Phosphate transporters in marine phytoplankton and their viruses: cross-domain commonalities in viral-host gene exchanges.

PubMed

Monier, Adam; Welsh, Rory M; Gentemann, Chelle; Weinstock, George; Sodergren, Erica; Armbrust, E Virginia; Eisen, Jonathan A; Worden, Alexandra Z

2012-01-01

Phosphate (PO(4)) is an important limiting nutrient in marine environments. Marine cyanobacteria scavenge PO(4) using the high-affinity periplasmic phosphate binding protein PstS. The pstS gene has recently been identified in genomes of cyanobacterial viruses as well. Here, we analyse genes encoding transporters in genomes from viruses that infect eukaryotic phytoplankton. We identified inorganic PO(4) transporter-encoding genes from the PHO4 superfamily in several virus genomes, along with other transporter-encoding genes. Homologues of the viral pho4 genes were also identified in genome sequences from the genera that these viruses infect. Genome sequences were available from host genera of all the phytoplankton viruses analysed except the host genus Bathycoccus. Pho4 was recovered from Bathycoccus by sequencing a targeted metagenome from an uncultured Atlantic Ocean population. Phylogenetic reconstruction showed that pho4 genes from pelagophytes, haptophytes and infecting viruses were more closely related to homologues in prasinophytes than to those in what, at the species level, are considered to be closer relatives (e.g. diatoms). We also identified PHO4 superfamily members in ocean metagenomes, including new metagenomes from the Pacific Ocean. The environmental sequences grouped with pelagophytes, haptophytes, prasinophytes and viruses as well as bacteria. The analyses suggest that multiple independent pho4 gene transfer events have occurred between marine viruses and both eukaryotic and bacterial hosts. Additionally, pho4 genes were identified in available genomes from viruses that infect marine eukaryotes but not those that infect terrestrial hosts. Commonalities in marine host-virus gene exchanges indicate that manipulation of host-PO(4) uptake is an important adaptation for viral proliferation in marine systems. Our findings suggest that PO(4) -availability may not serve as a simple bottom-up control of marine phytoplankton. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

The role of Epstein–Barr virus in epithelial malignancies

PubMed Central

Tsao, Sai-Wah; Tsang, Chi Man; To, Ka-Fai; Lo, Kwok-Wai

2015-01-01

The close association of Epstein–Barr virus (EBV) infection with non-keratinizing nasopharyngeal carcinomas and a subset of gastric carcinomas suggests that EBV infection is a crucial event in these cancers. The difficulties encountered in infecting and transforming primary epithelial cells in experimental systems suggest that the role of EBV in epithelial malignancies is complex and multifactorial in nature. Genetic alterations in the premalignant epithelium may support the establishment of latent EBV infection, which is believed to be an initiation event. Oncogenic properties have been reported in multiple EBV latent genes. The BamH1 A rightwards transcripts (BARTs) and the BART-encoded microRNAs (miR-BARTs) are highly expressed in EBV-associated epithelial malignancies and may induce malignant transformation. However, enhanced proliferation may not be the crucial function of EBV infection in epithelial malignancies, at least in the early stages of cancer development. EBV-encoded gene products may confer anti-apoptotic properties and promote the survival of infected premalignant epithelial cells harbouring genetic alterations. Multiple EBV-encoded microRNAs have been reported to have immune evasion functions. Genetic alterations in host cells, as well as inflammatory stroma, could modulate the expression of EBV genes and alter the growth properties of infected premalignant epithelial cells, encouraging their selection during carcinogenesis. PMID:25251730

DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

PubMed

Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

2012-05-01

We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days.

PubMed

de Fabritus, Lauriane; Nougairède, Antoine; Aubry, Fabien; Gould, Ernest A; de Lamballerie, Xavier

2016-01-01

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.

An infectious bat chimeric influenza virus harboring the entry machinery of a influenza A virus

PubMed Central

Juozapaitis, Mindaugas; Moreira, Étori Aguiar; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

2017-01-01

In 2012 the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the HA and NA proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event. PMID:25055345

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

PubMed

Juozapaitis, Mindaugas; Aguiar Moreira, Étori; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

2014-07-23

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

Molecular Mechanism of Arenavirus Assembly and Budding

PubMed Central

Urata, Shuzo; Yasuda, Jiro

2012-01-01

Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2. PMID:23202453

Discovery of Herpes B Virus-Encoded MicroRNAs▿

PubMed Central

Besecker, Michael I.; Harden, Mallory E.; Li, Guanglin; Wang, Xiu-Jie; Griffiths, Anthony

2009-01-01

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with ∼80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs. PMID:19144716

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

PubMed

Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

2017-09-15

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses. Copyright © 2017 American Society for Microbiology.

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.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

PubMed

Gan, Jin; Qiao, Niu; Strahan, Roxanne; Zhu, Caixia; Liu, Lei; Verma, Subhash C; Wei, Fang; Cai, Qiliang

2016-11-01

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

PubMed Central

Iwasaki, Masaharu; Caì, Yíngyún; de la Torre, Juan C.

2018-01-01

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. PMID:29462184

Biology of Epstein-Barr virus during infectious mononucleosis.

PubMed

Sitki-Green, Diane L; Edwards, Rachel Hood; Covington, Mary M; Raab-Traub, Nancy

2004-02-01

Infectious mononucleosis is the clinical manifestation of primary infection with Epstein-Barr virus (EBV). We monitored primary infection during convalescence and during the establishment of persistent infection. The profiles of EBV strains in the oral cavity and in peripheral blood were determined by use of a heteroduplex tracking assay specific for the EBV gene encoding latent membrane protein 1. Multiple EBV strains were detected in most patients and persisted in and were possibly transmitted among 3 distinct compartments of infection, including the oral cavity, peripheral blood lymphocytes, and the cell-free fraction of the blood plasma. We also tracked transmission of multiple strains from an asymptomatic carrier to a patient diagnosed with primary EBV infection. These data reveal that primary EBV infection is complex, with transmission of multiple strains and clear differences in relative abundance of strains in distinct compartments.

Virus-encoded miRNAs in Ebola virus disease.

PubMed

Duy, Janice; Honko, Anna N; Altamura, Louis A; Bixler, Sandra L; Wollen-Roberts, Suzanne; Wauquier, Nadia; O'Hearn, Aileen; Mucker, Eric M; Johnson, Joshua C; Shamblin, Joshua D; Zelko, Justine; Botto, Miriam A; Bangura, James; Coomber, Moinya; Pitt, M Louise; Gonzalez, Jean-Paul; Schoepp, Randal J; Goff, Arthur J; Minogue, Timothy D

2018-04-24

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Creation of a Recombinant Rift Valley Fever Virus with a Two-Segmented Genome ▿ †

PubMed Central

Brennan, Benjamin; Welch, Stephen R.; McLees, Angela; Elliott, Richard M.

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed. PMID:21795328

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein*

PubMed Central

Yang, Yin; Wu, Songfang; Wang, Yu; Pan, Shuang; Lan, Bei; Liu, Yaohui; Zhang, Liming; Leng, Qianli; Chen, Da; Zhang, Cuizhu; He, Bin; Cao, Youjia

2015-01-01

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance. PMID:25907557

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

PubMed Central

Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

1998-01-01

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

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.

Modified vaccinia virus Ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques.

PubMed

Florek, Nicholas W; Weinfurter, Jason T; Jegaskanda, Sinthujan; Brewoo, Joseph N; Powell, Tim D; Young, Ginger R; Das, Subash C; Hatta, Masato; Broman, Karl W; Hungnes, Olav; Dudman, Susanne G; Kawaoka, Yoshihiro; Kent, Stephen J; Stinchcomb, Dan T; Osorio, Jorge E; Friedrich, Thomas C

2014-11-01

Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4(+) and CD8(+) T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Modified Vaccinia Virus Ankara Encoding Influenza Virus Hemagglutinin Induces Heterosubtypic Immunity in Macaques

PubMed Central

Florek, Nicholas W.; Weinfurter, Jason T.; Jegaskanda, Sinthujan; Brewoo, Joseph N.; Powell, Tim D.; Young, Ginger R.; Das, Subash C.; Hatta, Masato; Broman, Karl W.; Hungnes, Olav; Dudman, Susanne G.; Kawaoka, Yoshihiro; Kent, Stephen J.; Stinchcomb, Dan T.

2014-01-01

ABSTRACT Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. IMPORTANCE Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus. PMID:25210172

Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

PubMed Central

Geisbert, Joan B; Shedlock, Devon J; Xu, Ling; Lamoreaux, Laurie; Custers, Jerome H. H. V; Popernack, Paul M; Yang, Zhi-Yong; Pau, Maria G; Roederer, Mario; Koup, Richard A; Goudsmit, Jaap; Jahrling, Peter B; Nabel, Gary J

2006-01-01

Background Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. Methods and Findings To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously. Conclusions Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate. PMID:16683867

Role of Viral miRNAs and Epigenetic Modifications in Epstein-Barr Virus-Associated Gastric Carcinogenesis.

PubMed

Giudice, Aldo; D'Arena, Giovanni; Crispo, Anna; Tecce, Mario Felice; Nocerino, Flavia; Grimaldi, Maria; Rotondo, Emanuela; D'Ursi, Anna Maria; Scrima, Mario; Galdiero, Massimiliano; Ciliberto, Gennaro; Capunzo, Mario; Franci, Gianluigi; Barbieri, Antonio; Bimonte, Sabrina; Montella, Maurizio

2016-01-01

MicroRNAs are short (21-23 nucleotides), noncoding RNAs that typically silence posttranscriptional gene expression through interaction with target messenger RNAs. Currently, miRNAs have been identified in almost all studied multicellular eukaryotes in the plant and animal kingdoms. Additionally, recent studies reported that miRNAs can also be encoded by certain single-cell eukaryotes and by viruses. The vast majority of viral miRNAs are encoded by the herpesviruses family. These DNA viruses including Epstein-Barr virus encode their own miRNAs and/or manipulate the expression of cellular miRNAs to facilitate respective infection cycles. Modulation of the control pathways of miRNAs expression is often involved in the promotion of tumorigenesis through a specific cascade of transduction signals. Notably, latent infection with Epstein-Barr virus is considered liable of causing several types of malignancies, including the majority of gastric carcinoma cases detected worldwide. In this review, we describe the role of the Epstein-Barr virus in gastric carcinogenesis, summarizing the functions of the Epstein-Barr virus-encoded viral proteins and related epigenetic alterations as well as the roles of Epstein-Barr virus-encoded and virally modulated cellular miRNAs.

Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

NASA Astrophysics Data System (ADS)

Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

1988-02-01

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

Giant viruses: The difficult breaking of multiple epistemological barriers.

PubMed

Claverie, Jean-Michel; Abergel, Chantal

2016-10-01

The discovery of the first "giant virus", Mimivirus, in 2003 could solely have been that of an exceptional freak, a blind alley of evolution as occasionally encountered in biology, albeit without conceptual significance. On the contrary, once broken this epistemological barrier, additional unrelated families of giant viruses such as the Pandoraviruses, the Pithoviruses and most recently Mollivirus, were quickly unraveled, suggesting that an entire chapter of microbiology had been ignored since Pasteur and Ivanovski. In this article, we examine to what extent the giant viruses challenge previous definitions of viruses, the diversity of forms they could take, and how they might have evolved from extinct ancestral cellular lineages. Inspired by the epistemology of Gaston Bachelard, we will also suggest the reasons for which giant viruses laid hidden in plain sight for more than a century. Finally, we propose a new definition for "viruses" that paradoxically emphasize the fact that they do not encode a single universally shared macromolecule or biochemical function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion

PubMed Central

French, Roy

2016-01-01

ABSTRACT Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases. PMID:27681136

Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences

PubMed Central

Brander, Christian; Yang, Otto O.; Jones, Norman G.; Lee, Yun; Goulder, Philip; Johnson, R. Paul; Trocha, Alicja; Colbert, David; Hay, Christine; Buchbinder, Susan; Bergmann, Cornelia C.; Zweerink, Hans J.; Wolinsky, Steven; Blattner, William A.; Kalams, Spyros A.; Walker, Bruce D.

1999-01-01

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences. PMID:10559335

Development of replication-competent viral vectors for HIV vaccine delivery

PubMed Central

Parks, Christopher L.; Picker, Louis J.; King, C. Richter

2014-01-01

Purpose of review Briefly describe some of the replication-competent (RC) vectors being investigated for development of candidate HIV vaccines focusing primarily on technologies that have advanced to testing in macaques or have entered clinical trials. Recent findings RC viral vectors have advanced to the stage were decisions can be made regarding future development of HIV vaccines. The viruses being used as RC vector platforms vary considerably, and their unique attributes make it possible to test multiple vaccine design concepts and also mimic various aspects of an HIV infection. RC viral vectors encoding SIV or HIV proteins can be used to safely immunize macaques, and in some cases, there is evidence of significant vaccine efficacy in challenge protection studies. Several live HIV vaccine vectors are in clinical trials to evaluate immunogenicity, safety, the effect of mucosal delivery, and potential effects of pre-existing immunity. Summary A variety of DNA and RNA viruses are being used to develop RC viral vectors for HIV vaccine delivery. Multiple viral vector platforms have proven to be safe and immunogenic with evidence of efficacy in macaques. Some of the more advanced HIV vaccine prototypes based on vesicular stomatitis virus, vaccinia virus, measles virus, and Sendai virus are in clinical trials. PMID:23925000

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus.

PubMed

Lai, Qingying; Wu, Wenbi; Li, Ao; Wang, Wei; Yuan, Meijin; Yang, Kai

2018-05-01

Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K ( ac98 ) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene ( ac100 ) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. Copyright © 2018 Lai et al.

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus

PubMed Central

Lai, Qingying; Li, Ao; Wang, Wei; Yuan, Meijin

2018-01-01

ABSTRACT Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. PMID:29444944

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms.

PubMed

Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Hankaniemi, Minna M; Larsson, Pär G; Domsgen, Erna; Stone, Virginia M; Määttä, Juha A E; Hyöty, Heikki; Hytönen, Vesa P; Flodström-Tullberg, Malin

2018-05-01

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A pro and 3C pro ), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A pro cause direct damage to the infected heart and tools to investigate 2A pro and 3C pro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A pro and 3C pro ; Two monoclonal 2A pro antibodies and one 3C pro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C pro antibody detects all of the EV species B (EV-B) viruses tested and that the 2A pro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A pro and 3C pro , and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice.

PubMed

Sakala, Isaac G; Chaudhri, Geeta; Scalzo, Anthony A; Eldi, Preethi; Newsome, Timothy P; Buller, Robert M; Karupiah, Gunasegaran

2015-12-01

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.

Virus-encoded chemokine receptors--putative novel antiviral drug targets.

PubMed

Rosenkilde, Mette M

2005-01-01

Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have as such a paramount role in the antiviral immune responses. It is therefore not surprising that viruses have found ways to exploit and subvert the chemokine system by means of molecular mimicry. By ancient acts of molecular piracy and by induction and suppression of endogenous genes, viruses have utilized chemokines and their receptors to serve a variety of roles in viral life-cycle. This review focuses on the pharmacology of virus-encoded chemokine receptors, yet also the family of virus-encoded chemokines and chemokine-binding proteins will be touched upon. Key properties of the virus-encoded receptors, compared to their closest endogenous homologs, are interactions with a wider range of chemokines, which can act as agonists, antagonists and inverse agonists, and the exploitation of many signal transduction pathways. High constitutive activity is another key property of some--but not all--of these receptors. The chemokine receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies--will be highlighted here together with the potentials of the virus-encoded chemokines and chemokine-binding proteins as novel anti-inflammatory biopharmaceutical strategies.

Multiple invasions of an infectious retrovirus in cat genomes

PubMed Central

Shimode, Sayumi; Nakagawa, So; Miyazawa, Takayuki

2015-01-01

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times. PMID:25641657

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

PubMed

Valles, Steven M; Bell, Susanne; Firth, Andrew E

2014-01-01

Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

PubMed Central

Meseda, Clement A.; Campbell, Joseph; Kumar, Arunima; Garcia, Alonzo D.; Merchlinsky, Michael; Weir, Jerry P.

2013-01-01

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model. PMID:23785523

RNA viruses and microRNAs: challenging discoveries for the 21st century

PubMed Central

Swaminathan, Gokul; Martin-Garcia, Julio

2013-01-01

RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay. PMID:24046280

Epstein-Barr virus (EBV)-encoded dUTPase and chronic restraint induce impaired learning and memory and sickness responses.

PubMed

Aubrecht, Taryn G; Weil, Zachary M; Ariza, Maria Eugenia; Williams, Marshall; Reader, Brenda F; Glaser, Ronald; Sheridan, John F; Nelson, Randy J

2014-10-01

Most adult humans have been infected with Epstein-Barr virus (EBV) and carry the latent virus. The EBV genome codes for several proteins that form an early antigen complex important for viral replication; one of these proteins is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The EBV-encoded dUTPase can induce sickness responses in mice. Because stress can increase latent virus reactivation, we hypothesized that chronic restraint would exacerbate sickness behaviors elicited by EBV-encoded dUTPase. Male Swiss-Webster mice were injected daily for 15 days with either saline or EBV-encoded dUTPase. Additionally, half of the mice from each condition were either restrained for 3h daily or left undisturbed. Restraint stress impaired learning and memory in the passive avoidance chamber; impaired learning and memory was due to EBV-encoded dUTPase injected into restrained mice. EBV-encoded dUTPase induced sickness responses and restraint stress interacts with EBV-encoded dUTPase to exacerbate the sickness response. These data support a role for EBV-encoded dUTPase and restraint stress in altering the pathophysiology of EBV independent of viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Identification of a novel circular DNA virus in pig feces

USDA-ARS?s Scientific Manuscript database

Metagenomic analysis of fecal samples collected from a swine with diarrhea detected sequences encoding a replicase (Rep) protein typically found in small circular Rep-encoding ssDNA (CRESS-DNA) viruses. The complete 3,062 nucleotide genome was generated and found to encode two bi-directionally trans...

Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

NASA Astrophysics Data System (ADS)

Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

2004-11-01

Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

PubMed Central

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine. PMID:23284733

Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

PubMed

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease.

PubMed

Richner, Justin M; Jagger, Brett W; Shan, Chao; Fontes, Camila R; Dowd, Kimberly A; Cao, Bin; Himansu, Sunny; Caine, Elizabeth A; Nunes, Bruno T D; Medeiros, Daniele B A; Muruato, Antonio E; Foreman, Bryant M; Luo, Huanle; Wang, Tian; Barrett, Alan D; Weaver, Scott C; Vasconcelos, Pedro F C; Rossi, Shannan L; Ciaramella, Giuseppe; Mysorekar, Indira U; Pierson, Theodore C; Shi, Pei-Yong; Diamond, Michael S

2017-07-13

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted. Copyright © 2017 Elsevier Inc. All rights reserved.

Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging.

PubMed

Logan, Grace; Newman, Joseph; Wright, Caroline F; Lasecka-Dykes, Lidia; Haydon, Daniel T; Cottam, Eleanor M; Tuthill, Tobias J

2017-10-18

Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad spectrum activity. Copyright © 2017 Logan et al.

The Fecal Virome of Pigs on a High-Density Farm ▿ †

PubMed Central

Shan, Tongling; Li, Linlin; Simmonds, Peter; Wang, Chunlin; Moeser, Adam; Delwart, Eric

2011-01-01

Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials. PMID:21900163

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR

PubMed Central

Nishiyama, Shoko; Slack, Olga A. L.; Lokugamage, Nandadeva; Hill, Terence E.; Juelich, Terry L.; Zhang, Lihong; Smith, Jennifer K.; Perez, David; Gong, Bin; Freiberg, Alexander N.; Ikegami, Tetsuro

2016-01-01

ABSTRACT Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker. PMID:27248570

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

PubMed

Nishiyama, Shoko; Slack, Olga A L; Lokugamage, Nandadeva; Hill, Terence E; Juelich, Terry L; Zhang, Lihong; Smith, Jennifer K; Perez, David; Gong, Bin; Freiberg, Alexander N; Ikegami, Tetsuro

2016-11-16

Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila.

PubMed

Lamiable, Olivier; Kellenberger, Christine; Kemp, Cordula; Troxler, Laurent; Pelte, Nadège; Boutros, Michael; Marques, Joao Trindade; Daeffler, Laurent; Hoffmann, Jules A; Roussel, Alain; Imler, Jean-Luc

2016-01-19

Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.

The great billion-year war between ribosome- and capsid-encoding organisms (cells and viruses) as the major source of evolutionary novelties.

PubMed

Forterre, Patrick; Prangishvili, David

2009-10-01

Our conceptions on the origin, nature, and role of viruses have been shaken recently by several independent lines of research. There are many reasons to believe now that viruses are more ancient than modern cells and have always been more abundant and diverse than their cellular targets. Viruses can be defined as capsid-encoding organisms that transform their "host" cell into a viral factory. If capsid-encoding organisms (viruses) and ribosome-encoding organisms (cells) are the major types of living entities on our planet, it seems logical to conclude that their conflict has been a major engine of biological evolution (in the framework of natural selection). In particular, many novelties first selected in the viral world might have been transferred to cells as a consequence of the continuous flow of viral genes into cellular genomes. We discuss recent observations and hypotheses suggesting that viruses have played a major role at different stages of biological evolution, such as the RNA to DNA transition, the origin of the eukaryotic nucleus, or, alternatively, the origin of unique features in multicellular macrobes.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

Survey of Navy Funded Marine Mammal Research and Studies FY 00-01

DTIC Science & Technology

2001-05-10

protein of canine distemper virus as a reporter system in order to evaluate 103 the humoral response to DNA-mediated vaccination in cetaceans. If...PCR/ RT PCR, DNA cloning and sequencing, etc. Efforts are ongoing to design and clone a vector encoding Canine Distemper Virus, a virus closely...alternative plasmid as our reporter gene delivery vector. This alternate plasmid will encode for Canine Distemper virus genes, closely related to

Bcl-2 Blocks a Caspase-Dependent Pathway of Apoptosis Activated by Herpes Simplex Virus 1 Infection in HEp-2 Cells

PubMed Central

Galvan, Veronica; Brandimarti, Renato; Munger, Joshua; Roizman, Bernard

2000-01-01

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate. PMID:10644366

Identification and in vitro characterization of a Marek’s disease virus encoded ribonucleotide reductase

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acid...

Genetic and Functional Diversification of Small RNA Pathways in Plants

PubMed Central

Gustafson, Adam M; Kasschau, Kristin D; Lellis, Andrew D; Zilberman, Daniel; Jacobsen, Steven E

2004-01-01

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense. PMID:15024409

Morphology and genome organization of the virus PSV of the hyperthermophilic archaeal genera Pyrobaculum and Thermoproteus: a novel virus family, the Globuloviridae.

