 after exposure to A/Duck. Virus-specific antibodies in sera of these mice were detectable by ELISA already after a single intranasal dose of A/Duck virus. Not only antibodies specific to the surface glycoprotein hemagglutinin (HA) were induced, but also antibodies specific to the NP and M1 of IAV were detected by Western blot and their titers increased after the second exposure of mice to this virus. Importantly, antibodies neutralizing virus A/Duck were proved in mouse immune sera after the second dose of virus and a slight increase of mRNA expression of immune mediators tumor necrosis factor alpha (TNF-α) and IP10 has been observed in lungs of these mice 48 hr after the infection. These observations correspond to the limited replication ability of the virus in mice and provided an important information about its ability to induce virus-specific antibodies, including those neutralizing virus, even without the previous virus adaptation to the new mammalian host. Such antibodies could consequently influence the immune potential of exposed individuals and their defensive capability against the newly emerged, even more virulent IAV.

Photodynamic treatment of herpes simplex virus during its replicative cycle.

PubMed Central

Khan, N C; Melnick, J L; Biswal, N

1977-01-01

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation. PMID:189063

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis

PubMed Central

Zhang, Zhenlu; He, Guijuan; Catanzaro, Nicholas; Wu, Zujian; Xie, Lianhui

2018-01-01

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection. PMID:29649282