PubMed

Häring, Monika; Peng, Xu; Brügger, Kim; Rachel, Reinhard; Stetter, Karl O; Garrett, Roger A; Prangishvili, David

2004-06-01

A novel virus, termed Pyrobaculum spherical virus (PSV), is described that infects anaerobic hyperthermophilic archaea of the genera Pyrobaculum and Thermoproteus. Spherical enveloped virions, about 100 nm in diameter, contain a major multimeric 33-kDa protein and host-derived lipids. A viral envelope encases a superhelical nucleoprotein core containing linear double-stranded DNA. The PSV infection cycle does not cause lysis of host cells. The viral genome was sequenced and contains 28337 bp. The genome is unique for known archaeal viruses in that none of the genes, including that encoding the major structural protein, show any significant sequence matches to genes in public sequence databases. Exceptionally for an archaeal double-stranded DNA virus, almost all the recognizable genes are located on one DNA strand. The ends of the genome consist of 190-bp inverted repeats that contain multiple copies of short direct repeats. The two DNA strands are probably covalently linked at their termini. On the basis of the unusual morphological and genomic properties of this DNA virus, we propose to assign PSV to a new viral family, the Globuloviridae.

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.

Production of pseudoinfectious yellow fever virus with a two-component genome.

PubMed

Shustov, Alexandr V; Mason, Peter W; Frolov, Ilya

2007-11-01

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

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 ...

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

PubMed

Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

2014-10-01

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs. © FASEB.

Epstein-Barr Virus Hijacks DNA Damage Response Transducers to Orchestrate Its Life Cycle.

PubMed

Hau, Pok Man; Tsao, Sai Wah

2017-11-16

The Epstein-Barr virus (EBV) is a ubiquitous virus that infects most of the human population. EBV infection is associated with multiple human cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, a subset of gastric carcinomas, and almost all undifferentiated non-keratinizing nasopharyngeal carcinoma. Intensive research has shown that EBV triggers a DNA damage response (DDR) during primary infection and lytic reactivation. The EBV-encoded viral proteins have been implicated in deregulating the DDR signaling pathways. The consequences of DDR inactivation lead to genomic instability and promote cellular transformation. This review summarizes the current understanding of the relationship between EBV infection and the DDR transducers, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), and discusses how EBV manipulates the DDR signaling pathways to complete the replication process of viral DNA during lytic reactivation.

An Adult Case of Chronic Active Epstein-Barr Virus Infection with Interstitial Pneumonitis

PubMed Central

Joo, Eun-Jeong; Ha, Young Eun; Jung, Dong Sik; Cheong, Hae Suk; Wi, Yu Mi; Song, Jae-Hoon

2011-01-01

Chronic active Epstein-Barr virus (CAEBV) infection is characterized by persistent infectious mononucleosis-like symptoms, an unusual pattern of Epstein-Barr virus (EBV) antibodies, detection of the EBV genome in affected tissues or peripheral blood, and chronic illness that cannot be attributed to any other known disease. This is the first reported Korean case of an immunocompetent adult with CAEBV-associated interstitial pneumonitis. A 28-year-old female was admitted with a fever that persisted for 3 weeks. She had multiple lymphadenopathy, hepatosplenomegaly, pancytopenia, and elevated serum aminotransferase levels. Serology for antibodies was positive and chest computed tomography showed diffuse ground glass opacities in both lungs. Histopathology of the lung tissue showed lymphocyte infiltration, and EBV DNA was detected in those lymphocytes using in situ hybridization with an EBV-encoded RNA probe. After 1 month of hospitalization, she improved without specific treatment. PMID:22205850

An adult case of chronic active Epstein-Barr virus infection with interstitial pneumonitis.

PubMed

Joo, Eun-Jeong; Ha, Young Eun; Jung, Dong Sik; Cheong, Hae Suk; Wi, Yu Mi; Song, Jae-Hoon; Peck, Kyong Ran

2011-12-01

Chronic active Epstein-Barr virus (CAEBV) infection is characterized by persistent infectious mononucleosis-like symptoms, an unusual pattern of Epstein-Barr virus (EBV) antibodies, detection of the EBV genome in affected tissues or peripheral blood, and chronic illness that cannot be attributed to any other known disease. This is the first reported Korean case of an immunocompetent adult with CAEBV-associated interstitial pneumonitis. A 28-year-old female was admitted with a fever that persisted for 3 weeks. She had multiple lymphadenopathy, hepatosplenomegaly, pancytopenia, and elevated serum aminotransferase levels. Serology for antibodies was positive and chest computed tomography showed diffuse ground glass opacities in both lungs. Histopathology of the lung tissue showed lymphocyte infiltration, and EBV DNA was detected in those lymphocytes using in situ hybridization with an EBV-encoded RNA probe. After 1 month of hospitalization, she improved without specific treatment.

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

PubMed Central

Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

2007-01-01

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

Identification of Western Equine Encephalitis Virus Structural Proteins That Confer Protection after DNA Vaccination▿

PubMed Central

Gauci, Penelope J.; Wu, Josh Q. H.; Rayner, George A.; Barabé, Nicole D.; Nagata, Leslie P.; Proll, David F.

2010-01-01

DNA vaccines encoding different portions of the structural proteins of western equine encephalitis virus were tested for the efficacy of their protection in a 100% lethal mouse model of the virus. The 6K-E1 structural protein encoded by the DNA vaccine conferred complete protection against challenge with the homologous strain and limited protection against challenge with a heterologous strain. PMID:19923571

T7 phage displaying latent membrane protein 1 of Epstein-Barr virus elicits humoral and cellular immune responses in rats.

PubMed

Gao, J; Liu, Z; Huang, M; Li, X; Wang, Z

2011-01-01

The latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) has become a potential target in EBV-associated tumor prevention and treatment due to its multiple biological effects. In this study, the recombinant T7 phage displaying full-length LMP1 protein was cloned and used as an immunogen to immunize rats. Results of flow cytometry, Western blot analysis, and ELISA confirmed that both humoral and cellular immune responses were elicited in the immunized rats. Our data suggested that T7 phage was an efficient antigen carrier. The recombinant T7-LMP1 phage reconstitutes the antigenic and immunogenic properties of LMP1 and can serve as a vaccine against EBV.

Resistance of virus to extinction on bottleneck passages: study of a decaying and fluctuating pattern of fitness loss

NASA Technical Reports Server (NTRS)

Lazaro, Ester; Escarmis, Cristina; Perez-Mercader, Juan; Manrubia, Susanna C.; Domingo, Esteban

2003-01-01

RNA viruses display high mutation rates and their populations replicate as dynamic and complex mutant distributions, termed viral quasispecies. Repeated genetic bottlenecks, which experimentally are carried out through serial plaque-to-plaque transfers of the virus, lead to fitness decrease (measured here as diminished capacity to produce infectious progeny). Here we report an analysis of fitness evolution of several low fitness foot-and-mouth disease virus clones subjected to 50 plaque-to-plaque transfers. Unexpectedly, fitness decrease, rather than being continuous and monotonic, displayed a fluctuating pattern, which was influenced by both the virus and the state of the host cell as shown by effects of recent cell passage history. The amplitude of the fluctuations increased as fitness decreased, resulting in a remarkable resistance of virus to extinction. Whereas the frequency distribution of fitness in control (independent) experiments follows a log-normal distribution, the probability of fitness values in the evolving bottlenecked populations fitted a Weibull distribution. We suggest that multiple functions of viral genomic RNA and its encoded proteins, subjected to high mutational pressure, interact with cellular components to produce this nontrivial, fluctuating pattern.

Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs.

PubMed

Akmal, Mohd; Baig, Mirza S; Khan, Jawaid A

2017-12-10

Cotton leaf curl disease (CLCuD), a major factor resulting in the enormous yield losses in cotton crop, is caused by a distinct monopartite begomovirus in association with Cotton leaf curl Multan betasatellite (CLCuMB). Micro(mi)RNAs are known to regulate gene expression in eukaryotes, including antiviral defense in plants. In a previous study, we had computationally identified a set of cotton miRNAs, which were shown to have potential targets in the genomes of Cotton leaf curl Multan virus (CLCuMuV) and CLCuMB at multiple loci. In the current study, effect of Gossypium arboreum-encoded miRNAs on the genome of CLCuMuV and CLCuMB was investigated in planta. Two computationally predicted cotton-encoded miRNAs (miR398 and miR2950) that showed potential to bind multiple Open Reading Frames (ORFs; C1, C4, V1, and non- coding intergenic region) of CLCuMuV, and (βC1) of CLCuMB were selected. Functional validation of miR398 and miR2950 was done by overexpression approach in G. hirsutum var. HS6. A total of ten in vitro cotton plants were generated from independent events and subjected to biological and molecular analyses. Presence of the respective Precursor (pre)-miRNA was confirmed through PCR and Southern blotting, and their expression level was assessed by semi quantitative RT-PCR, Real Time quantitative PCR and northern hybridization in the PCR-positive lines. Southern hybridization revealed 2-4 copy integration of T-DNA in the genome of the transformed lines. Remarkably, expression of pre-miRNAs was shown up to 5.8-fold higher in the transgenic (T 0 ) lines as revealed by Real Time PCR. The virus resistance was monitored following inoculation of the transgenic cotton lines with viruliferous whitefly (Bemisia tabaci) insect vector. After inoculation, four of the transgenic lines remained apparently symptom free. While a very low titre of viral DNA could be detected by Rolling circle amplification, betasatellite responsible for symptom induction could not be detected in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization.

PubMed Central

Haseloff, J; Goelet, P; Zimmern, D; Ahlquist, P; Dasgupta, R; Kaesberg, P

1984-01-01

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution. PMID:6611550

Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

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

Eisenmesser, Elan Z.; Capodagli, Glenn; Armstrong, Geoffrey S.

Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove multiple proteins involved in cellular signaling such as ubiquitin (Ub) and interferon stimulated gene produce 15 (ISG15). Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the dynamicmore » plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHV vOTU, both alone and in complex with Ub, thereby discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.« less

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

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

Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi

2007-01-20

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV genemore » products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.« less

The Retrovirus pol Gene Encodes a Product Required for DNA Integration: Identification of a Retrovirus int Locus

NASA Astrophysics Data System (ADS)

Panganiban, Antonito T.; Temin, Howard M.

1984-12-01

We mutagenized cloned spleen necrosis virus DNA to identify a region of the retrovirus genome encoding a polypeptide required for integration of viral DNA. Five plasmids bearing different lesions in the 3' end of the pol gene were examined for the ability to integrate or replicate following transfection of chicken embryo fibroblasts. Transfection with one of these DNAs resulted in the generation of mutant virus incapable of integrating but able to replicate at low levels; this phenotype is identical to that of mutants bearing alterations in the cis-acting region, att. To determine whether the 3' end of the pol gene encodes a protein that interacts with att, we did a complementation experiment. Cells were first infected with an att- virus and then superinfected with the integration-deficient virus containing a lesion in the pol gene and a wild-type att site. The results showed that the att- virus provided a trans-acting function allowing integration of viral DNA derived from the mutant bearing a wild-type att site. Thus, the 3' end of the pol gene serves as an ``int'' locus and encodes a protein mediating integration of retrovirus DNA through interaction with att.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

PubMed

Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

2010-05-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

PubMed

Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

2016-05-11

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

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

A novel Sulfolobus virus with an exceptional capsid architecture.

PubMed

Wang, Haina; Guo, Zhenqian; Feng, Hongli; Chen, Yufei; Chen, Xiuqiang; Li, Zhimeng; Hernández-Ascencio, Walter; Dai, Xin; Zhang, Zhenfeng; Zheng, Xiaowei; Mora-López, Marielos; Fu, Yu; Zhang, Chuanlun; Zhu, Ping; Huang, Li

2017-12-06

A novel archaeal virus, denoted Sulfolobus ellipsoid virus 1 (SEV1), was isolated from an acidic hot spring in Costa Rica. The morphologically unique virion of SEV1 contains a protein capsid with 16 regularly spaced striations and an 11-nm-thick envelope. The capsid exhibits an unusual architecture in which the viral DNA, probably in the form of a nucleoprotein filament, wraps around the longitudinal axis of the virion in a plane to form a multilayered disk-like structure with a central hole, and 16 of these structures are stacked to generate a spool-like capsid. SEV1 harbors a linear double-stranded DNA genome of ∼23 kb, which encodes 38 predicted open reading frames (ORFs). Among the few ORFs with a putative function is a gene encoding a protein-primed DNA polymerase. Six-fold symmetrical virus-associated pyramids (VAPs) appear on the surface of the SEV1-infected cells, which are ruptured to allow the formation of a hexagonal opening and subsequent release of the progeny virus particles. Notably, the SEV1 virions acquire the lipid membrane in the cytoplasm of the host cell. The lipid composition of the viral envelope correlates with that of the cell membrane. These results suggest the use of a unique mechanism by SEV1 in membrane biogenesis. IMPORTANCE Investigation of archaeal viruses has greatly expanded our knowledge of the virosphere and its role in the evolution of life. Here we show that Sulfolobus ellipsoid virus 1 (SEV1), an archaeal virus isolated from a hot spring in Costa Rica, exhibits a novel viral shape and an unusual capsid architecture. The SEV1 DNA wraps multiple times in a plane around the longitudinal axis of the virion to form a disk-like structure, and 16 of these structures are stacked to generate a spool-like capsid. The virus acquires its envelope intracellularly and exits the host cell by creating a hexagonal hole on the host cell surface. These results shed significant light on the diversity of viral morphogenesis. Copyright © 2017 American Society for Microbiology.

Mouse superkiller‐2‐like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses

PubMed Central

Goubau, Delphine; van der Veen, Annemarthe G.; Chakravarty, Probir; Lin, Rongtuan; Rogers, Neil; Rehwinkel, Jan; Deddouche, Safia; Rosewell, Ian; Hiscott, John

2015-01-01

Abstract IFN‐α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2‐like DExH‐box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN‐α/β by retinoic acid inducible gene 1‐like receptors (RLRs) that detect the presence of RNA viruses in a cell‐intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN‐α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60‐deficient mice revealed no impairment in IFN‐α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60‐deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus‐1. These results put in question the reported role of DDX60 as a broad‐acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses. PMID:26457795

Role of Soybean mosaic virus-encoded proteins in seed and aphid transmission in soybean

USDA-ARS?s Scientific Manuscript database

Soybean mosaic virus (SMV) is seed and aphid transmitted and can cause significant reductions in yield and seed quality in soybean, Glycine max. The roles in seed and aphid transmission of selected SMV-encoded proteins were investigated by constructing chimeric recombinants between SMV 413 (efficien...

Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.

PubMed

Okamoto, Toru; Campbell, Stephanie; Mehta, Ninad; Thibault, John; Colman, Peter M; Barry, Michele; Huang, David C S; Kvansakul, Marc

2012-11-01

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

PubMed

Ribaudo, Michael; Barik, Sailen

2017-11-06

Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

PubMed

Balasuriya, U B R; Nadler, S A; Wilson, W C; Pritchard, L I; Smythe, A B; Savini, G; Monaco, F; De Santis, P; Zhang, N; Tabachnick, W J; Maclachlan, N J

2008-01-01

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

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

Carte, Jason; Wang, Ruiying; Li, Hong

An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targetingmore » RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.« less

Broad cross-reactive T cell receptor repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

PubMed Central

Clute, Shalyn C.; Naumov, Yuri N.; Watkin, Levi B.; Aslan, Nuray; Sullivan, John L.; Thorley-Lawson, David A.; Luzuriaga, Katherine; Welsh, Raymond M.; Puzone, Roberto; Celada, Franco; Selin, Liisa K.

2013-01-01

Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show here, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M158-66 epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1280-288 epitope (GLCTLVAML), that under some conditions heterologous immunity can lead to a significant broadening rather than a narrowing of the T cell receptor repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad rather than narrow T cell repertoire if there is a lack of dominant high affinity clones, and this hypothesis is supported by computer simulation. PMID:21048112

Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses

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

Chatterjee, Srirupa; Basler, Christopher F.; Amarasinghe, Gaya K.

The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most openmore » reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures.« less

Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

PubMed Central

Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

2010-01-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

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

Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana

2007-03-30

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Ourmore » study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.« less

Spontaneous Regression of Pulmonary Nodules Presenting as Epstein-Barr Virus-related Atypical Infectious Mononucleosis.

PubMed

Shinozuka, Jun; Awaguni, Hitoshi; Tanaka, Shin-Ichiro; Makino, Shigeru; Maruyama, Rikken; Inaba, Tohru; Imashuku, Shinsaku

2016-07-01

Pulmonary nodules associated with Epstein-Barr virus (EBV)-related atypical infectious mononucleosis have rarely been described. A 12-year-old Japanese boy, upon admission, revealed multiple small round nodules (a total of 7 nodules in 4 to 8 mm size) in the lungs on computed tomography. The hemorrhagic pharyngeal tonsils with hot signals on 18F-fluorodeoxyglucose-positron emission tomography-computed tomography were biopsied revealing the presence of EBV-encoded small nuclear RNA (EBER)-positive cells; however, no lymphoma was noted. The patient was diagnosed as having atypical EBV-infectious mononucleosis associated with primary EBV infection. Pulmonary nodules markedly reduced in numbers and sizes spontaneously over a 2-year period. Differential diagnosis of pulmonary nodules in childhood should include atypical EBV infection.

Generation of Recombinant Ebola Viruses Using Reverse Genetics.

PubMed

Groseth, Allison

2017-01-01

Reverse genetics systems encompass a wide array of tools aimed at recapitulating some or all of the virus life cycle. In their most complete form, full-length clone systems allow us to use plasmid-encoded versions of the ribonucleoprotein (RNP) components to initiate the transcription and replication of a plasmid-encoded version of the complete viral genome, thereby initiating the complete virus life cycle and resulting in infectious virus. As such this approach is ideal for the generation of tailor-made recombinant filoviruses, which can be used to study virus biology. In addition, the generation of tagged and particularly fluorescent or luminescent viruses can be applied as tools for both diagnostic applications and for screening to identify novel countermeasures. Here we describe the generation and basic characterization of recombinant Ebola viruses rescued from cloned cDNA using a T7-driven system.

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

NASA Astrophysics Data System (ADS)

Mousnier, Aurélie; Bell, Andrew S.; Swieboda, Dawid P.; Morales-Sanfrutos, Julia; Pérez-Dorado, Inmaculada; Brannigan, James A.; Newman, Joseph; Ritzefeld, Markus; Hutton, Jennie A.; Guedán, Anabel; Asfor, Amin S.; Robinson, Sean W.; Hopkins-Navratilova, Iva; Wilkinson, Anthony J.; Johnston, Sebastian L.; Leatherbarrow, Robin J.; Tuthill, Tobias J.; Solari, Roberto; Tate, Edward W.

2018-06-01

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

PubMed

Han, Yingying; Xia, Hengchuan; Tang, Qi; Lü, Peng; Ma, Shangshang; Yang, Yanhua; Shao, Dandan; Ma, Quanbing; Chen, Keping

2014-07-01

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV), P26 and P9, are self-interacting.

PubMed

Stewart, Lucy R; Hwang, Min Sook; Falk, Bryce W

2009-11-01

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26-P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9-P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.

Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses.

PubMed

Goubau, Delphine; van der Veen, Annemarthe G; Chakravarty, Probir; Lin, Rongtuan; Rogers, Neil; Rehwinkel, Jan; Deddouche, Safia; Rosewell, Ian; Hiscott, John; Reis E Sousa, Caetano

2015-12-01

IFN-α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/β by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2014-01-01

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

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

PubMed Central

Ryerson, Melissa R.; Richards, Monique M.; Hawkins, Christine J.

2017-01-01

ABSTRACT Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1–procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions. IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules. PMID:28904196

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil.

PubMed

Fontenele, Rafaela S; Abreu, Rayane A; Lamas, Natalia S; Alves-Freitas, Dione M T; Vidal, Andreza H; Poppiel, Raul R; Melo, Fernando L; Lacorte, Cristiano; Martin, Darren P; Campos, Magnolia A; Varsani, Arvind; Ribeiro, Simone G

2018-04-02

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae : passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus . Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12-30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891-921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7-3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina , Arabidopsis thaliana , and Passiflora edulis .

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil

PubMed Central

Fontenele, Rafaela S.; Abreu, Rayane A.; Lamas, Natalia S.; Alves-Freitas, Dione M. T.; Vidal, Andreza H.; Melo, Fernando L.; Lacorte, Cristiano; Martin, Darren P.; Campos, Magnolia A.; Ribeiro, Simone G.

2018-01-01

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12–30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891–921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7–3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis. PMID:29614801

NF-κB deregulation in Hodgkin lymphoma.

PubMed

Weniger, Marc A; Küppers, Ralf

2016-08-01

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) show constitutive activity of both the canonical and non-canonical NF-κB signaling pathways. The central pathogenetic role of this activity is indicated from studies with HL cell lines, which undergo apoptosis upon NF-κB inhibition. Multiple factors contribute to the strong NF-κB activity of HRS cells. This includes interaction with other cells in the lymphoma microenvironment through CD30, CD40, BCMA and other receptors, but also recurrent somatic genetic lesions in various factors of the NF-κB pathway, including destructive mutations in negative regulators of NF-κB signaling (e.g. TNFAIP3, NFKBIA), and copy number gains of genes encoding positive regulators (e.g. REL, MAP3K14). In Epstein-Barr virus-positive cases of classical HL, the virus-encoded latent membrane protein 1 causes NF-κB activation by mimicking an active CD40 receptor. NF-κB activity is also seen in the tumor cells of the rare nodular lymphocyte predominant form of HL, but the causes for this activity are largely unclear. Copyright © 2016 Elsevier Ltd. All rights reserved.

Various plus unique: Viral protein U as a plurifunctional protein for HIV-1 replication.

PubMed

Soper, Andrew; Juarez-Fernandez, Guillermo; Aso, Hirofumi; Moriwaki, Miyu; Yamada, Eri; Nakano, Yusuke; Koyanagi, Yoshio; Sato, Kei

2017-04-01

Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, encodes four accessory genes, one of which is viral protein U (Vpu). Recently, the study of Vpu has been of great interest. For instance, various cellular proteins are degraded (e.g. CD4) and down-modulated (e.g. tetherin) by Vpu. Vpu also antagonizes the function of tetherin and inhibits NF-κB. Moreover, Vpu is a viroporin forming ion channels and may represent a promising target for anti-HIV-1 drugs. In this review, we summarize the domains/residues that are responsible for Vpu's functions, describe the current understanding of the role of Vpu in HIV-1-infected cells, and review the effect of Vpu on HIV-1 in replication and pathogenesis. Future investigations that simultaneously assess a combination of Vpu functions are required to clearly delineate the most important functions for viral replication. Impact statement Viral protein U (Vpu) is a unique protein encoded by human immunodeficiency virus type 1 (HIV-1) and related lentiviruses, playing multiple roles in viral replication and pathogenesis. In this review, we briefly summarize the most up-to-date knowledge of HIV-1 Vpu.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

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

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul

2008-10-10

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing eithermore » 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.« less

Cell and molecular biology of simian virus 40: implications for human infections and disease

NASA Technical Reports Server (NTRS)

Butel, J. S.; Lednicky, J. A.

1999-01-01

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology.

Cell and molecular biology of simian virus 40: implications for human infections and disease.

PubMed

Butel, J S; Lednicky, J A

1999-01-20

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology.

Poxvirus-induced alteration of arachidonate metabolism.

PubMed Central

Palumbo, G J; Glasgow, W C; Buller, R M

1993-01-01

Recent evidence suggests that orthopoxviruses have an obligate requirement for arachidonic acid metabolites during replication in vivo and in vitro. Our report indicates that a virus family (Poxviridae) possesses multiple genes that function to regulate arachidonate metabolism. Analyses of BS-C-1 cells infected with cowpox virus or vaccinia virus detected enhanced arachidonate product formation from both the cyclooxygenase (specifically prostaglandins E2 and F2 alpha) and lipoxygenase (specifically 15-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid) pathways. In contrast, human parainfluenza type 3 or herpes simplex virus type 1 infections did not increase arachidonate metabolism. Results were consistent with a virus early-gene product either directly mediating or inducing a host factor that mediated the up-regulation of arachidonate metabolism, although vaccinia growth factor was not responsible. In addition, the cowpox virus 38-kDa protein-encoding gene, which is associated with inhibition of an inflammatory response, correlated with inhibition of formation of a product biochemically characteristic of (14R,15S)-dihydroxyeicosatetraenoic acid. We propose that orthopoxvirus-induced up-regulation of arachidonic acid metabolism during infection renders the infected cells susceptible to generation of inflammatory mediators from both the cyclooxygenase and the lipoxygenase pathways, and poxviruses, therefore, possess at least one gene (38K) that can alter the lipoxygenase-metabolite spectrum. PMID:8383332

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

PubMed

Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

2016-11-28

H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection.

PubMed

Park, Sang Mi; Lee, Jung Suk; Jegal, Sung; Jeon, Bo Young; Jung, Min; Park, Yoon Sik; Han, Sang Lyul; Shin, Yoon Sup; Her, Nam Han; Lee, Jang Ha; Lee, Mi Yeon; Ryu, Ki Hyun; Yang, Seung Gyun; Harn, Chee Hark

2005-08-01

In watermelon, grafting of seedlings to rootstocks is necessary because watermelon roots are less viable than the rootstock. Moreover, commercially important watermelon varieties require disease-resistant rootstocks to reduce total watermelon yield losses due to infection with viruses such as cucumber green mottle mosaic virus (CGMMV). Therefore, we undertook to develop a CGMMV-resistant watermelon rootstock using a cDNA encoding the CGMMV coat protein gene (CGMMV-CP), and successfully transformed a watermelon rootstock named 'gongdae'. The transformation rate was as low as 0.1-0.3%, depending on the transformation method used (ordinary co-culture vs injection, respectively). However, watermelon transformation was reproducibly and reliably achieved using these two methods. Southern blot analysis confirmed that the CGMMV-CP gene was inserted into different locations in the genome either singly or multiple copies. Resistance testing against CGMMV showed that 10 plants among 140 T1 plants were resistant to CGMMV infection. This is the first report of the development by genetic engineering of watermelons resistant to CGMMV infection.

Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.

PubMed

Dheekollu, Jayaraju; Malecka, Kimberly; Wiedmer, Andreas; Delecluse, Henri-Jacques; Chiang, Alan K S; Altieri, Dario C; Messick, Troy E; Lieberman, Paul M

2017-01-31

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.

The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

PubMed Central

Herzog, Etienne; Guerra-Peraza, Orlene; Hohn, Thomas

2000-01-01

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly. PMID:10666237

[Entification of the Rubella virus genotype 1H in Western Siberia].

PubMed

Seregin, S V; Babkin, I V; Petrova, I D; Iashina, L N; Malkova, E M; Petrov, V S

2011-01-01

Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes all three Rubella structural proteins (C, E2, and E1), was implemented. This analysis provides characterization of this strain and classifies it as 1H genotype, thereby correcting previous classification of this strain based upon shorter nucleotide sequence, only encoding E1 protein. Therefore, this study identified the genotype of the Rubella virus not previously detected in Western Siberia (and even entire Russian Federation), which highlights the importance of more extensive characterization of genetic variability of the Rubella virus, especially with regard to potential influence of vaccination on the Rubella virus mutagenesis.

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

PubMed Central

Palanisamy, Navaneethan; Goedecke, Ulrike; Jäger, Nils; Pöhlmann, Stefan; Winkler, Michael

2014-01-01

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels. PMID:24842154

Wheat streak mosaic virus-encoded NIa-Pro and coat protein are involved in virus superinfection exclusion

USDA-ARS?s Scientific Manuscript database

Cross protection or superinfection exclusion (SE) is defined as the phenomenon whereby initial infection by one virus prevents subsequent infection by closely related viruses. The mechanisms of SE are just beginning to be understood. Wheat streak mosaic virus (WSMV; genus: Tritimovirus; family: Poty...

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques

PubMed Central

Mutschler, James P.; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W.; Osorio, Jorge E.

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a “mosaic” hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses. PMID:28771513

Genome segregation and packaging machinery in Acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.

PubMed

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi; Kondabagil, Kiran

2014-06-01

Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques.

PubMed

Florek, Nicholas W; Kamlangdee, Attapon; Mutschler, James P; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W; Osorio, Jorge E; Friedrich, Thomas C

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a "mosaic" hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.

Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

PubMed Central

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

2014-01-01

ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses. PMID:24623441

Early function of the Abutilon mosaic virus AC2 gene as a replication brake.

PubMed

Krenz, Björn; Deuschle, Kathrin; Deigner, Tobias; Unseld, Sigrid; Kepp, Gabi; Wege, Christina; Kleinow, Tatjana; Jeske, Holger

2015-04-01

The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

Diversity, origins and virulence of Avipoxviruses in Hawaiian Forest Birds

USGS Publications Warehouse

Jarvi, S.I.; Triglia, D.; Giannoulis, A.; Farias, M.; Bianchi, K.; Atkinson, C.T.

2008-01-01

We cultured avian pox (Avipoxvirus spp.) from lesions collected on Hawai'i, Maui, Moloka'i, and 'Oahu in the Hawaiian Islands from 15 native or non-native birds representing three avian orders. Phylogenetic analysis of a 538 bp fragment of the gene encoding the virus 4b core polypeptide revealed two distinct variant clusters, with sequences from chickens (fowlpox) forming a third distinct basal cluster. Pox isolates from one of these two clusters appear closely related to canarypox and other passerine pox viruses, while the second appears more specific to Hawai'i. There was no evidence that birds were infected simultaneously with multiple pox virus variants based on evaluation of multiples clones from four individuals. No obvious temporal or geographic associations were observed and strict host specificity was not apparent among the 4b-defined field isolates. We amplified a 116 bp 4b core protein gene fragment from an 'Elepaio (Chasiempis sandwichensis) collected in 1900 on Hawai'i Island that clustered closely with the second of the two variants, suggesting that this variant has been in Hawai'i for at least 100 years. The high variation detected between the three 4b clusters provides evidence for multiple, likely independent introductions, and does not support the hypothesis of infection of native species through introduction of infected fowl. Preliminary experimental infections in native Hawai'i 'Amakihi (Hemignathus virens) suggest that the 4b-defined variants may be biologically distinct, with one variant appearing more virulent. These pox viruses may interact with avian malaria (Plasmodium relictum), another introduced pathogen in Hawaiian forest bird populations, through modulation of host immune responses. ?? 2007 Springer Science+Business Media B.V.

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

PubMed Central

van de Ven, W J; Vermorken, A J; Onnekink, C; Bloemers, H P; Bloemendal, H

1978-01-01

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction. Images PMID:702639

Ankyrin-repeat containing proteins of microbes: a conserved structure with functional diversity

PubMed Central

Al-Khodor, Souhaila; Price, Christopher T.; Kalia, Awdhesh; Kwaik, Yousef Abu

2009-01-01

Summary The ankyrin repeat (ANK) is the most common protein-protein interaction motif in nature and predominantly found in eukaryotic proteins. The genome sequencing of various pathogenic or symbiotic bacteria and eukaryotic viruses identified numerous genes encoding ANK-containing proteins that were proposed to have been acquired from eukaryotes by horizontal gene transfer. However, the recent discovery of additional ANK-containing proteins encoded in the genomes of archaea and free-living bacteria suggests either a more ancient origin of the ANK motif or multiple convergent evolution events. Many bacterial pathogens employ various types of secretion systems to deliver ANK-containing proteins into eukaryotic cells where they mimic or manipulate various host functions. Understanding the molecular and biochemical functions of this family of proteins will enhance our understanding of important host-microbe interactions. PMID:19962898

Effect of multiple infections with white spot syndrome virus and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.): mortality and viral replication.

PubMed

Jang, I K; Qiao, G; Kim, S-K

2014-10-01

Multiple infections are commonly found in practical shrimp culture and may cause more serious consequences than infections by one pathogen only. Therefore, this study was conducted to evaluate the effect of multiple infections with white spot syndrome virus (WSSV) and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.), mortality, WSSV replication in vivo and host immune response. In the WSSV single-infection group (WSSV load, 2 × 10(2) copies μL(-1)), mean cumulative mortality was 29.2%. In the V. anguillarum single-infection group, cumulative mortality was 12.5% when shrimp were challenged by 10(5) CFU mL(-1) of bacteria. In the co- and super-infection groups, 37.5% and 50% cumulative mortalities, respectively, were observed at a lower bacterial concentration of 10(3) CFU mL(-1), suggesting that shrimp with multiple infections died earlier and more frequently than singly infected shrimp. WSSV load after injection was tracked over time by TaqMan quantitative PCR. WSSV load increased more rapidly in the multiple-infection groups than in the single-infection group. Additionally, mRNA expression of the genes encoding prophenoloxidase 1 and 2, which are closely involved in innate immunity in shrimp, was down-regulated more extensively in multiple-infection groups than in single-infection groups, as indicated by quantitative reverse-transcription PCR. © 2013 John Wiley & Sons Ltd.

Marek’s Disease Virus Encoded Ribonucleotide Reductase Large Subunit is not Essential for In Vitro Replication

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) infected cells express a viral ribonucleotide reductase (RR) that is distinguishable from that present in uninfected cells by monoclonal antibody T81. Open reading frames UL39 and UL40 of the MDV genome encode the large (RR1) and small (RR2) subunits of RR enzyme, respe...

Both Homo and Heterodimers of Marek's Disease Virus Encoded Meq Protein Contribute to Transformation of Lymphocytes in Chickens

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) elicits T-cell lymphomas in chickens. The MDV genome encodes an oncoprotein, Meq, with similarity to the Jun/Fos family of proteins. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. We have previously shown that Meq homodime...

Octapartite negative-sense RNA genome of High Plains wheat mosaic virus encodes two suppressors of RNA silencing.

PubMed

Gupta, Adarsh K; Hein, Gary L; Graybosch, Robert A; Tatineni, Satyanarayana

2018-05-01

High Plains wheat mosaic virus (HPWMoV, genus Emaravirus; family Fimoviridae), transmitted by the wheat curl mite (Aceria tosichella Keifer), harbors a monocistronic octapartite single-stranded negative-sense RNA genome. In this study, putative proteins encoded by HPWMoV genomic RNAs 2-8 were screened for potential RNA silencing suppression activity by using a green fluorescent protein-based reporter agroinfiltration assay. We found that proteins encoded by RNAs 7 (P7) and 8 (P8) suppressed silencing induced by single- or double-stranded RNAs and efficiently suppressed the transitive pathway of RNA silencing. Additionally, a Wheat streak mosaic virus (WSMV, genus Tritimovirus; family Potyviridae) mutant lacking the suppressor of RNA silencing (ΔP1) but having either P7 or P8 from HPWMoV restored cell-to-cell and long-distance movement in wheat, thus indicating that P7 or P8 rescued silencing suppressor-deficient WSMV. Furthermore, HPWMoV P7 and P8 substantially enhanced the pathogenicity of Potato virus X in Nicotiana benthamiana. Collectively, these data demonstrate that the octapartite genome of HPWMoV encodes two suppressors of RNA silencing. Published by Elsevier Inc.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

PubMed Central

Soler, Nicolas; Marguet, Evelyne; Cortez, Diego; Desnoues, Nicole; Keller, Jenny; van Tilbeurgh, Herman; Sezonov, Guennadi; Forterre, Patrick

2010-01-01

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication. PMID:20403814

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

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

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

Viral genome methylation as an epigenetic defense against geminiviruses.

PubMed

Raja, Priya; Sanville, Bradley C; Buchmann, R Cody; Bisaro, David M

2008-09-01

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

PubMed Central

Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

2016-01-01

Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites.

PubMed

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-04-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass ( Zostera marina ) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae . They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites

PubMed Central

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-01-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses. PMID:29628822

Genetics, recombination and clinical features of human rhinovirus species C (HRV-C) infections; interactions of HRV-C with other respiratory viruses.

PubMed

Wisdom, Anne; Kutkowska, Aldona E; McWilliam Leitch, E Carol; Gaunt, Eleanor; Templeton, Kate; Harvala, Heli; Simmonds, Peter

2009-12-30

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6-12 months; 80% of infections in those <2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5'untranslated region (5'UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5'UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other respiratory virus infections. HRV-C's disease associations, its prevalence and evidence for interfering effects on other respiratory viruses mandates incorporation of rhinoviruses into future diagnostic virology screening.

Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity

PubMed Central

Jurak, Igor; Silverstein, Leah B.; Sharma, Mayuri

2012-01-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection. PMID:22787211

Herpes simplex virus is equipped with RNA- and protein-based mechanisms to repress expression of ATRX, an effector of intrinsic immunity.

PubMed

Jurak, Igor; Silverstein, Leah B; Sharma, Mayuri; Coen, Donald M

2012-09-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3' untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.

The kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea

PubMed Central

Deeg, Christoph M; Chow, Cheryl-Emiliane T

2018-01-01

Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses. PMID:29582753

Genome-Wide Analysis of Polymorphisms Associated with Cytokine Responses in Smallpox Vaccine Recipients

PubMed Central

Kennedy, Richard B.; Ovsyannikova, Inna G.; Pankratz, V. Shane; Haralambieva, Iana H.; Vierkant, Robert A.; Poland, Gregory A.

2014-01-01

The role that genetics plays in response to infection or disease is becoming increasingly clear as we learn more about immunogenetics and host-pathogen interactions. Here we report a genome-wide analysis of the effects of host genetic variation on cytokine responses to vaccinia virus stimulation in smallpox vaccine recipients. Our data show that vaccinia stimulation of immune individuals results in secretion of inflammatory and Th1 cytokines. We identified multiple SNPs significantly associated with variations in cytokine secretion. These SNPs are found in genes with known immune function, as well as in genes encoding for proteins involved in signal transduction, cytoskeleton, membrane channels and ion transport, as well as others with no previously identified connection to immune responses. The large number of significant SNP associations implies that cytokine secretion in response to vaccinia virus is a complex process controlled by multiple genes and gene families. Follow-up studies to replicate these findings and then pursue mechanistic studies will provide a greater understanding of how genetic variation influences vaccine responses. PMID:22610502

An autopsy case of chronic active Epstein-Barr virus infection (CAEBV): distribution of central nervous system (CNS) lesions.

PubMed

Kobayashi, Zen; Tsuchiya, Kuniaki; Takahashi, Makoto; Yokota, Osamu; Sasaki, Atsushi; Bhunchet, Ekapot; Arai, Tetsuaki; Akiyama, Haruhiko; Kamoshita, Masaharu; Kotera, Minoru; Mizusawa, Hidehiro

2008-12-15

A 27-year-old Japanese man developed recurrent respiratory and central nervous system (CNS) symptoms, and hemophagocytic syndromes with a clinical course of 6 years. CT demonstrated multiple nodular lesions in the bilateral lungs, and MRI revealed multiple abnormal intensity areas in the brain and spinal cord. Cerebrospinal fluid (CSF) examination disclosed mild pleocytosis and the presence of Epstein-Barr virus (EBV)-DNA detected by polymerase chain reaction (PCR). The patient died of a hemorrhagic shock associated with a hemophagocytic syndrome. A postmortem study revealed massive hemorrhage in the abdominal cavity and iliopsoas muscles, as well as diffuse infiltration of lymphocytes and/or macrophages into the lungs, liver, kidneys, spleen, cardiac muscle, bone marrow, and CNS. The severe involvement was demonstrated in the CNS, especially in the spinal cord and brainstem. The CD3 positive cells of the brainstem were EBV-encoded RNA 1 positive. This is the first autopsy case of chronic active EBV infection (CAEBV) in which severe and extensive CNS involvement was demonstrated.

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

PubMed Central

Farza, H; Hadchouel, M; Scotto, J; Tiollais, P; Babinet, C; Pourcel, C

1988-01-01

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication. Images PMID:2845128

Herpes simplex virus 1 microRNAs expressed abundantly during latent infection are not essential for latency in mouse trigeminal ganglia

PubMed Central

Kramer, Martha F.; Jurak, Igor; Pesola, Jean M.; Boissel, Sandrine; Knipe, David M.; Coen, Donald M.

2013-01-01

Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it. PMID:21782205

Live-Cell Imaging of Filoviruses.

PubMed

Schudt, Gordian; Dolnik, Olga; Becker, Stephan

2017-01-01

Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.

Ectromelia virus encodes an anti-apoptotic protein that regulates cell death.

PubMed

Mehta, Ninad; Taylor, John; Quilty, Douglas; Barry, Michele

2015-01-15

Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many viruses including poxviruses, encode proteins to prevent or delay apoptosis. Here we show that ectromelia virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Complete genome sequence of keunjorong mosaic virus, a potyvirus from Cynanchum wilfordii.

PubMed

Nam, Moon; Lee, Joo-Hee; Choi, Hong Soo; Lim, Hyoun-Sub; Moon, Jae Sun; Lee, Su-Heon

2013-08-01

We have determined the complete genome sequence of keunjorong mosaic virus (KjMV). The KjMV genome is composed of 9,611 nucleotides, excluding the 3'-terminal poly(A) tail. It contains two open reading frames (ORFs), with the large one encoding a polyprotein of 3,070 amino acids and the small overlapping ORF encoding a PIPO protein of 81 amino acids. The KjMV genome shared the highest nucleotide sequence identity (57.5  %) with pepper mottle virus and freesia mosaic virus, two members of the genus Potyvirus. Based on the phylogenetic relatedness to known potyviruses, KjMV appears to be a member of a new species in the genus Potyvirus.

The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that interacts with host protein BAG6

USDA-ARS?s Scientific Manuscript database

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few have been studied in some detail. Here we rep...

Marek’s disease virus encoded ribonucleotide reductase large subunit is essential for in vivo replication and plays a critical role in viral pathogenesis.

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus encodes a ribonucleotide reductase (RR) that consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme and both subunits are necessary for enzyme activity. It is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleo...

Potential role for a B-catenin coactivator (high mobility group AT-hook 1 protein) during the latency-reactivation cycle of bovine herpesverus 1

USDA-ARS?s Scientific Manuscript database

The latency-related (LR)-RNA encoded by bovine herpes virus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to play a dominant role during the latency-reactivation cycle because a mutant virus containing stop codons...

The L83L ORF of African swine fever virus strain Georgia encodes for a non-essential gene that interacts with host protein IL-1ß

USDA-ARS?s Scientific Manuscript database

African swine fever virus (ASFV) causes a contagious and frequently lethal disease of pigs that produces significant economic consequences to the swine industry. ASFV genome encodes for more than 150 genes, but only a few of them have been studied in detail. Here we report the characterization of op...

Virus engineering: Fighting HIV at its own game

NASA Astrophysics Data System (ADS)

Lin, Shixian; Chen, Peng R.

2014-07-01

Live-attenuated viruses used in vaccines can regain their virulence, which for deadly viruses such as HIV is an unacceptable risk. Now, attenuated HIV-1 viruses, which include mutations that genetically encode unnatural amino acids and prevent them from replicating in normal cells, have been constructed.

Designed Proteins Induce the Formation of Nanocage-containing Extracellular Vesicles

PubMed Central

Votteler, Jörg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.

2017-01-01

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids, and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3–5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here, we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as Enveloped Protein Nanocages (EPNs). Robust EPN biogenesis required protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induced EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical and electron cryomicroscopic (cryo-EM) analyses revealed that one design, EPN-01, comprised small (~100 nm) vesicles containing multiple protein nanocages that closely matched the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV-G) could fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These studies show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a novel class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells. PMID:27919066

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR.

PubMed

Periyannan Rajeswari, Prem Kumar; Soderberg, Lovisa M; Yacoub, Alia; Leijon, Mikael; Andersson Svahn, Helene; Joensson, Haakan N

2017-08-01

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads. Copyright © 2017. Published by Elsevier B.V.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice.

PubMed

Gummow, Jason; Li, Yanrui; Yu, Wenbo; Garrod, Tamsin; Wijesundara, Danushka; Brennan, Amelia J; Mullick, Ranajoy; Voskoboinik, Ilia; Grubor-Bauk, Branka; Gowans, Eric J

2015-08-01

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

PubMed

Kang, Sung-Hwan; Atallah, Osama O; Sun, Yong-Duo; Folimonova, Svetlana Y

2018-01-15

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host. Copyright © 2017 Elsevier Inc. All rights reserved.

Virocell Metabolism: Metabolic Innovations During Host-Virus Interactions in the Ocean.

PubMed

Rosenwasser, Shilo; Ziv, Carmit; Creveld, Shiri Graff van; Vardi, Assaf

2016-10-01

Marine viruses are considered to be major ecological, evolutionary, and biogeochemical drivers of the marine environment, responsible for nutrient recycling and determining species composition. Viruses can re-shape their host's metabolic network during infection, generating the virocell-a unique metabolic state that supports their specific requirement. Here we discuss the concept of 'virocell metabolism' and its formation by rewiring of host-encoded metabolic networks, or by introducing virus-encoded auxiliary metabolic genes which provide the virocell with novel metabolic capabilities. The ecological role of marine viruses is commonly assessed by their relative abundance and phylogenetic diversity, lacking the ability to assess the dynamics of active viral infection. The new ability to define a unique metabolic state of the virocell will expand the current virion-centric approaches in order to quantify the impact of marine viruses on microbial food webs. Copyright © 2016. Published by Elsevier Ltd.

Ac102 Participates in Nuclear Actin Polymerization by Modulating BV/ODV-C42 Ubiquitination during Autographa californica Multiple Nucleopolyhedrovirus Infection.

PubMed

Zhang, Yongli; Hu, Xue; Mu, Jingfang; Hu, Yangyang; Zhou, Yuan; Zhao, He; Wu, Chunchen; Pei, Rongjuan; Chen, Jizheng; Chen, Xinwen; Wang, Yun

2018-06-15

As a virus-encoded actin nucleation promoting factor (NPF), P78/83 induces actin polymerization to assist in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) propagation. According to our previous study, although P78/83 actively undergoes ubiquitin-independent proteasomal degradation, AcMNPV encodes budded virus/occlusion derived virus (BV/ODV)-C42 (C42), which allows P78/83 to function as a stable NPF by inhibiting its degradation during viral infection. However, whether there are other viral proteins involved in regulating P78/83-induced actin polymerization has yet to be determined. In this study, we found that Ac102, an essential viral gene product previously reported to play a key role in mediating the nuclear accumulation of actin during AcMNPV infection, is a novel regulator of P78/83-induced actin polymerization. By characterizing an ac102 knockout bacmid, we demonstrated that Ac102 participates in regulating nuclear actin polymerization as well as the morphogenesis and distribution of capsid structures in the nucleus. These regulatory effects are heavily dependent on an interaction between Ac102 and C42. Further investigation revealed that Ac102 binds to C42 to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable NPF to induce actin polymerization. Thus, Ac102 and C42 form a regulatory cascade to control viral NPF activity, representing a sophisticated mechanism for AcMNPV to orchestrate actin polymerization in both a ubiquitin-dependent and ubiquitin-independent manner. IMPORTANCE Actin is one of the most functionally important proteins in eukaryotic cells. Morphologically, actin can be found in two forms: a monomeric form called globular actin (G-actin) and a polymeric form called filamentous actin (F-actin). G-actin can polymerize to form F-actin, and nucleation promoting factor (NPF) is the initiator of this process. Many viral pathogens harness the host actin polymerization machinery to assist in virus propagation. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) induces actin polymerization in host cells. P78/83, a viral NPF, is responsible for this process. Previously, we identified that BV/ODV-C42 (C42) binds to P78/83 and protects it from degradation. In this report, we determined that another viral protein, Ac102, is involved in modulating C42 ubiquitination and, consequently, ensures P78/83 activity as an NPF to initiate actin polymerization. This regulatory cascade represents a novel mechanism by which a virus can harness the cellular actin cytoskeleton to assist in viral propagation. Copyright © 2018 American Society for Microbiology.

Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus.

PubMed

Sun, Yinyan; Qi, Yonghe; Liu, Chenxuan; Gao, Wenqing; Chen, Pan; Fu, Liran; Peng, Bo; Wang, Haimin; Jing, Zhiyi; Zhong, Guocai; Li, Wenhui

2014-01-01

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.

Pathogenicity evaluation of different Newcastle disease virus chimeras in 4-week-old chickens

USDA-ARS?s Scientific Manuscript database

Infection with a virulent strain of Newcastle disease virus is considered one of the most important threats to the poultry industry worldwide. The causative virus, Newcastle disease virus, belongs to the Paramyxoviridae family, genus Avulavirus, and its genome encodes for 6 structural proteins: nu...

Residue 41 of the Eurasian Avian-Like Swine Influenza A Virus Matrix Protein Modulates Virion Filament Length and Efficiency of Contact Transmission

PubMed Central

Campbell, Patricia J.; Kyriakis, Constantinos S.; Marshall, Nicolle; Suppiah, Suganthi; Seladi-Schulman, Jill; Danzy, Shamika; Lowen, Anice C.

2014-01-01

ABSTRACT Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity. IMPORTANCE We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model. PMID:24760887

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.

Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

PubMed Central

Lihoradova, Olga A.; Indran, Sabarish V.; Kalveram, Birte; Lokugamage, Nandadeva; Head, Jennifer A.; Gong, Bin; Tigabu, Bersabeh; Juelich, Terry L.; Freiberg, Alexander N.; Ikegami, Tetsuro

2013-01-01

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever. PMID:23638202

Characterization of Rift Valley fever virus MP-12 strain encoding NSs of Punta Toro virus or sandfly fever Sicilian virus.

PubMed

Lihoradova, Olga A; Indran, Sabarish V; Kalveram, Birte; Lokugamage, Nandadeva; Head, Jennifer A; Gong, Bin; Tigabu, Bersabeh; Juelich, Terry L; Freiberg, Alexander N; Ikegami, Tetsuro

2013-01-01

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.

US9, a stable lysine-less herpes simplex virus 1 protein, is ubiquitinated before packaging into virions and associates with proteasomes

PubMed Central

Brandimarti, Renato; Roizman, Bernard

1997-01-01

The US9 gene of herpes simplex virus 1 encodes a virion tegument protein with a predicted Mr of 10,000. Earlier studies have shown that the gene is not essential for viral replication in cells in culture. We report that (i) US9 forms in denaturing polyacrylamide gels multiple overlapping bands ranging in Mr from 12,000 to 25,000; (ii) the protein recovered from infected cells or purified virions reacts with anti-ubiquitin antibodies; (iii) autoradiographic images of US9 protein immunoprecipitated from cells infected with [35S]methionine-labeled virus indicate that the protein is stable for at least 4 h after entry into cells (the protein was also stable for at least 4 h after a 1-h labeling interval 12 h after infection); (iv) antibody to subunit 12 of proteasomes pulls down US9 protein from herpes simplex virus-infected cell lysates; and (v) the US9 gene is highly conserved among the members of the alpha subfamily of herpes viruses, and the US9 gene product lacks lysines. We conclude that US9 is a lysine-less, ubiquitinated protein that interacts with the ubiquitin-dependent pathway for degradation of proteins and that this function may be initiated at the time of entry of the virus into the cell. PMID:9391137

The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon▿

PubMed Central

Graef, Katy M.; Vreede, Frank T.; Lau, Yuk-Fai; McCall, Amber W.; Carr, Simon M.; Subbarao, Kanta; Fodor, Ervin

2010-01-01

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence. PMID:20538852

Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

PubMed

Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

2011-09-01

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

Functional characterization of Bombyx mori nucleopolyhedrovirus mutant lacking late expression factor 9.

PubMed

Zhang, Y; Shi, Y; Yu, H; Li, J; Quan, Y; Shu, T; Nie, Z; Zhang, Y; Yu, W

Baculoviridae is a family of invertebrate viruses with large double-stranded DNA genomes. Proteins encoded by some late expression factor (lef ) genes are involved in the regulation of viral gene expression. Lef-9 is one of four transcription-specific Lefs, which are components of the virus-encoded RNA polymerase, and can initiate and transcribe late and very late genes. As a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus (BmNPV), Lef-9 may be involved in the regulation of viral propagation. However, the underlying mechanism remains unclear. To determine the role of lef-9 in baculovirus infection, lef-9-knockout virus (lef-9-KO-Bacmid virus) was constructed using the Red recombination system, and the Bac-to-Bac system was used to prepare lef-9-repaired virus (lef-9-Re-Bacmid virus). The lef-9-KO virus did not produce infectious viruses or show infection activity, while the lef-9-repaired virus recovered both. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the transcription levels in wild-type-Bacmid, lef-9-KO-Bacmid, and lef-9-Re-Bacmid viruses showed that the lef-9-KO bacmid had little effect on viral genome replication. However, the transcription levels of the early and late viral genes, lef-3, ie-1, vp39, and p10, were significantly lower in BmN cells transfected with lef-9-KO-Bacmids than in the controls. Electron microscopy showed no visible enveloped virions in cells transfected with lef-9-KO-Bacmids, while many mature virions in cells transfected with lef-9-Re-Bacmid and wt-Bacmid were present. Thus, lef-9 was not essential for viral genome replication, but significantly affected viral gene transcription and expression in all periods of cell life cycle.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

PubMed

Vasilakis, Nikos; Castro-Llanos, Fanny; Widen, Steven G; Aguilar, Patricia V; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J; Wood, Thomas G; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J; Holmes, Edward C; Walker, Peter J; Tesh, Robert B

2014-04-01

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru

PubMed Central

Castro-Llanos, Fanny; Widen, Steven G.; Aguilar, Patricia V.; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J.; Wood, Thomas G.; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J.; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2014-01-01

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family. PMID:24421116

The Complete Sequence of the First Spodoptera frugiperda Betabaculovirus Genome: A Natural Multiple Recombinant Virus

PubMed Central

Cuartas, Paola E.; Barrera, Gloria P.; Belaich, Mariano N.; Barreto, Emiliano; Ghiringhelli, Pablo D.; Villamizar, Laura F.

2015-01-01

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness. PMID:25609309

The complete sequence of the first Spodoptera frugiperda Betabaculovirus genome: a natural multiple recombinant virus.

PubMed

Cuartas, Paola E; Barrera, Gloria P; Belaich, Mariano N; Barreto, Emiliano; Ghiringhelli, Pablo D; Villamizar, Laura F

2015-01-20

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.

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

NASA Technical Reports Server (NTRS)

Butel, J. S.

2000-01-01

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

An overview of live attenuated recombinant pseudorabies viruses for use as novel vaccines

USDA-ARS?s Scientific Manuscript database

Pseudorabies virus (PRV) is a double-stranded, DNA-based swine virus with a genome approximating 150 kb in size. In cell culture, PRV has many non-essential genes which can be replaced with genes encoding heterologous antigens but without deleterious effects on virus propagation. Recombinant PRVs ex...

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

PubMed

Kong, Wei; Brovold, Matthew; Koeneman, Brian A; Clark-Curtiss, Josephine; Curtiss, Roy

2012-11-20

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

PubMed Central

Kong, Wei; Brovold, Matthew; Koeneman, Brian A.; Clark-Curtiss, Josephine; Curtiss, Roy

2012-01-01

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases. PMID:23129620

The complete nucleotide sequence and genome organization of a novel betaflexivirus infecting Citrullus lanatus.

PubMed

Xin, Min; Zhang, Peipei; Liu, Wenwen; Ren, Yingdang; Cao, Mengji; Wang, Xifeng

2017-10-01

The complete nucleotide sequence of a novel positive single-stranded (+ss) RNA virus, tentatively named watermelon virus A (WVA), was determined using a combination of three methods: RNA sequencing, small RNA sequencing, and Sanger sequencing. The full genome of WVA is comprised of 8,372 nucleotides (nt), excluding the poly (A) tail, and contains four open reading frames (ORFs). The largest ORF, ORF1 encodes a putative replication-associated polyprotein (RP) with three conserved domains. ORF2 and ORF4 encode a movement protein (MP) and coat protein (CP), respectively. The putative product encoded by ORF3, of an estimated molecular mass of 25 kDa, has no significant similarity with other proteins. Identity and phylogenetic analysis indicate that WVA is a new virus, closely related to members of the family Betaflexiviridae. However, the final taxonomic allocation of WVA within the family is yet to be determined.

A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae▿

PubMed Central

Woolford, Lucy; Rector, Annabel; Van Ranst, Marc; Ducki, Andrea; Bennett, Mark D.; Nicholls, Philip K.; Warren, Kristin S.; Swan, Ralph A.; Wilcox, Graham E.; O'Hara, Amanda J.

2007-01-01

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families. PMID:17898069

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

PubMed

Santana, Vinicius C; Diniz, Mariana O; Cariri, Francisco A M O; Ventura, Armando M; Cunha-Neto, Edécio; Almeida, Rafael R; Campos, Marco A; Lima, Graciela K; Ferreira, Luís C S

2013-01-01

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: A soluble, secreted CD30 homologue

PubMed Central

Panus, Joanne Fanelli; Smith, Craig A.; Ray, Caroline A.; Smith, Terri Davis; Patel, Dhavalkumar D.; Pickup, David J.

2002-01-01

Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus. Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene, vCD30, encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus. The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51–59% identity with highly conserved extracellular segments of both mouse and human CD30. A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions. Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses. Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases. PMID:12034885

Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein.

PubMed

Chen, Huiqing; Li, Mei; Huang, Guoping; Mai, Weijun; Chen, Keping; Zhou, Yajing

2014-08-01

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

Isolation and characterization of a novel mycovirus from Penicillium digitatum

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

Niu, Yuhui; Zhang, Tingfu; Zhu, Ying

A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211 bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed sphericalmore » particles ~35 nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. - Highlights: • A novel victorivirus designated Penicillium digitatum virus 1 (PdV1) was isolated fromP. digitatum. • The dsRNA genome of PdV1 are 5211 bp long, two ORFs encoding CP and RdRp, and are encased in virions of ~35 nm in diameter. • PdV1 infection led to hypovirulent effect on P. digitatum. PdV1 may potentially be used for citrus green mold biocontrol. • Our study provides a research basis for establishing a model system for the study of P. digitatum–mycovirus interactions.« less

Mapping of herpes simplex virus-1 neurovirulence to. gamma. sub 1 34. 5, a gene nonessential for growth in culture

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

Chou, J.; Roizman, B.; Kern, E.R.

1990-11-30

The gene designated {gamma}{sub 1}34.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to reactmore » with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the {gamma}{sub 1}34.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.« less

Viruses and viruslike particles of eukaryotic algae.

PubMed Central

Van Etten, J L; Lane, L C; Meints, R H

1991-01-01

Until recently there was little interest or information on viruses and viruslike particles of eukaryotic algae. However, this situation is changing. In the past decade many large double-stranded DNA-containing viruses that infect two culturable, unicellular, eukaryotic green algae have been discovered. These viruses can be produced in large quantities, assayed by plaque formation, and analyzed by standard bacteriophage techniques. The viruses are structurally similar to animal iridoviruses, their genomes are similar to but larger (greater than 300 kbp) than that of poxviruses, and their infection process resembles that of bacteriophages. Some of the viruses have DNAs with low levels of methylated bases, whereas others have DNAs with high concentrations of 5-methylcytosine and N6-methyladenine. Virus-encoded DNA methyltransferases are associated with the methylation and are accompanied by virus-encoded DNA site-specific (restriction) endonucleases. Some of these enzymes have sequence specificities identical to those of known bacterial enzymes, and others have previously unrecognized specificities. A separate rod-shaped RNA-containing algal virus has structural and nucleotide sequence affinities to higher plant viruses. Quite recently, viruses have been associated with rapid changes in marine algal populations. In the next decade we envision the discovery of new algal viruses, clarification of their role in various ecosystems, discovery of commercially useful genes in these viruses, and exploitation of algal virus genetic elements in plant and algal biotechnology. Images PMID:1779928

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

PubMed Central

Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

1999-01-01

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

African Swine Fever Virus: a new old enemy of Europe

PubMed

Cisek, Agata A; Dąbrowska, Iwona; Gregorczyk, Karolina P; Wyżewski, Zbigniew

2016-10-01

African swine fever (ASF) is a highly contagious viral disease of swine with a mortality rate approaching 100 percent. African Swine Fever Virus (ASFV) is a double-stranded DNA virus with a complex molecular structure. Its large genome, encoding multiple virulence factors, allows for efficient replication, which takes place predominantly in the cytoplasm of monocytes and macrophages. Also, ASFV has the ability to interfere with cell signalling pathways, which leads to various modulations in the synthesis profiles of interferon and other cytokines. Sustained viremia favours the persistence of virions in blood and tissues of the convalescents, and the extended circulation of ASFV within the herd. ASFV has been spreading in the Caucasus since 2007, and in 2014 reached the eastern territory of the European Union. Outbreaks pose an economical threat to native pig rearing, especially since a single point source may easily develop into an epizootic event. There is currently no effective vaccine nor treatment for ASF, and eradication is possible only by prevention or the slaughter of diseased animals. This review paper summarizes the current state of knowledge about ASFV.

[Study of the functional role of mutation in the guinea pig-adapted Ebola virus genome on a Drosophila melanogaster model].

PubMed

Shelemba-Chepurnova, A A; Omel'ianchuk, L V; Chepurnov, A A

2011-01-01

Ebola virus virulence in guinea pigs, which appears through virus adaptation to this animal host, correlates with substitutions in the gene encoding vp24 protein. In particular, the substitution His-->Tyr186 was found when obtaining strain 8 ms. An attempt was made to clarify the functional role of this substitution in a transgenic fruit fly model. Using the drosophila transformation technique provided transgenic strains that contained genomic insertions of wild-type Ebola virus vp24 gene and the mutant gene with the His-->Tyr substitution at the above position. Thus, the drosophila strains carrying the sequences encoding for the vp24 proteins of Ebola virus Zaire and 8 ms in pUAST vector were obtained. This makes it possible to study the expression of transgenic constructs in various D. melanogaster organs and tissues.

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses

PubMed Central

Nogales, Aitor; Martinez-Sobrido, Luis

2016-01-01

ABSTRACT Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo. PMID:28003482

NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses.

PubMed

Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-03-01

Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo . Copyright © 2017 American Society for Microbiology.

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

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

Klein, George, E-mail: Georg.Klein@ki.se; Klein, Eva; Kashuba, Elena

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore asmore » a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.« less

Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism.

PubMed

Ciancanelli, Michael J; Volchkova, Valentina A; Shaw, Megan L; Volchkov, Viktor E; Basler, Christopher F

2009-08-01

The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (C(ko)) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the C(ko) mutation proved attenuating but the G121E mutant virus replicated identically to the C(ko) virus. In cells infected with the wild-type and C(ko) viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

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

PubMed Central

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

2017-01-01

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

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

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

Jiang, Hongbing; Franz, Carl J.; Wu, Guang

2014-02-15

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated intomore » Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.« less

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

PubMed Central

Sanchez, A; Trappier, S G; Mahy, B W; Peters, C J; Nichol, S T

1996-01-01

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild. Images Fig. 2 Fig. 3 PMID:8622982

The not so universal tree of life or the place of viruses in the living world

PubMed Central

Brüssow, Harald

2009-01-01

Darwin provided a great unifying theory for biology; its visual expression is the universal tree of life. The tree concept is challenged by the occurrence of horizontal gene transfer and—as summarized in this review—by the omission of viruses. Microbial ecologists have demonstrated that viruses are the most numerous biological entities on earth, outnumbering cells by a factor of 10. Viral genomics have revealed an unexpected size and distinctness of the viral DNA sequence space. Comparative genomics has shown elements of vertical evolution in some groups of viruses. Furthermore, structural biology has demonstrated links between viruses infecting the three domains of life pointing to a very ancient origin of viruses. However, presently viruses do not find a place on the universal tree of life, which is thus only a tree of cellular life. In view of the polythetic nature of current life definitions, viruses cannot be dismissed as non-living material. On earth we have therefore at least two large DNA sequence spaces, one represented by capsid-encoding viruses and another by ribosome-encoding cells. Despite their probable distinct evolutionary origin, both spheres were and are connected by intensive two-way gene transfers. PMID:19571246

Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

PubMed Central

Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

2014-01-01

We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication

PubMed Central

Zhu, Mengxiao; Deng, Riqiang

2016-01-01

ABSTRACT An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae. IMPORTANCE Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a better general understanding of virus-encoded miRNAs. In brief, our study implied that AcMNPV-miR-1 constrains viral replication and cellular infection but enhances larval infection. PMID:27147751

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

PubMed Central

Alejo, Alí; Ruiz-Argüello, M. Begoña; Ho, Yin; Smith, Vincent P.; Saraiva, Margarida; Alcami, Antonio

2006-01-01

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. PMID:16581912

The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses

PubMed Central

Shukla, Avi; Chatterjee, Anirvan

2018-01-01

Abstract Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near–linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption. PMID:29308275

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

PubMed

Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

2006-04-11

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

Immune Protection of Nonhuman Primates Against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

DTIC Science & Technology

2006-06-01

21. Geisbert TW, Hensley LE , Larsen T, Young HA, Reed DS, et al. (2003) Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: Evidence that...Shedlock DJ, Xu L, et al. (2006) Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified...CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 9 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

Near-Complete Genome Sequence of a Novel Single-Stranded RNA Virus Discovered in Indoor Air

PubMed Central

2018-01-01

ABSTRACT Viral metagenomic analysis of heating, ventilation, and air conditioning (HVAC) filters recovered the near-complete genome sequence of a novel virus, named HVAC-associated RNA virus 1 (HVAC-RV1). The HVAC-RV1 genome is most similar to those of picorna-like viruses identified in arthropods but encodes a small domain observed only in negative-sense single-stranded RNA viruses. PMID:29567746

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

Subversion of cytokine networks by virally encoded decoy receptors

PubMed Central

Epperson, Megan L.; Lee, Chung A.; Fremont, Daved H.

2012-01-01

Summary During the course of evolution, viruses have captured or created a diverse array of open reading frames that encode for proteins that serve to evade and sabotage the host innate and adaptive immune responses, which would otherwise lead to their elimination. These viral genomes are some of the best textbooks of immunology ever written. The established arsenal of immunomodulatory proteins encoded by viruses is large and growing and includes specificities for virtually all known inflammatory pathways and targets. The focus of this review is on herpes and poxvirus-encoded cytokine and chemokine binding proteins that serve to undermine the coordination of host immune surveillance. Structural and mechanistic studies of these decoy receptors have provided a wealth of information, not only about viral pathogenesis but also about the inner workings of cytokine signaling networks. PMID:23046131

SARS-like cluster of circulating bat coronavirus pose threat for human emergence

PubMed Central

Menachery, Vineet D.; Yount, Boyd L.; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E.; Plante, Jessica A.; Graham, Rachel L.; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F.; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Shi, Zhengli-Li; Baric, Ralph S.

2016-01-01

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARS-like CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations. PMID:26552008

Novel double-stranded RNA viruses of plant-feeding insects encode a serine-alanine-proline rich protein and a polymerase distantly related to fungal viruses

USDA-ARS?s Scientific Manuscript database

Novel double stranded RNAs (~8 kbp) were isolated from the three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genome organization of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and ...

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

USDA-ARS?s Scientific Manuscript database

Novel double-stranded RNAs (~8 kbp) were isolated from three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genomes of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and Circulifer tenell...

Enhanced susceptibility of cancer cells to oncolytic rhabdo-virotherapy by expression of Nodamura virus protein B2 as a suppressor of RNA interference.

PubMed

Bastin, Donald; Aitken, Amelia S; Pelin, Adrian; Pikor, Larissa A; Crupi, Mathieu J F; Huh, Michael S; Bourgeois-Daigneault, Marie-Claude; Bell, John C; Ilkow, Carolina S

2018-06-19

Antiviral responses are barriers that must be overcome for efficacy of oncolytic virotherapy. In mammalian cells, antiviral responses involve the interferon pathway, a protein-signaling cascade that alerts the immune system and limits virus propagation. Tumour-specific defects in interferon signaling enhance viral infection and responses to oncolytic virotherapy, but many human cancers are still refractory to oncolytic viruses. Given that invertebrates, fungi and plants rely on RNA interference pathways for antiviral protection, we investigated the potential involvement of this alternative antiviral mechanism in cancer cells. Here, we detected viral genome-derived small RNAs, indicative of RNAi-mediated antiviral responses, in human cancer cells. As viruses may encode suppressors of the RNA interference pathways, we engineered an oncolytic vesicular stomatitis virus variant to encode the Nodamura virus protein B2, a known inhibitor of RNAi-mediated immune responses. B2-expressing oncolytic virus showed enhanced viral replication and cytotoxicity, impaired viral genome cleavage and altered microRNA processing in cancer cells. Our data establish the improved therapeutic potential of our novel virus which targets the RNAi-mediated antiviral defense of cancer cells.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

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

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clonesmore » prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.« less

A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.

PubMed

Menachery, Vineet D; Yount, Boyd L; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E; Plante, Jessica A; Graham, Rachel L; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F; Randell, Scott H; Lanzavecchia, Antonio; Marasco, Wayne A; Shi, Zhengli-Li; Baric, Ralph S

2015-12-01

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.

Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health.

PubMed

Chen, Yan Ping; Pettis, Jeffery S; Corona, Miguel; Chen, Wei Ping; Li, Cong Jun; Spivak, Marla; Visscher, P Kirk; DeGrandi-Hoffman, Gloria; Boncristiani, Humberto; Zhao, Yan; vanEngelsdorp, Dennis; Delaplane, Keith; Solter, Leellen; Drummond, Francis; Kramer, Matthew; Lipkin, W Ian; Palacios, Gustavo; Hamilton, Michele C; Smith, Barton; Huang, Shao Kang; Zheng, Huo Qing; Li, Ji Lian; Zhang, Xuan; Zhou, Ai Fen; Wu, Li You; Zhou, Ji Zhong; Lee, Myeong-L; Teixeira, Erica W; Li, Zhi Guo; Evans, Jay D

2014-07-01

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV-host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide.

Identification of an essential virulence gene of cyprinid herpesvirus 3.

PubMed

Boutier, Maxime; Gao, Yuan; Vancsok, Catherine; Suárez, Nicolás M; Davison, Andrew J; Vanderplasschen, Alain

2017-09-01

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Novel rod-shaped viruses isolated from garlic, Allium sativum, possessing a unique genome organization.

PubMed

Sumi, S; Tsuneyoshi, T; Furutani, H

1993-09-01

Rod-shaped flexuous viruses were partially purified from garlic plants (Allium sativum) showing typical mosaic symptoms. The genome was shown to be composed of RNA with a poly(A) tail of an estimated size of 10 kb as shown by denaturing agarose gel electrophoresis. We constructed cDNA libraries and screened four independent clones, which were designated GV-A, GV-B, GV-C and GV-D, using Northern and Southern blot hybridization. Nucleotide sequence determination of the cDNAs, two of which correspond to nearly one-third of the virus genomic RNA, shows that all of these viruses possess an identical genomic structure and that also at least four proteins are encoded in the viral cDNA, their M(r)s being estimated to be 15K, 27K, 40K and 11K. The 15K open reading frame (ORF) encodes the core-like sequence of a zinc finger protein preceded by a cluster of basic amino acid residues. The 27K ORF probably encodes the viral coat protein (CP), based on both the existence of some conserved sequences observed in many other rod-shaped or flexuous virus CPs and an overall amino acid sequence similarity to potexvirus and carlavirus CPs. The 11K ORF shows significant amino acid sequence similarities to the corresponding 12K proteins of the potexviruses and carlaviruses. On the other hand, the 40K ORF product does not resemble any other plant virus gene products reported so far. The genomic organization in the 3' region of the garlic viruses resembles, but clearly differs from, that of carlaviruses. Phylogenetic analysis based upon the amino acid sequence of the viral capsid protein also indicates that the garlic viruses have a unique and distinct domain different from those of the potexvirus and carlavirus groups. The results suggest that the garlic viruses described here belong to an unclassified and new virus group closely related to the carlaviruses.

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

PubMed

Noel, Eric A; Kang, Ming; Adamec, Jiri; Van Etten, James L; Oyler, George A

2014-12-01

The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Immunization against Small Ruminant Lentiviruses

PubMed Central

Reina, Ramsés; de Andrés, Damián; Amorena, Beatriz

2013-01-01

Multisystemic disease caused by Small Ruminant Lentiviruses (SRLV) in sheep and goats leads to production losses, to the detriment of animal health and welfare. This, together with the lack of treatments, has triggered interest in exploring different strategies of immunization to control the widely spread SRLV infection and, also, to provide a useful model for HIV vaccines. These strategies involve inactivated whole virus, subunit vaccines, DNA encoding viral proteins in the presence or absence of plasmids encoding immunological adjuvants and naturally or artificially attenuated viruses. In this review, we revisit, comprehensively, the immunization strategies against SRLV and analyze this double edged tool individually, as it may contribute to either controlling or enhancing virus replication and/or disease. PMID:23917352

Overproduction, purification, and biochemical characterization of the dual specificity H1 protein phosphatase encoded by variola major virus.

PubMed

Tropea, Joseph E; Phan, Jason; Waugh, David S

2006-11-01

Smallpox, a highly contagious infectious disease caused by the variola major virus, has an overall mortality rate of about 30%. Because there currently is no specific treatment for smallpox, and the only prevention is vaccination, there is an urgent need for the development of effective antiviral drugs. The dual specificity protein phosphatase encoded by the smallpox virus (H1) is essential for the production of infectious viral particles, making it a promising molecular target for antiviral therapeutics. Here, we report the molecular cloning, overproduction, purification, and initial biochemical characterization of H1 phosphatase, thereby paving the way for the discovery of small molecule inhibitors.

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

PubMed Central

Watters, Kelly; Inankur, Bahar; Gardiner, Jaye C.; Warrick, Jay; Sherer, Nathan M.; Yin, John

2017-01-01

ABSTRACT The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/β, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes. IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes. PMID:28179529

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression

PubMed Central

Li, Ao; Zhao, Haizhou; Lai, Qingying; Huang, Zhihong; Yuan, Meijin

2015-01-01

ABSTRACT Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein, P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited similar distribution patterns in infected cells, and coimmunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes was downregulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished from Sf9 cells transfected with the pk1 deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1 dependent. Replacement of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1 deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insights into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics. IMPORTANCE Diverse posttranslational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein, P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9's PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase, PK1, was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1 dependent. Mutation of these Ser/Thr sites reduced very late viral gene transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a code exists in the sophisticated PTM of viral protamine-like proteins and participates in viral gene transcription. PMID:25972542

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging

PubMed Central

Pocock, Ginger M.; Zimdars, Laraine L.; Yuan, Ming; Eliceiri, Kevin W.; Ahlquist, Paul; Sherer, Nathan M.

2017-01-01

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include “burst” RNA nuclear export dynamics regulated by HIV-1’s Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element–specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. PMID:27903772

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

PubMed

Sailaja, B; Anjum, Najreen; Patil, Yogesh K; Agarwal, Surekha; Malathi, P; Krishnaveni, D; Balachandran, S M; Viraktamath, B C; Mangrauthia, Satendra K

2013-12-01

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

Xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus.

PubMed

Szczepanek, Steven M; Barrette, Roger W; Rood, Debra; Alejo, Diana; Silbart, Lawrence K

2012-04-01

Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.

Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

PubMed Central

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

2012-01-01

ABSTRACT Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens. PMID:22893382

Histopathological evaluation of the diversity of cells susceptible to H5N1 virulent avian influenza virus.

PubMed

Ogiwara, Haru; Yasui, Fumihiko; Munekata, Keisuke; Takagi-Kamiya, Asako; Munakata, Tsubasa; Nomura, Namiko; Shibasaki, Futoshi; Kuwahara, Kazuhiko; Sakaguchi, Nobuo; Sakoda, Yoshihiro; Kida, Hiroshi; Kohara, Michinori

2014-01-01

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Histopathological Evaluation of the Diversity of Cells Susceptible to H5N1 Virulent Avian Influenza Virus

PubMed Central

Ogiwara, Haru; Yasui, Fumihiko; Munekata, Keisuke; Takagi-Kamiya, Asako; Munakata, Tsubasa; Nomura, Namiko; Shibasaki, Futoshi; Kuwahara, Kazuhiko; Sakaguchi, Nobuo; Sakoda, Yoshihiro; Kida, Hiroshi; Kohara, Michinori

2015-01-01

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. PMID:24200852

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides.

PubMed

Laassri, Majid; Chizhikov, Vladimir; Mikheev, Maxim; Shchelkunov, Sergei; Chumakov, Konstantin

2003-09-01

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

The Role of microRNAs in the Pathogenesis of Herpesvirus Infection.

PubMed

Piedade, Diogo; Azevedo-Pereira, José Miguel

2016-06-02

MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein-Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis.

The Role of microRNAs in the Pathogenesis of Herpesvirus Infection

PubMed Central

Piedade, Diogo; Azevedo-Pereira, José Miguel

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein–Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis. PMID:27271654

Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

NASA Astrophysics Data System (ADS)

Krupovic, Mart; Koonin, Eugene V.

2014-06-01

Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

Involvement of RNA2-encoded proteins in the specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index.

PubMed

Belin, C; Schmitt, C; Demangeat, G; Komar, V; Pinck, L; Fuchs, M

2001-12-05

The nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by the nematode Xiphinema index. To identify the RNA2-encoded proteins involved in X. index-mediated spread of GFLV, chimeric RNA2 constructs were engineered by replacing the 2A, 2B(MP), and/or 2C(CP) sequences of GFLV with their counterparts in Arabis mosaic virus (ArMV), a closely related nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index. Among the recombinant viruses obtained from transcripts of GFLV RNA1 and chimeric RNA2, only those which contained the 2C(CP) gene (504 aa) and 2B(MP) contiguous 9 C-terminal residues of GFLV were transmitted by X. index as efficiently as natural and synthetic wild-type GFLV, regardless of the origin of the 2A and 2B(MP) genes. As expected, ArMV was not transmitted probably because it is not retained by X. index. These results indicate that the determinants responsible for the specific spread of GFLV by X. index are located within the 513 C-terminal residues of the polyprotein encoded by RNA2. Copyright 2001 Elsevier Science.

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction.

PubMed

Chareza, Sarah; Slavkovic Lukic, Dragana; Liu, Yang; Räthe, Ann-Mareen; Münk, Carsten; Zabogli, Elisa; Pistello, Mauro; Löchelt, Martin

2012-03-15

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable. Copyright © 2012 Elsevier Inc. All rights reserved.

In vivo expression of human cytomegalovirus (HCMV) microRNAs during latency.

PubMed

Meshesha, Mesfin K; Bentwich, Zvi; Solomon, Semaria A; Avni, Yonat Shemer

2016-01-01

Viral encoded microRNAs play key roles in regulating gene expression and the life cycle of human herpes viruses. Latency is one of the hallmarks of the human cytomegalovirus (HCMV or HHV5) life cycle, and its control may have immense practical applications. The present study aims to identify HCMV encoded microRNAs during the latency phase of the virus. We used a highly sensitive real time PCR (RTPCR) assay that involves a pre-amplification step before RTPCR. It can detect HCMV encoded microRNAs (miRNAs) during latency in purified monocytes and PBMCs from HCMV IgG positive donors and in latently infected monocytic THP-1 cell lines. During the latency phase, only eight HCMV encoded microRNAs were detected in PBMCs, monocytes and in the THP-1 cells. Five originated from the UL region of the virus genome and three from the US region. Reactivation of the virus from latency, in monocytes obtained from the same donor, using dexamethasone restored the expression of all known HCMV encoded miRNAs including those that were absent during latency. We observed a shift in the abundance of the two arms of mir-US29 between the productive and latency stages of the viral life cycle, suggesting that the star "passenger" form of this microRNA is preferentially expressed during latency. As a whole, our study demonstrates that HCMV expresses during the latency phase, both in vivo and in vitro, only a subset of its microRNAs, which may indicate that they play an important role in maintenance and reactivation of latency. Copyright © 2015 Elsevier B.V. All rights reserved.

CYTOKINE - The Official Journal of the International Cytokine Society. Volume 11, Number 11

DTIC Science & Technology

1999-12-09

in the host. Two herpes viruses capable of infecting humans harbor homologs of IL-10. Epstein Barr virus (EBV) encoded IL-10 was the first viral DL...tumor metastasis. We have previously shown that IL4 enhances CD44 expression and hyaluronan (HA) binding ability in an Epstein Barr -vinis (EBV) positive... viruses utilize the same strategy to protect virus -infected cells from immune defenses and thus enhance, survival of virus and virus -infected cells

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

PubMed

Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

2018-03-01

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation.

PubMed

Sánchez-Navarro, Jesús A; Carmen Herranz, María; Pallás, Vicente

2006-03-01

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.

Natural anti-carbohydrate antibodies contributing to evolutionary survival of primates in viral epidemics?

PubMed

Galili, Uri

2016-11-01

Humans produce multiple natural antibodies against carbohydrate antigens on gastrointestinal bacteria. Two such antibodies appeared in primates in recent geological times. Anti-Gal, abundant in humans, apes and Old-World monkeys, appeared 20-30 million years ago (mya) following inactivation of the α1,3GT gene (GGTA1). This gene encodes in other mammals the enzyme α1,3galactosyltransferase (α1,3GT) that synthesizes α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) which bind anti-Gal. Anti-Neu5Gc, found only in humans, appeared in hominins <6 mya, following elimination of N-glycolylneuraminic-acid (Neu5Gc) because of inactivation of CMAH, the gene encoding hydroxylase that converts N-acetylneuraminic-acid (Neu5Ac) into Neu5Gc. These antibodies, were initially produced in few individuals that acquired random mutations inactivating the corresponding genes and eliminating α-gal epitopes or Neu5Gc, which became nonself antigens. It is suggested that these evolutionary selection events were induced by epidemics of enveloped viruses, lethal to ancestral Old World primates or hominins. Such viruses presented α-gal epitopes or Neu5Gc, synthesized in primates that conserved active GGTA1 or CMAH, respectively, and were lethal to their hosts. The natural anti-Gal or anti-Neu5Gc antibodies, produced in offspring lacking the corresponding carbohydrate antigens, neutralized and destroyed viruses presenting α-gal epitopes or Neu5Gc. These antibodies further induced rapid, effective immune responses against virus antigens, thus preventing infections from reaching lethal stages. These epidemics ultimately resulted in extinction of primate populations synthesizing these carbohydrate antigens and their replacement with offspring populations lacking the antigens and producing protective antibodies against them. Similar events could mediate the elimination of various carbohydrate antigens, thus preventing the complete extinction of other vertebrate species. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

PubMed

Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

2016-06-14

Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting filamentous freshwater cyanobacteria, revealing that their gene content is unlike that of other cyanophages. In addition to sharing many gene homologues with freshwater cyanobacteria, cyanophage N-1 encodes a CRISPR array and expresses it upon infection. Also, both viruses contain a DNA polymerase B-encoding gene with high similarity to genes found in proteobacterial plasmids of filamentous cyanobacteria. The observation that phages can acquire CRISPRs from their hosts suggests that phages can also move them among hosts, thereby conferring resistance to competing phages. The presence in these cyanophages of CRISPR and DNA polymerase B sequences, as well as a suite of other host-related genes, illustrates the long and complex evolutionary history of these viruses and their hosts. Copyright © 2016 Chénard et al.

Coat protein expression strategy of oat blue dwarf virus.

PubMed

Edwards, Michael C; Weiland, John J

2014-02-01

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system. Published by Elsevier Inc.

Evolution and Structural Organization of the C Proteins of Paramyxovirinae

PubMed Central

Karlin, David G.

2014-01-01

The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations. PMID:24587180

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements.

PubMed

Smith, C D; Baglia, L A; Curristin, S M; Ruddell, A

1994-10-01

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

Upregulation of voltage-gated Ca2+ channels in mouse astrocytes infected with Theiler's murine encephalomyelitis virus (TMEV).

PubMed

Rubio, N; Almanza, A; Mercado, F; Arévalo, M-Á; Garcia-Segura, L M; Vega, R; Soto, E

2013-09-05

Theiler's murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice through a CD4(+) Th1 T cell-mediated immunopathological process. TMEV infection produces a syndrome in mice that resembles multiple sclerosis. In this work, we focused on the increased expression of the genes encoding voltage-gated Ca(2+) channel subunits in SJL/J mouse astrocytes infected in culture with a BeAn strain of TMEV. Affymetrix DNA murine genome U74v2 DNA microarray hybridized with cRNA from mock- and TMEV-infected astrocytes revealed the upregulation of four sequences encoding Ca(2+)-binding and Ca(2+) channel subunit proteins. The DNA hybridization results were further validated using conventional RT-PCR and quantitative RT-PCR, demonstrating the increased expression of mRNA encoding channel subunit proteins. Western blotting also showed the increased synthesis of L- and N-type channel subunit specific proteins after infection. The reduced expression and the functional upregulation of functional voltage-gated Ca(2+) channels in mock- and TMEV-infected cells, respectively, was demonstrated using voltage clamp experiments. TMEV infection in mouse astrocytes induced a Ca(2+) current with a density proportional to the amount of viral particles used for infection. The use of Ca(2+) channel blockers, nimodipine and ω-conotoxin-GVIA, showed that both functional L- and N-type Ca(2+) channels were upregulated in infected astrocytes. The upregulation of Ca(2+) channels in astrocytes after TMEV infection provides insight into the molecular processes and potential role of astrocyte Ca(2+) dysregulation in the pathophysiology of encephalomyelitis and is important for the development of novel therapeutic strategies leading to prevention of neurodegeneration. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

The Transactivation Domain of Marek's Disease Virus (MDV) Meq Oncoprotein Does Not Affect Tumor Incidence But Plays a Role in Tumor Phenotype

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus encoded oncoprotein, Meq, is responsible for the tumorigenic phenotype of the virus. We have previously shown that replacement of the meq gene in the very virulent strain Md5 with that of vaccine strain CVI988/Rispens results in virus attenuation in chickens. To determine the...

Characterization of the Triticum Mosaic Virus Genome and Interactions between Triticum Mosaic Virus and Wheat Streak Mosaic Virus

USDA-ARS?s Scientific Manuscript database

The complete genome sequence of Triticum mosaic virus (TriMV) has been determined to be 10,266 nucleotides encoding a large polyprotein of 3,112 amino acids. The proteins of TriMV possess only 33-44% (with NIb protein) and 15-29% (with P1 protein) amino acid identity with the reported members of Pot...

Nucleotide Sequence of the Hantaan Virus S RNA Segment and Expression of Encoded Proteins

DTIC Science & Technology

1987-11-03

human vaccinia vaccination ). A second dose of virus was given in the same ...vaccinia vector. A necessary first step in vaccine investigation woul d be to determine if animals infected with the two HTV recombinant viruses can ...vaccinia virus (Buller et al., 1985). Mice were infected by tail scarification since it is identical to the method used to vaccinate 169 humans

Multiple enzyme activities of flavivirus proteins.

PubMed

Padmanabhan, R; Mueller, N; Reichert, E; Yon, C; Teramoto, T; Kono, Y; Takhampunya, R; Ubol, S; Pattabiraman, N; Falgout, B; Ganesh, V K; Murthy, K

2006-01-01

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis.

PubMed

Nakahara, Kenji S; Kitazawa, Hiroaki; Atsumi, Go; Choi, Sun Hee; Suzuki, Yuji; Uyeda, Ichiro

2011-07-18

Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

PubMed Central

Azevedo, Jacinthe; Garcia, Damien; Pontier, Dominique; Ohnesorge, Stephanie; Yu, Agnes; Garcia, Shahinez; Braun, Laurence; Bergdoll, Marc; Hakimi, Mohamed Ali; Lagrange, Thierry; Voinnet, Olivier

2010-01-01

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans. PMID:20439431

Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

PubMed

Farré, Domènec; Martínez-Vicente, Pablo; Engel, Pablo; Angulo, Ana

2017-05-01

Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multicolor quantum dot-encoded microspheres for the fluoroimmunoassays of chicken newcastle disease and goat pox virus.

PubMed

Yuan, Pingfan; Ma, Qiang; Meng, Rizeng; Wang, Chao; Dou, Wenchao; Wang, Guannan; Su, Xingguang

2009-05-01

Semiconductor nanocrystals (or quantum dots, QDs) have the potential to overcome some of the limitations encountered by traditional fluorophores in fluorescence labeling applications. The unique spectroscopic properties of QDs make them hold immense promise as versatile labels for biological applications. In this work, we employ the layer-by-layer (LbL) method for the construction of bio-functional multicolor QD-encoded microspheres. Polystyrene microspheres with diameter of 3 microm were used as templates for the deposition of different sized CdTe QDs/polyelectrolyte multilayers. Two different antigens, Chicken newcastle disease (CND) antigen and goat pox virus (GPV) antigen, were conjugated to two kinds of biofunctional multicolor microspheres with different optical encoding. The multicolor microspheres can capture corresponding antibodies labeled with QDs, QDs-CND antibody and QDs-GPV antibody in the fluoroimmunoassays. The microspheres can be distinguished from each other based on their optical encoding.

Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.

PubMed

Das, Subash C; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K

2009-03-01

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-DeltaM-Mtc and VSV-DeltaM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-DeltaM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-DeltaM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses

USDA-ARS?s Scientific Manuscript database

Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four short open reading frames in its 5' one third, three of which encode proteins with homology to picornavirus-like jelly-r...

Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

PubMed

Kalveram, Birte; Ikegami, Tetsuro

2013-04-01

Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

PubMed

Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

2016-11-29

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

A novel totivirus-like virus isolated from bat guano.

PubMed

Yang, Xinglou; Zhang, Yunzhi; Ge, Xingyi; Yuan, Junfa; Shi, Zhengli

2012-06-01

Previous metagenomic analysis indicated that numerous insect viruses exist in bat guano. In this study, we isolated a novel double-stranded RNA virus, a tentative member of the family Totiviridae, designated Tianjin totivirus (ToV-TJ), from bat feces. The virus is an icosahedral particle with a diameter of 40-43 nm, and it causes cytopathic effect in Sf9, Hz, and C6/36 cell lines. Full-length genomic sequence analysis showed that ToV-TJ shares high similarity with the totivirus OMRV-AK4, which was recently isolated from mosquitoes in Japan. The full-length genome of the ToV-TJ was 7611 bp and contained two predicted non-overlapping open reading frames (ORFs): ORF1, encoding the capsid protein (CP), and ORF2, encoding an RNA-dependent RNA polymerase. Bioassay of ToV-TJ by feeding on the larvae of Spodoptera exigua and Helicoverpa armigera (Hubner) suggests that this virus is not infectious for these two larvae in vivo. Sequences similar to that of ToV-TJ have been detected in bat feces sampled in Yunnan and Hainan Provinces, suggesting that this virus is widely distributed.

Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery.

PubMed

Kaulfuß, Meike; Wensing, Ina; Windmann, Sonja; Hrycak, Camilla Patrizia; Bayer, Wibke

2017-02-06

In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines that were designed to induce either strong Friend virus GagL 85-93 -specific CD8 + T cell or antibody responses, respectively. To optimize the immunization outcome we evaluated vaccination strategies using combinations of these vaccines. While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine effectivity. We demonstrate that the co-immunization with GagL 85-93 /leader-gag encoding vectors together with envelope-encoding vectors abrogates the induction of GagL 85-93 -specific CD8 + T cells, and in successive immunization protocols the immunization with the GagL 85-93 /leader-gag encoding vector had to precede the immunization with an envelope encoding vector for the efficient induction of GagL 85-93 -specific CD8 + T cells. Importantly, the antibody response to envelope was in fact enhanced when the mice were adenovirus-experienced from a prior immunization, highlighting the expedience of this approach. To circumvent the immunosuppressive effect of envelope on immune responses to simultaneously or subsequently administered immunogens, we developed a two immunizations-based vaccination protocol that induces strong immune responses and confers robust protection of highly Friend virus-susceptible mice from a lethal Friend virus challenge.

Persistence of Epstein-Barr virus in self-reactive memory B cells.

PubMed

Tracy, Sean I; Kakalacheva, Kristina; Lünemann, Jan D; Luzuriaga, Katherine; Middeldorp, Jaap; Thorley-Lawson, David A

2012-11-01

Epstein-Barr virus infection has been epidemiologically associated with the development of multiple autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis. Currently, there is no known mechanism that can account for these associations. The germinal-center (GC) model of EBV infection and persistence proposes that EBV gains access to the memory B cell compartment via GC reactions by driving infected cells to differentiate using the virus-encoded LMP1 and LMP2a proteins, which act as functional homologues of CD40 and the B cell receptor, respectively. The ability of LMP2a, when expressed in mice, to allow escape of autoreactive B cells suggests that it could perform a similar role in infected GC B cells, permitting the survival of potentially pathogenic autoreactive B cells. To test this hypothesis, we cloned and expressed antibodies from EBV(+) and EBV(-) memory B cells present during acute infection and profiled their self- and polyreactivity. We find that EBV does persist within self- and polyreactive B cells but find no evidence that it favors the survival of pathogenic autoreactive B cells. On the contrary, EBV(+) memory B cells express lower levels of self-reactive and especially polyreactive antibodies than their uninfected counterparts do. Our work suggests that EBV has only a modest effect on the GC process, which allows it to access and persist within a subtly unique niche of the memory compartment characterized by relatively low levels of self- and polyreactivity. We suggest that this might reflect an active process where EBV and its human host have coevolved so as to minimize the virus's potential to contribute to autoimmune disease.

Domain Organization and Evolution of the Highly Divergent 5′ Coding Region of Genomes of Arteriviruses, Including the Novel Possum Nidovirus

PubMed Central

Gulyaeva, Anastasia; Hoogendoorn, Erik; Giles, Julia; Samborskiy, Dmitry

2017-01-01

ABSTRACT In five experimentally characterized arterivirus species, the 5′-end genome coding region encodes the most divergent nonstructural proteins (nsp's), nsp1 and nsp2, which include papain-like proteases (PLPs) and other poorly characterized domains. These are involved in regulation of transcription, polyprotein processing, and virus-host interaction. Here we present results of a bioinformatics analysis of this region of 14 arterivirus species, including that of the most distantly related virus, wobbly possum disease virus (WPDV), determined by a modified 5′ rapid amplification of cDNA ends (RACE) protocol. By combining profile-profile comparisons and phylogeny reconstruction, we identified an association of the four distinct domain layouts of nsp1-nsp2 with major phylogenetic lineages, implicating domain gain, including duplication, and loss in the early nsp1 evolution. Specifically, WPDV encodes highly divergent homologs of PLP1a, PLP1b, PLP1c, and PLP2, with PLP1a lacking the catalytic Cys residue, but does not encode nsp1 Zn finger (ZnF) and “nuclease” domains, which are conserved in other arteriviruses. Unexpectedly, our analysis revealed that the only catalytically active nsp1 PLP of equine arteritis virus (EAV), known as PLP1b, is most similar to PLP1c and thus is likely to be a PLP1b paralog. In all non-WPDV arteriviruses, PLP1b/c and PLP1a show contrasting patterns of conservation, with the N- and C-terminal subdomains, respectively, being enriched with conserved residues, which is indicative of different functional specializations. The least conserved domain of nsp2, the hypervariable region (HVR), has its size varied 5-fold and includes up to four copies of a novel PxPxPR motif that is potentially recognized by SH3 domain-containing proteins. Apparently, only EAV lacks the signal that directs −2 ribosomal frameshifting in the nsp2 coding region. IMPORTANCE Arteriviruses comprise a family of mammalian enveloped positive-strand RNA viruses that include some of the most economically important pathogens of swine. Most of our knowledge about this family has been obtained through characterization of viruses from five species: Equine arteritis virus, Simian hemorrhagic fever virus, Lactate dehydrogenase-elevating virus, Porcine respiratory and reproductive syndrome virus 1, and Porcine respiratory and reproductive syndrome virus 2. Here we present the results of comparative genomics analyses of viruses from all known 14 arterivirus species, including the most distantly related virus, WPDV, whose genome sequence was completed in this study. Our analysis focused on the multifunctional 5′-end genome coding region that encodes multidomain nonstructural proteins 1 and 2. Using diverse bioinformatics techniques, we identified many patterns of evolutionary conservation that are specific to members of distinct arterivirus species, both characterized and novel, or their groups. They are likely associated with structural and functional determinants important for virus replication and virus-host interaction. PMID:28053107

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

PubMed Central

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

2017-01-01

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

A Recombinant Rabies Virus Encoding Two Copies of the Glycoprotein Gene Confers Protection in Dogs against a Virulent Challenge

PubMed Central

Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F.; Niu, Xuefeng; Guo, Xiaofeng

2014-01-01

The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines PMID:24498294

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Ohkawa, T; Majima, K; Maeda, S

1994-01-01

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

PubMed

Kuhl, U; Lassner, D; Dorner, A; Rohde, M; Escher, F; Seeberg, B; Hertel, E; Tschope, C; Skurk, C; Gross, U M; Schultheiss, H-P; Poller, W

2013-09-01

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

PubMed

van Kasteren, Puck B; Bailey-Elkin, Ben A; James, Terrence W; Ninaber, Dennis K; Beugeling, Corrine; Khajehpour, Mazdak; Snijder, Eric J; Mark, Brian L; Kikkert, Marjolein

2013-02-26

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

Ebolavirus comparative genomics

DOE PAGES

Jun, Se-Ran; Leuze, Michael R.; Nookaew, Intawat; ...

2015-07-14

The 2014 Ebola outbreak in West Africa is the largest documented for this virus. We examine the dynamics of this genome, comparing more than one hundred currently available ebolavirus genomes to each other and to other viral genomes. Based on oligomer frequency analysis, the family Filoviridae forms a distinct group from all other sequenced viral genomes. All filovirus genomes sequenced to date encode proteins with similar functions and gene order, although there is considerable divergence in sequences between the three genera Ebolavirus, Cuevavirus, and Marburgvirus within the family Filoviridae. Whereas all ebolavirus genomes are quite similar (multiple sequences of themore » same strain are often identical), variation is most common in the intergenic regions and within specific areas of the genes encoding the glycoprotein (GP), nucleoprotein (NP), and polymerase (L). We predict regions that could contain epitope-binding sites, which might be good vaccine targets. In conclusion, this information, combined with glycosylation sites and experimentally determined epitopes, can identify the most promising regions for the development of therapeutic strategies.« less

Neuronal network imaging in acute slices using Ca2+ sensitive bioluminescent reporter.

PubMed

Tricoire, Ludovic; Lambolez, Bertrand

2014-01-01

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.

Protection of Chickens against Avian Influenza with Non-Replicating Adenovirus-Vectored Vaccine

PubMed Central

Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Shi, Z.

2009-01-01

Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding an H7 AI hemagglutinin (AdChNY94.H7). Chickens vaccinated in ovo with an Ad vector encoding an AI H5 (AdTW68.H5) previously described, which were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Antibody responses to Ad vector in ovo vaccination follow a dose-response kinetic. The use of a synthetic AI H5 gene codon optimized to match the chicken cell tRNA pool was more potent than the cognate H5 gene. The use of Ad-vectored vaccines to increase resistance of chicken populations against multiple AI strains could reduce the risk of an avian-originating influenza pandemic in humans. PMID:18384919

Identification of herpes simplex virus type 1 proteins encoded within the first 1.5 kb of the latency-associated transcript.

PubMed

Henderson, Gail; Jaber, Tareq; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

2009-09-01

Expression of the first 1.5 kb of the latency-associated transcript (LAT) that is encoded by herpes simplex virus type 1 (HSV-1) is sufficient for wild-type (wt) levels of reactivation from latency in small animal models. Peptide-specific immunoglobulin G (IgG) was generated against open reading frames (ORFs) that are located within the first 1.5 kb of LAT coding sequences. Cells stably transfected with LAT or trigeminal ganglionic neurons of mice infected with a LAT expressing virus appeared to express the L2 or L8 ORF. Only L2 ORF expression was readily detected in trigeminal ganglionic neurons of latently infected mice.

A Survey of Antiviral Drugs for Bioweapons: Review

DTIC Science & Technology

2005-01-01

person . An attack with these viruses would result in high morbidity and mortality and cause widespread panic. With the exception of smallpox and...infected cells and are not dependent upon the host cell nucleus. Possible targets for these viruses are the DNA polymerase, virus -encoded immune modulators... person to person . An attack with these viruses would result in high morbidity and mortality and cause widespread panic. With the

Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines

PubMed Central

Yao, Yongxiu; Mingay, Louise J.; McCauley, John W.; Barclay, Wendy S.

2001-01-01

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells. PMID:11333926

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

PubMed

Yadav, Pragya D; Shete, Anita M; Nyayanit, Dimpal A; Albarino, Cesar G; Jain, Shilpi; Guerrero, Lisa W; Kumar, Sandeep; Patil, Deepak Y; Nichol, Stuart T; Mourya, Devendra T

2018-06-25

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.

Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63.

PubMed Central

Kinchington, P R; Vergnes, J P; Defechereux, P; Piette, J; Turse, S E

1994-01-01

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes. Images PMID:8189496

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Bromovirus movement protein genes play a crucial role in host specificity.

PubMed Central

Mise, K; Allison, R F; Janda, M; Ahlquist, P

1993-01-01

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small. Images PMID:7682628

Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

PubMed

Phillpotts, R J; Lescott, T L; Jacobs, S C

2000-10-01

Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.

Multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children

PubMed Central

Martin, Emily T.; Kuypers, Jane; Wald, Anna; Englund, Janet A.

2011-01-01

Please cite this paper as: Martin et al. (2012) Multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children. Influenza and Other Respiratory Viruses 6(1), 71–77. Background  Molecular testing for viral pathogens has resulted in increasing detection of multiple viruses in respiratory secretions of ill children. The clinical impact of multiple virus infections on clinical presentation and outcome is unclear. Objectives  To compare clinical characteristics and viral load between children with multiple virus versus single virus illnesses. Patients/methods  Eight hundred and ninety‐three residual nasal wash samples from children treated for respiratory illness at Children’s Hospital, Seattle, from September 2003 to September 2004 were evaluated by quantitative PCR for respiratory syncytial virus (RSV), human metapneumovirus (hMPV), influenza (Flu), parainfluenza, adenoviruses, and coronaviruses (CoV). Illness severity and patient characteristics were abstracted from medical charts. Results  Coinfections were identified in 103 (18%) of 566 virus‐positive samples. Adenovirus was most commonly detected in coinfections (52%), followed by CoV (50%). Illnesses with a single virus had increased risk of oxygen requirement (P = 0·02), extended hospital stays (P = 0·002), and admissions to the inpatient (P = 0·02) or intensive care units (P = 0·04). For Adv and PIV‐1, multiple virus illnesses had a significantly lower viral load (log10 copies/ml) than single virus illnesses (4·2 versus 5·6, P = 0·007 and 4·2 versus 6·9, P < 0·001, respectively). RSV, Flu‐A, PIV‐3, and hMPV viral loads were consistently high whether or not another virus was detected. Conclusions  Illnesses with multiple virus detections were correlated with less severe disease. The relationship between viral load and multiple virus infections was virus specific, and this may serve as a way to differentiate viruses in multiple virus infections. PMID:21668660

Intranasal vaccination with replication defective adenovirus-5 encoding influenza hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs

USDA-ARS?s Scientific Manuscript database

Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic loss. To combat IAV infection the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines. These vaccines can provide sterilizing immunity towards homologous virus but often have l...

Rift Valley fever virus incorporates the 78kDa glycoprotein into virions matured in C6/36 2 mosquito cells

USDA-ARS?s Scientific Manuscript database

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment codi...

Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus

USDA-ARS?s Scientific Manuscript database

The complete genome sequence of a virus recently detected in switchgrass (Panicum virgatum) was determined and was found to be closely related to Maize rayado fino virus (MRFV), genus Marafivirus, family Tymoviridae. The genomic RNA is 6408 nucleotides long, excluding the poly (A) tail, and encodes...

Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex.

PubMed

Dong, Zhan-Qi; Zhang, Jun; Chen, Xue-Mei; He, Qian; Cao, Ming-Ya; Wang, La; Li, Hai-Qing; Xiao, Wen-Fu; Pan, Cai-Xia; Lu, Cheng; Pan, Min-Hui

2014-05-12

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF79 (Bm79) encodes an occlusion-derived virus (ODV)-specific envelope protein, which is a homologue of the per os infectivity factor 4 (PIF4) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To investigate the role of ORF79 in the BmNPV life cycle, a Bm79 knockout virus (vBm(Bm79KO)) was constructed through homologous recombination in Escherichia coli. Viral DNA replication, budded virus (BV) production and polyhedra formation were unaffected by the absence of BM79. However, results of the larval bioassay demonstrated that the Bm79 deletion resulted in a complete loss of per os infection. Immunofluorescence analysis showed that BM79 localized at the innernuclear membrane of infected cells through its N-terminal sorting motif (SM). Further bimolecular fluorescence protein complementation and co-immunoprecipitation assays demonstrated the interaction of BM79 with PIF1, PIF2, PIF3 and ODV-E66. Thus, BM79 plays an important role in per os infection and is associated with the viral PIF complex of BmNPV. Copyright © 2014 Elsevier B.V. All rights reserved.

Isolation and Characterization of Metallosphaera turreted icosahedral virus (MTIV), a founding member of a new family of archaeal viruses.

PubMed

Wagner, Cassia; Reddy, Vijay; Asturias, Francisco; Khoshouei, Maryam; Johnson, John E; Manrique, Pilar; Munson-McGee, Jacob; Baumeister, Wolfgang; Lawrence, C Martin; Young, Mark J

2017-08-02

Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis Each strain encodes for a 9.8-9.9 kb, linear dsDNA genome with large inverted terminal repeats. Each genome encodes for 21 ORFs. Between the strains the ORFs display high homology, but they are quite distinct from other known viral genes. The 70-nm diameter virion is built upon on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryo-tomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 from each of the 5-fold axes and 30 hexameric units positioned on icosahedral 2-fold axes. Both the virion structural properties and genome content support MTIV as the founding member of a new family of archaeal viruses. Importance: Many archaeal viruses are quite different than viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group of viruses. As the first known virus infecting the Metallosphaera , MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution. Copyright © 2017 American Society for Microbiology.

[The great virus comeback].

PubMed

Forterre, Patrick

2013-01-01

Viruses have been considered for a long time as by-products of biological evolution. This view is changing now as a result of several recent discoveries. Viral ecologists have shown that viral particles are the most abundant biological entities on our planet, whereas metagenomic analyses have revealed an unexpected abundance and diversity of viral genes in the biosphere. Comparative genomics have highlighted the uniqueness of viral sequences, in contradiction with the traditional view of viruses as pickpockets of cellular genes. On the contrary, cellular genomes, especially eukaryotic ones, turned out to be full of genes derived from viruses or related elements (plasmids, transposons, retroelements and so on). The discovery of unusual viruses infecting archaea has shown that the viral world is much more diverse than previously thought, ruining the traditional dichotomy between bacteriophages and viruses. Finally, the discovery of giant viruses has blurred the traditional image of viruses as small entities. Furthermore, essential clues on virus history have been obtained in the last ten years. In particular, structural analyses of capsid proteins have uncovered deeply rooted homologies between viruses infecting different cellular domains, suggesting that viruses originated before the last universal common ancestor (LUCA). These studies have shown that several lineages of viruses originated independently, i.e., viruses are polyphyletic. From the time of LUCA, viruses have coevolved with their hosts, and viral lineages can be viewed as lianas wrapping around the trunk, branches and leaves of the tree of life. Although viruses are very diverse, with genomes encoding from one to more than one thousand proteins, they can all be simply defined as organisms producing virions. Virions themselves can be defined as infectious particles made of at least one protein associated with the viral nucleic acid, endowed with the capability to protect the viral genome and ensure its delivery to the infected cell. These definitions, which clearly distinguish viruses from plasmids, suggest that infectious RNA molecules that only encode an RNA replicase presently classified among viruses by the ICTV (International Committee for the Taxonomy of Viruses) into families of Endornaviridae and Hypoviridae are in fact RNA plasmids. Since a viral genome should encode for at least one structural protein, these definitions also imply that viruses originated after the emergence of the ribosome in an RNA-protein cellular world. Although virions are the hallmarks of viruses, viruses and virions should not be confused. The infection transforms the ribocell (cell encoding ribosomes and dividing by binary fission) into a virocell (cell producing virions) or ribovirocell (cell that produces virions but can still divide by binary fission). In the ribovirocell, two different organisms, defined by their distinct evolutionary histories, coexist in symbiosis in the same cell. The virocells or ribovirocells are the living forms of the virus, which can be in fine considered to be a living organism. In the virocell, the metabolism is reorganized for the production of virions, while the ability to capture and store free energy is retained, as in other cellular organisms. In the virocell, viral genomes replicate, recombine and evolve, leading to the emergence of new viral proteins and potentially novel functions. Some of these new functions can be later on transferred to the cell, explaining how viruses can play a major (often underestimated) role in the evolution of cellular organisms. The virocell concept thus helps to understand recent hypotheses suggesting that viruses played a critical role in major evolutionary transitions, such as the origin of DNA genomes or else the origin of the eukaryotic nucleus. Finally, it is more and more recognized that viruses are the major source of variation and selection in living organisms (both viruses and cells), the two pillars of darwinism. One can thus conclude that the continuous interaction between viruses and cells, all along the history of life, has been, and still is, a major engine of biological evolution. © Société de Biologie, 2013.

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

PubMed Central

Manley, N R; O'Connell, M A; Sharp, P A; Hopkins, N

1989-01-01

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements. Images PMID:2778872

Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice.

PubMed

Liu, Shan; Jackson, Andrew; Beloor, Jagadish; Kumar, Priti; Sutton, Richard E

2015-09-01

Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.

Emergence of a deviating genotype VI pigeon paramyxovirus type-1 isolated from India.

PubMed

Ganar, Ketan; Das, Moushumee; Raut, Ashwin Ashok; Mishra, Anamika; Kumar, Sachin

2017-07-01

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1 (APMV-1), which infects pigeons. The virus causes high morbidity and mortality, creating an alarming state for the poultry industry. The present work describes the molecular and pathogenic characterization of a PPMV-1 strain isolated from pigeon in Bhopal, India. Complete genome sequence analysis revealed a genome of 15,192 nucleotides encoding six genes organized in the order 3'-N-P-M-F-HN-L-5'. The fusion gene sequence analysis showed the presence of multiple basic amino acids 112 R-R-Q-K-R-F 117 at the cleavage site corresponding to pathogenic strains. The mean death time and intracerebral pathogenicity index values indicated a mesogenic nature for the PPMV-1 isolate. On phylogenetic analysis, the strain clustered with genotype VI viruses, including isolates from pigeon and dove. The Bhopal strain showed significant intra and inter-genotype evolutionary distance, suggesting the emergence of a new sub-genotype, VIj.

Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere.

PubMed

Abrahão, Jônatas; Silva, Lorena; Silva, Ludmila Santos; Khalil, Jacques Yaacoub Bou; Rodrigues, Rodrigo; Arantes, Thalita; Assis, Felipe; Boratto, Paulo; Andrade, Miguel; Kroon, Erna Geessien; Ribeiro, Bergmann; Bergier, Ivan; Seligmann, Herve; Ghigo, Eric; Colson, Philippe; Levasseur, Anthony; Kroemer, Guido; Raoult, Didier; La Scola, Bernard

2018-02-27

Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44-1.51 Mb linear double-strand DNA coding for 1276-1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.

The roles of ebolavirus glycoproteins in viral pathogenesis.

PubMed

Ning, Yun-Jia; Deng, Fei; Hu, Zhihong; Wang, Hualin

2017-02-01

Ebolaviruses are highly dangerous pathogens exhibiting extreme virulence in humans and nonhuman primates. The majority of ebolavirus species, most notably Zaire ebolavirus, can cause Ebola virus disease (EVD), formerly known as Ebola hemorrhagic fever, in humans. EVD is associated with case-fatality rates as high as 90%, and there is currently no specific treatment or licensed vaccine available against EVD. Understanding the molecular biology and pathogenesis of ebolaviruses is important for the development of antiviral therapeutics. Ebolavirus encodes several forms of glycoproteins (GPs), which have some interesting characteristics, including the transcriptional editing coding strategy and extensive O-glycosylation modification, clustered in the mucin-like domain of GP1, full-length GP (GP 1,2 ), and shed GP. In addition to the canonical role of the spike protein, GP 1,2 , in viral entry, ebolavirus GPs appear to have multiple additional functions, likely contributing to the complex pathogenesis of the virus. Here, we review the roles of ebolavirus GPs in viral pathogenesis.

Israeli Acute Paralysis Virus: Epidemiology, Pathogenesis and Implications for Honey Bee Health

PubMed Central

Chen, Yan Ping; Pettis, Jeffery S.; Corona, Miguel; Chen, Wei Ping; Li, Cong Jun; Spivak, Marla; Visscher, P. Kirk; DeGrandi-Hoffman, Gloria; Boncristiani, Humberto; Zhao, Yan; vanEngelsdorp, Dennis; Delaplane, Keith; Solter, Leellen; Drummond, Francis; Kramer, Matthew; Lipkin, W. Ian; Palacios, Gustavo; Hamilton, Michele C.; Smith, Barton; Huang, Shao Kang; Zheng, Huo Qing; Li, Ji Lian; Zhang, Xuan; Zhou, Ai Fen; Wu, Li You; Zhou, Ji Zhong; Lee, Myeong-L.; Teixeira, Erica W.; Li, Zhi Guo; Evans, Jay D.

2014-01-01

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV–host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide. PMID:25079600

Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

PubMed

Howe, J G; Shu, M D

1988-08-01

Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

PubMed

Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

2014-02-13

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular and phylogenetic characterization of honey bee viruses, Nosema microsporidia, protozoan parasites, and parasitic mites in China.

PubMed

Yang, Bu; Peng, Guangda; Li, Tianbang; Kadowaki, Tatsuhiko

2013-02-01

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana-infecting SBV, and relatively homogenous populations of IAPV and A. meliifera-infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast-encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.

Effect of cytokine-encoding plasmid delivery on immune response to Japanese encephalitis virus DNA vaccine in mice.

PubMed

Bharati, Kaushik; Appaiahgari, Mohan Babu; Vrati, Sudhanshu

2005-01-01

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti-JEV humoral and cellular immune responses. We now show that intra-muscular co-administration of mice with pMEa and pGM-CSF, encoding murine granulocyte-macrophage colony-stimulating factor or pIL-2, encoding murine interleukin-2 given 4 days after pMEa, augmented anti-JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra-dermal co-administration of pMEa and pGM-CSF in mice using the gene gun, enhanced anti-JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.

Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector.

PubMed Central

Elfassi, E; Haseltine, W A; Dienstag, J L

1986-01-01

The genome of the hepatitis B virus (HBV) contains a sequence, designated X, capable of encoding a protein of 154 amino acids. To determine whether the putative protein synthesized from this region is antigenic, we examined the sera of HBV-infected patients for the ability to react with a hybrid protein that contained 133 amino acids encoded by the X region and portions of the bacterial ompF and beta-galactosidase genes. Some HBV-positive sera tested contained antibodies that specifically recognized the hybrid protein. All sera were from patients diagnosed as suffering from chronic active hepatitis. We conclude that the X region of HBV encodes a protein and that this protein is antigenic in some patients. Images PMID:3515347

Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.

PubMed

Sakaguchi, M; Urakawa, T; Hirayama, Y; Miki, N; Yamamoto, M; Zhu, G S; Hirai, K

1993-07-01

The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.

Virus versus Host Plant MicroRNAs: Who Determines the Outcome of the Interaction?

PubMed Central

Maghuly, Fatemeh; Ramkat, Rose C.; Laimer, Margit

2014-01-01

Considering the importance of microRNAs (miRNAs) in the regulation of essential processes in plant pathogen interactions, it is not surprising that, while plant miRNA sequences counteract viral attack via antiviral RNA silencing, viruses in turn have developed antihost defense mechanisms blocking these RNA silencing pathways and establish a counter-defense. In the current study, computational and stem-loop Reverse Transcription – Polymerase Chain Reaction (RT-PCR) approaches were employed to a) predict and validate virus encoded mature miRNAs (miRs) in 39 DNA-A sequences of the bipartite genomes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) isolates, b) determine whether virus encoded miRs/miRs* generated from the 5′/3′ harpin arms have the capacity to bind to genomic sequences of the host plants Jatropha or cassava and c) investigate whether plant encoded miR/miR* sequences have the potential to bind to the viral genomes. Different viral pre-miRNA hairpin sequences and viral miR/miR* length variants occurring as isomiRs were predicted in both viruses. These miRNAs were located in three Open Reading Frames (ORFs) and in the Intergenic Region (IR). Moreover, various target genes for miRNAs from both viruses were predicted and annotated in the host plant genomes indicating that they are involved in biotic response, metabolic pathways and transcription factors. Plant miRs/miRs* from conserved and highly expressed families were identified, which were shown to have potential targets in the genome of both begomoviruses, representing potential plant miRNAs mediating antiviral defense. This is the first assessment of predicted viral miRs/miRs* of ACMV and EACMV-UG and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in Euphorbiaceous crop plants. PMID:24896088

Isolation of a novel Rhabdovirus from an insectivorous bat (Pipistrellus kuhlii) in Italy.

PubMed

Lelli, Davide; Prosperi, Alice; Moreno, Ana; Chiapponi, Chiara; Gibellini, Anna Maria; De Benedictis, Paola; Leopardi, Stefania; Sozzi, Enrica; Lavazza, Antonio

2018-02-17

Rhabdoviridae is one of the most ecologically diverse families of RNA viruses which can infect a wide range of vertebrates and invertebrates. Bats, among mammals, are pointed to harbor a significantly higher proportion of unknown or emerging viruses with zoonotic potential. Herein, we report the isolation of a novel rhabdovirus, detected in the framework of a virological survey on bats implemented in North Italy. Virus isolation and identification were performed on samples of 635 bats by using cell cultures, negative staining electron microscopy and PCRs for different viruses. NGS was commonly performed on cell culture supernatants showing cytopathic effect or in case of samples resulted positive by at least one of the PCRs included in the diagnostic protocol. A rhabdovirus was isolated from different organs of a Pipistrellus kuhlii. Virus identification was obtained by electron microscopy and NGS sequencing. The complete genome size was 11,774 nt comprised 5 genes, encoding the canonical rhabdovirus structural proteins, and an additional transcriptional unit (U1) encoding a hypothetical small protein (157aa) (3'-N-P-M-G-U1-L-5'). The genome organization and phylogenetic analysis suggest that the new virus, named Vaprio virus (VAPV), belongs to the recently established genus Ledantevirus (subgroup B) and it is highly divergent to its closest known relative, Le Dantec virus (LDV) (human, 1965 Senegal). A specific RT-PCR amplifying a 350 bp fragment of the ORF 6 gene, encoding for L protein, was developed and used to test retrospectively a subset of 76 bats coming from the same area and period, revealing two more VAPV positive bats. VAPV is a novel isolate of chiropteran rhabdovirus. Genome organization and phylogenetic analyses demonstrated that VAPV should be considered a novel species within the genus Ledantevirus for which viral ecology and disease associations should be investigated.

A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production.

PubMed

Geisler, Christoph

2018-02-07

Adventitious viral contamination in cell substrates used for biologicals production is a major safety concern. A powerful new approach that can be used to identify adventitious viruses is a combination of bioinformatics tools with massively parallel sequencing technology. Typically, this involves mapping or BLASTN searching individual reads against viral nucleotide databases. Although extremely sensitive for known viruses, this approach can easily miss viruses that are too dissimilar to viruses in the database. Moreover, it is computationally intensive and requires reference cell genome databases. To avoid these drawbacks, we set out to develop an alternative approach. We reasoned that searching genome and transcriptome assemblies for adventitious viral contaminants using TBLASTN with a compact viral protein database covering extant viral diversity as the query could be fast and sensitive without a requirement for high performance computing hardware. We tested our approach on Spodoptera frugiperda Sf-RVN, a recently isolated insect cell line, to determine if it was contaminated with one or more adventitious viruses. We used Illumina reads to assemble the Sf-RVN genome and transcriptome and searched them for adventitious viral contaminants using TBLASTN with our viral protein database. We found no evidence of viral contamination, which was substantiated by the fact that our searches otherwise identified diverse sequences encoding virus-like proteins. These sequences included Maverick, R1 LINE, and errantivirus transposons, all of which are common in insect genomes. We also identified previously described as well as novel endogenous viral elements similar to ORFs encoded by diverse insect viruses. Our results demonstrate TBLASTN searching massively parallel sequencing (MPS) assemblies with a compact, manually curated viral protein database is more sensitive for adventitious virus detection than BLASTN, as we identified various sequences that encoded virus-like proteins, but had no similarity to viral sequences at the nucleotide level. Moreover, searches were fast without requiring high performance computing hardware. Our study also documents the enhanced biosafety profile of Sf-RVN as compared to other Sf cell lines, and supports the notion that Sf-RVN is highly suitable for the production of safe biologicals.

Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection

PubMed Central

Yamazaki, Tatsuya; Nagashima, Maria; Ninomiya, Daisuke; Ainai, Akira; Fujimoto, Akira; Ichimonji, Isao; Takagi, Hidekazu; Morita, Naoko; Murotani, Kenta; Hasegawa, Hideki; Chiba, Joe; Akashi-Takamura, Sachiko

2018-01-01

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection. PMID:29416543

VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets.

PubMed

Qureshi, Abid; Thakur, Nishant; Monga, Isha; Thakur, Anamika; Kumar, Manoj

2014-01-01

Viral microRNAs (miRNAs) regulate gene expression of viral and/or host genes to benefit the virus. Hence, miRNAs play a key role in host-virus interactions and pathogenesis of viral diseases. Lately, miRNAs have also shown potential as important targets for the development of novel antiviral therapeutics. Although several miRNA and their target repositories are available for human and other organisms in literature, but a dedicated resource on viral miRNAs and their targets are lacking. Therefore, we have developed a comprehensive viral miRNA resource harboring information of 9133 entries in three subdatabases. This includes 1308 experimentally validated miRNA sequences with their isomiRs encoded by 44 viruses in viral miRNA ' VIRMIRNA: ' and 7283 of their target genes in ' VIRMIRTAR': . Additionally, there is information of 542 antiviral miRNAs encoded by the host against 24 viruses in antiviral miRNA ' AVIRMIR': . The web interface was developed using Linux-Apache-MySQL-PHP (LAMP) software bundle. User-friendly browse, search, advanced search and useful analysis tools are also provided on the web interface. VIRmiRNA is the first specialized resource of experimentally proven virus-encoded miRNAs and their associated targets. This database would enhance the understanding of viral/host gene regulation and may also prove beneficial in the development of antiviral therapeutics. Database URL: http://crdd.osdd.net/servers/virmirna. © The Author(s) 2014. Published by Oxford University Press.

The Bovine Herpesvirus 4 Bo10 Gene Encodes a Nonessential Viral Envelope Protein That Regulates Viral Tropism through both Positive and Negative Effects▿

PubMed Central

Machiels, Bénédicte; Lété, Céline; de Fays, Katalin; Mast, Jan; Dewals, Benjamin; Stevenson, Philip G.; Vanderplasschen, Alain; Gillet, Laurent

2011-01-01

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG+) cells and more infectious for GAG-negative (GAG−) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21+ cell infection and inhibits CD21− cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor. PMID:21068242

Viral repression of fungal pheromone precursor gene expression.

PubMed

Zhang, L; Baasiri, R A; Van Alfen, N K

1998-02-01

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a G alpha(i) subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete.

Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

PubMed Central

Quemin, Emmanuelle R. J.; Pietilä, Maija K.; Oksanen, Hanna M.; Forterre, Patrick; Rijpstra, W. Irene C.; Schouten, Stefan; Bamford, Dennis H.; Prangishvili, David

2015-01-01

ABSTRACT Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions. PMID:26355093

Inhibition of Type 1 Cytokine–mediated Inflammation by a Soluble CD30 Homologue Encoded by Ectromelia (Mousepox) Virus

PubMed Central

Saraiva, Margarida; Smith, Philip; Fallon, Padraic G.; Alcami, Antonio

2002-01-01

CD30 is up-regulated in several human diseases and viral infections but its role in immune regulation is poorly understood. Here, we report the expression of a functional soluble CD30 homologue, viral CD30 (vCD30), encoded by ectromelia (mousepox) virus, a poxvirus that causes a severe disease related to human smallpox. We show that vCD30 is a 12-kD secreted protein that not only binds CD30L with high affinity and prevents its interaction with CD30, but it also induces reverse signaling in cells expressing CD30L. vCD30 blocked the generation of interferon γ–producing cells in vitro and was a potent inhibitor of T helper cell (Th)1- but not Th2-mediated inflammation in vivo. The finding of a CD30 homologue encoded by ectromelia virus suggests a role for CD30 in antiviral defense. Characterization of the immunological properties of vCD30 has uncovered a role of CD30–CD30L interactions in the generation of inflammatory responses. PMID:12235215

Giant Virus Megavirus chilensis Encodes the Biosynthetic Pathway for Uncommon Acetamido Sugars*

PubMed Central

Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G.

2014-01-01

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. PMID:25035429

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

PubMed Central

Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

1993-01-01

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

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

Feyereisen-Koener, J.M.

Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

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

Koener, J.F.; Leong, J.A.C.

A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits o...

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

PubMed

Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

2003-10-01

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

PubMed

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

PubMed Central

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus.

PubMed

Pappi, Polyxeni G; Dovas, Chrysostomos I; Efthimiou, Konstantinos E; Maliogka, Varvara I; Katis, Nikolaos I

2013-08-01

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

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

Tress, E.; Pierotti, M.; DeLeo, A.B.

1982-02-01

To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulsechase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65/sup gag/, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95/sup gag/, or its precursor, Pr75/sup gag/. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instancesmore » a monoclonal antibody, 35/56, which is specific for the NuLV G/sub IX/ antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same 35/56 phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.« less

Synthetic generation of influenza vaccine viruses for rapid response to pandemics.

PubMed

Dormitzer, Philip R; Suphaphiphat, Pirada; Gibson, Daniel G; Wentworth, David E; Stockwell, Timothy B; Algire, Mikkel A; Alperovich, Nina; Barro, Mario; Brown, David M; Craig, Stewart; Dattilo, Brian M; Denisova, Evgeniya A; De Souza, Ivna; Eickmann, Markus; Dugan, Vivien G; Ferrari, Annette; Gomila, Raul C; Han, Liqun; Judge, Casey; Mane, Sarthak; Matrosovich, Mikhail; Merryman, Chuck; Palladino, Giuseppe; Palmer, Gene A; Spencer, Terika; Strecker, Thomas; Trusheim, Heidi; Uhlendorff, Jennifer; Wen, Yingxia; Yee, Anthony C; Zaveri, Jayshree; Zhou, Bin; Becker, Stephan; Donabedian, Armen; Mason, Peter W; Glass, John I; Rappuoli, Rino; Venter, J Craig

2013-05-15

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase

PubMed Central

Kalveram, Birte

2013-01-01

Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses—i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus—has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

Inhibition of allergic encephalomyelitis in marmosets by vaccination with recombinant vaccinia virus encoding for myelin basic protein.

PubMed

Genain, C P; Gritz, L; Joshi, N; Panicali, D; Davis, R L; Whitaker, J N; Letvin, N L; Hauser, S L

1997-11-01

A primary demyelinating form of experimental allergic encephalomyelitis (EAE) resembling human multiple sclerosis (MS) occurs in Callithrix jacchus marmosets following immunization with human white matter. Participation of a T-cell immune response against myelin basic protein (MBP) in this disease model is supported by observations of increased reactivity against MBP in PBMC and of adoptive transfer of an inflammatory form of EAE by MBP-reactive T-cells. To evaluate the effects of ectopic presentation of MBP on marmoset EAE, animals were vaccinated prior to induction of EAE by subcutaneous injection of attenuated strains of vaccinia virus genetically engineered to contain either the entire coding sequence for human MBP (vT15) or the equine herpes virus glycoprotein gH gene (vAbT249). Vaccination with vT15 was followed by transient cytoplasmic and surface membrane expression of MBP in circulating PBMC (15-45 days). The onset of clinical EAE after immunization (pi) was markedly delayed in vT15-vaccinated animals (37-97 days pi, n = 4) compared to vAbT249-vaccinated controls (14-18 days pi, n = 3). Proliferative responses against MBP but not against vaccinia antigens or phytohemagglutinin were suppressed in protected animals. Thus, development of attenuated live viruses carrying genes for myelin antigens could be useful for induction of immunologic tolerance and for modulation of autoimmune demyelination.

Designs of Optoelectronic Trinary Signed-Digit Multiplication by use of Joint Spatial Encodings and Optical Correlation

NASA Astrophysics Data System (ADS)

Cherri, Abdallah K.

1999-02-01

Trinary signed-digit (TSD) symbolic-substitution-based (SS-based) optical adders, which were recently proposed, are used as the basic modules for designing highly parallel optical multiplications by use of cascaded optical correlators. The proposed multiplications perform carry-free generation of the multiplication partial products of two words in constant time. Also, three different multiplication designs are presented, and new joint spatial encodings for the TSD numbers are introduced. The proposed joint spatial encodings allow one to reduce the SS computation rules involved in optical multiplication. In addition, the proposed joint spatial encodings increase the space bandwidth product of the spatial light modulators of the optical system. This increase is achieved by reduction of the numbers of pixels in the joint spatial encodings for the input TSD operands as well as reduction of the number of pixels used in the proposed matched spatial filters for the optical multipliers.

Designs of optoelectronic trinary signed-digit multiplication by use of joint spatial encodings and optical correlation.

PubMed

Cherri, A K

1999-02-10

Trinary signed-digit (TSD) symbolic-substitution-based (SS-based) optical adders, which were recently proposed, are used as the basic modules for designing highly parallel optical multiplications by use of cascaded optical correlators. The proposed multiplications perform carry-free generation of the multiplication partial products of two words in constant time. Also, three different multiplication designs are presented, and new joint spatial encodings for the TSD numbers are introduced. The proposed joint spatial encodings allow one to reduce the SS computation rules involved in optical multiplication. In addition, the proposed joint spatial encodings increase the space-bandwidth product of the spatial light modulators of the optical system. This increase is achieved by reduction of the numbers of pixels in the joint spatial encodings for the input TSD operands as well as reduction of the number of pixels used in the proposed matched spatial filters for the optical multipliers.

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

PubMed

Tan, Wui Siew; Lewis, Christina L; Horelik, Nicholas E; Pregibon, Daniel C; Doyle, Patrick S; Yi, Hyunmin

2008-11-04

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

Near-Complete Genome Sequence of a Novel Single-Stranded RNA Virus Discovered in Indoor Air.

PubMed

Rosario, Karyna; Fierer, Noah; Breitbart, Mya

2018-03-22

Viral metagenomic analysis of heating, ventilation, and air conditioning (HVAC) filters recovered the near-complete genome sequence of a novel virus, named HVAC-associated R NA v irus 1 (HVAC-RV1). The HVAC-RV1 genome is most similar to those of picorna-like viruses identified in arthropods but encodes a small domain observed only in negative-sense single-stranded RNA viruses. Copyright © 2018 Rosario et al.

Use of Genetically-encoded Calcium Indicators for Live Cell Calcium Imaging and Localization in Virus-infected Cells

PubMed Central

Perry, Jacob L.; Ramachandran, Nina K.; Utama, Budi; Hyser, Joseph M.

2015-01-01

Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections. PMID:26344758

Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

PubMed

Bai, Xingwen; Bao, Huifang; Li, Pinghua; Wei, Wei; Zhang, Meng; Sun, Pu; Cao, Yimei; Lu, Zengjun; Fu, Yuanfang; Xie, Baoxia; Chen, Yingli; Li, Dong; Luo, Jianxun; Liu, Zaixin

2014-07-24

Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor

PubMed Central

2014-01-01

Background Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Results Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. Conclusion The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction. PMID:25056022

Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

PubMed Central

Chénard, Caroline; Wirth, Jennifer F.

2016-01-01

ABSTRACT   Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. PMID:27302758

Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein.

PubMed

Mohamed, Maizan; McLees, Angela; Elliott, Richard M

2009-08-01

Viruses in the genus Orthobunyavirus, family Bunyaviridae, have a genome comprising three segments (called L, M, and S) of negative-sense RNA. Serological studies have classified the >170 named virus isolates into 18 serogroups, with a few additional as yet ungrouped viruses. Until now, molecular studies and full-length S-segment nucleotide sequences were available for representatives of eight serogroups; in all cases, the S segment encodes two proteins, N (nucleocapsid) and NSs (nonstructural), in overlapping open reading frames (ORFs) that are translated from the same mRNA. The NSs proteins of Bunyamwera virus (BUNV) and California serogroup viruses have been shown to play a role in inhibiting host cell mRNA and protein synthesis, thereby preventing induction of interferon (IFN). We have determined full-length sequences of the S segments of representative viruses in the Anopheles A, Anopheles B, and Tete serogroups, and we report here that these viruses do not show evidence of having an NSs ORF. In addition, these viruses have rather longer N proteins than those in the other serogroups. Most of the naturally occurring viruses that lack the NSs protein behaved like a recombinant BUNV with the NSs gene deleted in that they failed to prevent induction of IFN-beta mRNA. However, Tacaiuma virus (TCMV) in the Anopheles A serogroup inhibited IFN induction in a manner similar to that of wild-type BUNV, suggesting that TCMV has evolved an alternative mechanism, not involving a typical NSs protein, to antagonize the host innate immune response.

Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length

PubMed Central

Grigorov, Boyan; Rabilloud, Jessica; Lawrence, Philip; Gerlier, Denis

2011-01-01

Background Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. Methods/Findings A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson's law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion Overall, performing the assay takes only 24–30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1. The step-by-step procedure we have set up can be, in principle, applicable to accurately quantify any virus including lentiviral vectors, provided that a virus encoded gene product can be detected by flow cytometry. PMID:21915289

A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models

PubMed Central

Raulji, Payal; Mohapatra, Subhra; Mohapatra, Shyam S

2015-01-01

Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections. PMID:26407080

Plasma viral microRNA profiles reveal potential biomarkers for chronic active Epstein-Barr virus infection.

PubMed

Kawano, Yoshihiko; Iwata, Seiko; Kawada, Jun-ichi; Gotoh, Kensei; Suzuki, Michio; Torii, Yuka; Kojima, Seiji; Kimura, Hiroshi; Ito, Yoshinori

2013-09-01

Chronic active Epstein-Barr virus (CAEBV) infection has high mortality and morbidity, and biomarkers for disease severity and prognosis are required. MicroRNAs (miRNAs) are small noncoding RNAs, and EBV encodes multiple miRNAs. Because plasma contains sufficiently stable miRNAs, circulating EBV-associated miRNA profiles were investigated as novel biomarkers in CAEBV infection. Plasma miRNA expression was assessed for 12 miRNAs encoded within 2 EBV open reading frames (BART and BHRF). Expression levels were investigated in 19 patients with CAEBV infection, 14 patients with infectious mononucleosis, and 11 healthy controls. Relative expression levels of plasma miRNAs were determined by TaqMan probe-based quantitative assay. Plasma miR-BART1-5p, 2-5p, 5, and 22 levels in patients with CAEBV infection were significantly greater than those in patients with infectious mononucleosis and in controls. Plasma miR-BART2-5p, 4, 7, 13, 15, and 22 levels were significantly elevated in patients with CAEBV infection with systemic symptoms, compared with levels in patients with no systemic symptoms. The levels of miR-BART2-5p, 13, and 15 showed clinical cutoff values associated with specific clinical conditions, in contrast to plasma EBV loads. Levels of specific plasma EBV miRNAs were elevated differentially in patients with CAEBV infection. Several EBV miRNAs, particularly miR-BART2-5p, 13, and 15, are potentially biomarkers of disease severity or prognosis.

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

Expression of Marek's disease virus oncoprotein Meq during infection in the natural host

USDA-ARS?s Scientific Manuscript database

Marek's disease virus (MDV, Gallid herpesvirus 2) causes a lymphoproliferative disease known as Marek's disease (MD), which is unique among alphaherpesviruses as the viral genome encodes an oncoprotein, Meq. Previous studies, using cultured fibroblasts and MDV-transformed lymphoblastoid cell lines, ...

Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease.

PubMed

Scholte, Florine E M; Zivcec, Marko; Dzimianski, John V; Deaton, Michelle K; Spengler, Jessica R; Welch, Stephen R; Nichol, Stuart T; Pegan, Scott D; Spiropoulou, Christina F; Bergeron, Éric

2017-09-05

Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and support the development of therapeutics and vaccines. Published by Elsevier Inc.

A Mouse Polyomavirus-encoded microRNA Targets the Cellular Apoptosis Pathway through Smad2 Inhibition

PubMed Central

Sung, Chang Kyoo; Yim, Hyungshin; Andrews, Erik; Benjamin, Thomas L.

2014-01-01

Some viruses and most eukaryotic cells have microRNAs that regulate the expression of many genes. Although many viral miRNAs have been identified, only a few have been included in in vivo functional studies. Here we show that a Py-encoded miRNA downregulates the expression of the pro-apoptotic factor Smad2, resulting in the suppression of the apoptosis pathway. To study the Py miRNA in an in vivo context, a miRNA-deficient mutant virus was created on the background of the LID virus strain which establishes a rapid and lethal infection in newborn mice. Apoptosis analysis on kidney tissues indicates that the pro-apoptotic pathway is targeted in the infected host as well. Suppression of apoptosis through targeting of Smad2 by the Py miRNA is expected to synergize with anti-apoptotic effects previously attributed to the polyoma tumor antigens in support of virus replication in the natural host. PMID:25146733

Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles [corrected].

PubMed

Strecker, Thomas; Eichler, Robert; Meulen, Jan ter; Weissenhorn, Winfried; Dieter Klenk, Hans; Garten, Wolfgang; Lenz, Oliver

2003-10-01

Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i). is abundant in viral particles, (ii). is strongly membrane associated, (iii). is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv). contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.

Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

PubMed

Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

2010-02-01

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

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.

Virus-encoded microRNAs

PubMed Central

Grundhoff, Adam; Sullivan, Christopher S.

2011-01-01

microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue

PubMed Central

Shinn, Helen Ki; Yan, Chunri; Kim, Tae-Hwan; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Jayoung; Cha, Eun-Jong

2016-01-01

Purpose: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. Methods: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Results: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). Conclusions: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections. PMID:27377944

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue.

PubMed

Yun, Seok Joong; Jeong, Pildu; Kang, Ho Won; Shinn, Helen Ki; Kim, Ye-Hwan; Yan, Chunri; Choi, Young-Ki; Kim, Dongho; Ryu, Dong Hee; Ha, Yun-Sok; Kim, Tae-Hwan; Kwon, Tae Gyun; Kim, Jung Min; Suh, Sang Heon; Kim, Seon-Kyu; Kim, Seon-Young; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Isaac Yi; Kim, Jayoung; Cha, Hee-Jae; Choi, Yung-Hyun; Cha, Eun-Jong; Kim, Wun-Jae

2016-06-01

Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections.

Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

PubMed Central

Howe, J G; Shu, M D

1988-01-01

Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein. Images PMID:2839701

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

PubMed

Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

2009-12-01

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

PubMed Central

Bogani, Federica; Boehmer, Paul E.

2008-01-01

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5′-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3′ side of abasic sites and 5′-dRP residues that remain after cleavage by 5′-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency. PMID:18695225

Characterization of Bombyx mori nucleopolyhedrovirus orf68 gene that encodes a novel structural protein of budded virus.

PubMed

Iwanaga, Masashi; Kurihara, Masaaki; Kobayashi, Masahiko; Kang, WonKyung

2002-05-25

All lepidopteran baculovirus genomes sequenced to date encode a homolog of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf68 gene, suggesting that it performs an important role in the virus life cycle. In this article we describe the characterization of BmNPV orf68 gene. Northern and Western analyses demonstrated that orf68 gene was expressed as a late gene and encoded a structural protein of budded virus (BV). Immunohistochemical analysis by confocal microscopy showed that ORF68 protein was localized mainly in the nucleus of infected cells. To examine the function of orf68 gene, we constructed orf68 deletion mutant (BmD68) and characterized it in BmN cells and larvae of B. mori. BV production was delayed in BmD68-infected cells. The larval bioassays also demonstrated that deletion of orf68 did not reduce the infectivity, but mutant virus took 70 h longer to kill the host than wild-type BmNPV. In addition, dot-blot analysis showed viral DNA accumulated more slowly in mutant infected cells. Further examination suggested that BmD68 was less efficient in entry and budding from cells, although it seemed to possess normal attachment ability. These results suggest that ORF68 is a BV-associated protein involved in secondary infection from cell-to-cell. (c) 2002 Elsevier Science (USA).

Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

DOEpatents

Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

2015-04-21

The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

Drug-Encoded Biomarkers for Monitoring Biological Therapies

PubMed Central

Bedenk, Kristina; Zhang, Qian; Frentzen, Alexa; Cappello, Joseph; Fischer, Utz; Szalay, Aladar A.

2015-01-01

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers. PMID:26348361

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.

Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB

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

Burles, Kristin, E-mail: burles@ualberta.ca; Buuren, Nicholas van; Barry, Michele

2014-11-15

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and thismore » inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. - Highlights: • Ectromelia virus encodes four Ank/F-box proteins, EVM002, EVM005, EVM154 and EVM165. • The Ank/F-box proteins inhibit NFκB nuclear translocation, dependent on the F-box. • The Ank/F-box proteins prevent IκBα degradation, suggesting they target the SCF. • Deletion of a single Ank/F-box gene from ECTV does not prevent viral NFκB inhibition. • This study identifies a new mechanism by which ectromelia virus inhibits NFκB.« less

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors*

PubMed Central

Pontejo, Sergio M.; Alejo, Ali; Alcami, Antonio

2015-01-01

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. PMID:25940088

Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0

PubMed Central

Swanson, Priscilla; Soriano, Vincent; Devare, Sushil G.; Hackett, John

2001-01-01

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O. PMID:11230396

Viral Repression of Fungal Pheromone Precursor Gene Expression

PubMed Central

Zhang, Lei; Baasiri, Rudeina A.; Van Alfen, Neal K.

1998-01-01

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a Gαi subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete. PMID:9447992

Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations

PubMed Central

Rodríguez-Cousiño, Nieves

2016-01-01

ABSTRACT Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed. IMPORTANCE Killer strains of S. cerevisiae secrete protein toxins that kill nonkiller yeasts. The “killer phenomenon” depends on two dsRNA viruses: L-A and M. M encodes the toxin, and L-A, the helper virus, provides the capsids for both viruses. Different killer toxins exist: K1, K2, K28, and Klus, encoded on different M viruses. Our data indicate that each M dsRNA depends on a specific helper virus; these helper viruses have nucleotide sequences that may be as much as 26% different, suggesting coevolution. In wine environments, K2 and Klus strains frequently coexist. We have previously characterized the association of Mlus and L-A-lus. Here we sequence and characterize L-A-2, the helper virus of M2, establishing the helper virus requirements of M2, which had not been completely elucidated. We also report the existence of two specific L-BC totiviruses in Klus and K2 strains with about 10% of their nucleotides different, suggesting different evolutionary histories from those of L-A viruses. PMID:27940540

Zika Virus - Multiple Languages

MedlinePlus

... Are Here: Home → Multiple Languages → All Health Topics → Zika Virus URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Zika Virus - Multiple Languages To use the sharing features on ...

A stretch of 11 amino acids in the betaB-betaC loop of the coat protein of grapevine fanleaf virus is essential for transmission by the nematode Xiphinema index.

PubMed

Schellenberger, Pascale; Andret-Link, Peggy; Schmitt-Keichinger, Corinne; Bergdoll, Marc; Marmonier, Aurélie; Vigne, Emmanuelle; Lemaire, Olivier; Fuchs, Marc; Demangeat, Gérard; Ritzenthaler, Christophe

2010-08-01

Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the betaB-betaC loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the betaE-alphaB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.

IMOJEV(®): a Yellow fever virus-based novel Japanese encephalitis vaccine.

PubMed

Appaiahgari, Mohan Babu; Vrati, Sudhanshu

2010-12-01

Japanese encephalitis (JE) is a disease of the CNS caused by Japanese encephalitis virus (JEV). The disease appears in the form of frequent outbreaks in most south- and southeast Asian countries and the virus has become endemic in several areas. There is no licensed therapy available and disease control by vaccination is considered to be most effective. Mouse brain-derived inactivated JE vaccines, although immunogenic, have several limitations in terms of safety, availability and requirement for multiple doses. Owing to these drawbacks, the WHO called for the development of novel, safe and more efficacious JE vaccines. Several candidate vaccines have been developed and at least three of them that demonstrated strong immunogenicity after one or two doses of the vaccine in animal models were subsequently tested in various clinical trials. One of these vaccines, IMOJEV(®) (JE-CV and previously known as ChimeriVax™-JE), is a novel recombinant chimeric virus vaccine, developed using the Yellow fever virus (YFV) vaccine vector YFV17D, by replacing the cDNA encoding the envelope proteins of YFV with that of an attenuated JEV strain SA14-14-2. IMOJEV was found to be safe, highly immunogenic and capable of inducing long-lasting immunity in both preclinical and clinical trials. Moreover, a single dose of IMOJEV was sufficient to induce protective immunity, which was similar to that induced in adults by three doses of JE-VAX(®), a mouse brain-derived inactivated JE vaccine. Recently, Phase III trials evaluating the immunogenicity and safety of the chimeric virus vaccine have been successfully completed in some JE-endemic countries and the vaccine manufacturers have filed an application for vaccine registration. IMOJEV may thus be licensed for use in humans as an improved alternative to the currently licensed JE vaccines.

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress.

PubMed

von Einem, Jens; Schumacher, Daniel; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2006-03-01

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

Rift Valley Fever Vaccine Virus Clone 13 Is Able to Cross the Ovine Placental Barrier Associated with Foetal Infections, Malformations, and Stillbirths.

PubMed

Makoschey, Birgit; van Kilsdonk, Emma; Hubers, Willem R; Vrijenhoek, Mieke P; Smit, Marianne; Wichgers Schreur, Paul J; Kortekaas, Jeroen; Moulin, Véronique

2016-03-01

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that affects domesticated ruminants and occasionally humans. Classical RVF vaccines are based on formalin-inactivated virus or the live-attenuated Smithburn strain. The inactivated vaccine is highly safe but requires multiple administrations and yearly re-vaccinations. Although the Smithburn vaccine provides solid protection after a single vaccination, this vaccine is not safe for pregnant animals. An alternative live-attenuated vaccine, named Clone 13, carries a large natural deletion in the NSs gene which encodes the major virulence factor of the virus. The Clone 13 vaccine was previously shown to be safe for young lambs and calves. Moreover, a study in pregnant ewes suggested that the vaccine could also be applied safely during gestation. To anticipate on a possible future incursion of RVFV in Europe, we have evaluated the safety of Clone 13 for young lambs and pregnant ewes. In line with the guidelines from the World Organisation for Animal health (Office International des Epizooties, OIE) and regulations of the European Pharmacopeia (EP), these studies were performed with an overdose. Our studies with lambs showed that Clone 13 dissemination within vaccinated animals is very limited. Moreover, the Clone 13 vaccine virus was not shed nor spread to in-contact sentinels and did not revert to virulence upon animal-to-animal passage. Importantly, a large experiment with pregnant ewes demonstrated that the Clone 13 virus is able to spread to the fetus, resulting in malformations and stillbirths. Altogether, our results suggest that Clone 13 can be applied safely in lambs, but that caution should be taken when Clone 13 is used in pregnant animals, particularly during the first trimester of gestation.

Characterization of Clade 7.2 H5 Avian Influenza Viruses That Continue To Circulate in Chickens in China

PubMed Central

Liu, Liling; Zeng, Xianying; Chen, Pucheng; Deng, Guohua; Li, Yanbing; Shi, Jianzhong; Gu, Chunyang; Kong, Huihui; Suzuki, Yasuo; Jiang, Yongping; Tian, Guobin

2016-01-01

ABSTRACT The H5N1 avian influenza viruses emerged in Southeast Asia in the late 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to the clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here, we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses had antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at a dosage of 106 50% egg infective doses (EID50). Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans. IMPORTANCE Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses into different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines for these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly 9 years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health. PMID:27558424

Characterization of Clade 7.2 H5 Avian Influenza Viruses That Continue To Circulate in Chickens in China.

PubMed

Liu, Liling; Zeng, Xianying; Chen, Pucheng; Deng, Guohua; Li, Yanbing; Shi, Jianzhong; Gu, Chunyang; Kong, Huihui; Suzuki, Yasuo; Jiang, Yongping; Tian, Guobin; Chen, Hualan

2016-11-01

The H5N1 avian influenza viruses emerged in Southeast Asia in the late 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to the clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here, we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses had antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at a dosage of 10 6 50% egg infective doses (EID 50 ). Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans. Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses into different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines for these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly 9 years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health. Copyright © 2016 Liu et al.

Alteration of a single amino acid in the basic domain of Marek's disease virus Meq oncoprotein plays an important role in T-cell transformation

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus encoded oncoprotein, Meq, has been shown to play a major role in transformation of T-lymphocytes. We have earlier shown that replacement of the meq gene in the very virulent strain Md5 with that of vaccine strain CVI988/Rispens resulted in virus attenuation in chickens. To dete...

Cell culture attenuation eliminates rMd5deltaMeq-induced bursal and thymic atrophy and renders the mutant virus as an effective and safe vaccine against Marek's disease

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a basic leucine zipper oncoprotein, meq, which structurally resembles jun/fos family of transcriptional activators. It has been clearly demonstrated that deletion of meq results in loss of transformation and oncogenic capacity of MDV. The rMd5'meq virus provided s...

Reverse Genetics for Mammalian Orthoreovirus.

PubMed

Stuart, Johnasha D; Phillips, Matthew B; Boehme, Karl W

2017-01-01

Reverse genetics allows introduction of specific alterations into a viral genome. Studies performed with mutant viruses generated using reverse genetics approaches have contributed immeasurably to our understanding of viral replication and pathogenesis, and also have led to development of novel vaccines and virus-based vectors. Here, we describe the reverse genetics system that allows for production and recovery of mammalian orthoreovirus, a double-stranded (ds) RNA virus, from plasmids that encode the viral genome.

RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

PubMed Central

Liesman, Rachael M.; Buchholz, Ursula J.; Luongo, Cindy L.; Yang, Lijuan; Proia, Alan D.; DeVincenzo, John P.; Collins, Peter L.; Pickles, Raymond J.

2014-01-01

Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease. PMID:24713657

The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

PubMed Central

McBride, Ruth; Fielding, Burtram C.

2012-01-01

A respiratory disease caused by a novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), was first reported in China in late 2002. The subsequent efficient human-to-human transmission of this virus eventually affected more than 30 countries worldwide, resulting in a mortality rate of ~10% of infected individuals. The spread of the virus was ultimately controlled by isolation of infected individuals and there has been no infections reported since April 2004. However, the natural reservoir of the virus was never identified and it is not known if this virus will re-emerge and, therefore, research on this virus continues. The SARS-CoV genome is about 30 kb in length and is predicted to contain 14 functional open reading frames (ORFs). The genome encodes for proteins that are homologous to known coronavirus proteins, such as the replicase proteins (ORFs 1a and 1b) and the four major structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). SARS-CoV also encodes for eight unique proteins, called accessory proteins, with no known homologues. This review will summarize the current knowledge on SARS-CoV accessory proteins and will include: (i) expression and processing; (ii) the effects on cellular processes; and (iii) functional studies. PMID:23202509

Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

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

Zheng Min; Guangxi Center for Animal Disease Control and Prevention, Nanning 530001; College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062

Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AALmore » and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.« less

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

PubMed Central

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

1995-01-01

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

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus.

PubMed

Schnitzler, P; Darai, G

1993-10-01

The gene encoding the major capsid protein (MCP) of fish lymphocystis disease virus (flounder isolate; FLCDV-f) has been identified by PCR using oligonucleotide primers corresponding to different regions of the MCP of Tipula iridescent virus (TIV), iridescent virus 22 (IV22) and Chilo iridescent virus (CIV). DNA fragments of 0.4 kbp, 0.5 kbp and 0.27 kbp in size were amplified using oligonucleotide primers corresponding to amino acids (aa) 146 to 153 (primer 1) and 274 to 268 (primer 6), or aa 146 to 153 (primer 1) and 313 to 304 (primer 8), or aa 304 to 312 (primer 7) and 385 to 381 (primer 9) of the MCP of TIV, respectively. The PCR products were used as hybridization probes for screening the gene library of FLCDV-f. The MCP gene of FLCDV-f(1377 bp; 459 aa; 51.4K) was identified within the DNA sequence of the EcoRI FLCDV-f DNA fragment C (11.2 kbp; 0.611 to 0.718 map units). A high degree of aa sequence identity/similarity was detected between the MCP of FLCDV-f and TIV (50.3%/33.8%), IV22 (49.1%/34.2%). CIV (53%/29.5%) and African swine fever virus (16%/38.1%).

Self-Association of Lymphocytic Choriomeningitis Virus Nucleoprotein Is Mediated by Its N-Terminal Region and Is Not Required for Its Anti-Interferon Function

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin

2012-01-01

Arenaviruses have a bisegmented, negative-strand RNA genome. Both the large (L) and small (S) genome segments use an ambisense coding strategy to direct the synthesis of two viral proteins. The L segment encodes the virus polymerase (L protein) and the matrix Z protein, whereas the S segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC). NPs are the most abundant viral protein in infected cells and virions and encapsidate genomic RNA species to form an NP-RNA complex that, together with the virus L polymerase, forms the virus ribonucleoprotein (RNP) core capable of directing both replication and transcription of the viral genome. RNP formation predicts a self-association property of NPs. Here we document self-association (homotypic interaction) of the NP of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), as well as those of the hemorrhagic fever (HF) arenaviruses Lassa virus (LASV) and Machupo virus (MACV). We also show heterotypic interaction between NPs from both closely (LCMV and LASV) and distantly (LCMV and MACV) genetically related arenaviruses. LCMV NP self-association was dependent on the presence of single-stranded RNA and mediated by an N-terminal region of the NP that did not overlap with the previously described C-terminal NP domain involved in either counteracting the host type I interferon response or interacting with LCMV Z. PMID:22258244

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

PubMed

Guo, Tong; Han, Wenyuan; She, Qunxin

2018-04-09

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

Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola.

PubMed

Zhong, Jie; Shang, Hong Hong; Zhu, Chuan Xia; Zhu, Jun Zi; Zhu, Hong Jian; Hu, Yan; Gao, Bi Da

2016-06-02

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host. Copyright © 2016. Published by Elsevier B.V.

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

PubMed

Kinney, R M; Esposito, J J; Johnson, B J; Roehrig, J T; Mathews, J H; Barrett, A D; Trent, D W

1988-12-01

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis

PubMed Central

Wang, Danher; Suhrbier, Andreas; Penn-Nicholson, Adam; Woraratanadharm, Jan; Gardner, Joy; Luo, Min; Le, Thuy T.; Anraku, Itaru; Sakalian, Michael; Einfeld, David; Dong, John Y.

2011-01-01

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates. PMID:21320541

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

PubMed Central

Scholthof, Karen-Beth G.

2015-01-01

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes. PMID:25634987

The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event.

PubMed

Rodríguez, Irene; Nogal, María L; Redrejo-Rodríguez, Modesto; Bustos, María J; Salas, María L

2009-12-01

The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.

The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event ▿

PubMed Central

Rodríguez, Irene; Nogal, María L.; Redrejo-Rodríguez, Modesto; Bustos, María J.; Salas, María L.

2009-01-01

The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event. PMID:19793823

Attenuation of Marek's disease virus lacking the Meq oncogene in cell culture

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a basic leucine zipper oncoprotein, meq, which structurally resembles the jun/fos family of transcriptional activators. It has been clearly demonstrated that deletion of meq results in loss of transformation and oncogenic capacity of MDV. Chickens vaccinated with ...

Effects of the HN gene c-terminal extensions on the Newcastle disease virus virulence

USDA-ARS?s Scientific Manuscript database

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open reading frame...

Deltabaculoviruses encode a functional type I budded virus envelope fusion protein

USDA-ARS?s Scientific Manuscript database

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cells in culture. An F protein homolog gene was absent in gammabaculoviruses. Here we show tha...

The virally encoded killer proteins from Ustilago maydis

USDA-ARS?s Scientific Manuscript database

Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a 'killer' phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in ...

Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

NASA Astrophysics Data System (ADS)

Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

1985-09-01

The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

Full-Genome Characterisation of Orungo, Lebombo and Changuinola Viruses Provides Evidence for Co-Evolution of Orbiviruses with Their Arthropod Vectors

PubMed Central

Mohd Jaafar, Fauziah; Belhouchet, Mourad; Belaganahalli, Manjunatha; Tesh, Robert B.; Mertens, Peter P. C.; Attoui, Houssam

2014-01-01

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein ‘outer-capsid protein 1′ (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 ‘subcore-shell protein’ as the third largest protein ‘VP3(T2)’ in the first of these groups, but the second largest protein ‘VP3(T2)’ in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors. PMID:24475112

Molecular biology and genetic diversity of Rift Valley fever virus

PubMed Central

Ikegami, Tetsuro

2013-01-01

Rift Valley fever virus (RVFV), a member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever (RVF), a mosquito-borne disease of ruminant animals and humans. The generation of a large sequence database has facilitated studies of the evolution and spread of the virus. Bayesian analyses indicate that currently circulating strains of RVFV are descended from an ancestral species that emerged from a natural reservoir in Africa when large-scale cattle and sheep farming were introduced during the 19th century. Viruses descended from multiple lineages persist in that region, through infection of reservoir animals and vertical transmission in mosquitoes, emerging in years of heavy rainfall to cause epizootics and epidemics. On a number of occasions, viruses from these lineages have been transported outside the enzootic region through the movement of infected animals or mosquitoes, triggering outbreaks in countries such as Egypt, Saudi Arabia, Mauritania and Madagascar, where RVF had not previously been seen. Such viruses could potentially become established in their new environments through infection of wild and domestic ruminants and other animals and vertical transmission in local mosquito species. Despite their extensive geographic dispersion, all strains of RVFV remain closely related at the nucleotide and amino acid level. The high degree of conservation of genes encoding the virion surface glycoproteins suggests that a single vaccine should protect against all currently circulating RVFV strains. Similarly, preservation of the sequence of the RNA-dependent RNA polymerase across viral lineages implies that antiviral drugs targeting the enzyme should be effective against all strains. Researchers should be encouraged to collect additional RVFV isolates and perform whole-genome sequencing and phylogenetic analysis, so as to enhance our understanding of the continuing evolution of this important virus. This review forms part of a series of invited papers in Antiviral Research on the genetic diversity of emerging viruses. PMID:22710362

Molecular biology and genetic diversity of Rift Valley fever virus.

PubMed

Ikegami, Tetsuro

2012-09-01

Rift Valley fever virus (RVFV), a member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever (RVF), a mosquito-borne disease of ruminant animals and humans. The generation of a large sequence database has facilitated studies of the evolution and spread of the virus. Bayesian analyses indicate that currently circulating strains of RVFV are descended from an ancestral species that emerged from a natural reservoir in Africa when large-scale cattle and sheep farming were introduced during the 19th century. Viruses descended from multiple lineages persist in that region, through infection of reservoir animals and vertical transmission in mosquitoes, emerging in years of heavy rainfall to cause epizootics and epidemics. On a number of occasions, viruses from these lineages have been transported outside the enzootic region through the movement of infected animals or mosquitoes, triggering outbreaks in countries such as Egypt, Saudi Arabia, Mauritania and Madagascar, where RVF had not previously been seen. Such viruses could potentially become established in their new environments through infection of wild and domestic ruminants and other animals and vertical transmission in local mosquito species. Despite their extensive geographic dispersion, all strains of RVFV remain closely related at the nucleotide and amino acid level. The high degree of conservation of genes encoding the virion surface glycoproteins suggests that a single vaccine should protect against all currently circulating RVFV strains. Similarly, preservation of the sequence of the RNA-dependent RNA polymerase across viral lineages implies that antiviral drugs targeting the enzyme should be effective against all strains. Researchers should be encouraged to collect additional RVFV isolates and perform whole-genome sequencing and phylogenetic analysis, so as to enhance our understanding of the continuing evolution of this important virus. This review forms part of a series of invited papers in Antiviral Research on the genetic diversity of emerging viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

Antiviral Defense Involves AGO4 in an Arabidopsis-Potexvirus Interaction.

PubMed

Brosseau, Chantal; El Oirdi, Mohamed; Adurogbangba, Ayooluwa; Ma, Xiaofang; Moffett, Peter

2016-11-01

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

Insights into the nuclear export of murine leukemia virus intron-containing RNA.

PubMed

Pessel-Vivares, Lucie; Houzet, Laurent; Lainé, Sébastien; Mougel, Marylène

2015-01-01

The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA.

MLV integration site selection is driven by strong enhancers and active promoters

PubMed Central

LaFave, Matthew C.; Varshney, Gaurav K.; Gildea, Derek E.; Wolfsberg, Tyra G.; Baxevanis, Andreas D.; Burgess, Shawn M.

2014-01-01

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors. PMID:24464997

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

PubMed Central

Oliveira, Sofia A.; Park, Se-Ho; Lee, Peter; Bendelac, Albert; Shenk, Thomas E.

2002-01-01

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient γc/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance. PMID:11752177

Proteomic composition of Nipah virus-like particles.

PubMed

Vera-Velasco, Natalia Mara; García-Murria, Maria Jesús; Sánchez Del Pino, Manuel M; Mingarro, Ismael; Martinez-Gil, Luis

2018-02-10

Virions are often described as virus-only entities with no cellular components with the exception of the lipids in their membranes. However, advances in proteomics are revealing substantial amounts of host proteins in the viral particles. In the case of Nipah virus (NiV), the viral components in the virion have been known for some time. Nonetheless, no information has been obtained regarding the cellular proteins in the viral particles. To address this question, we produced Virus-Like Particles (VLPs) for NiV by expressing the F, G and M proteins in human-derived cells. Next, the proteomic content in these VLPs was analyzed by LC-MS/MS. We identified 67 human proteins including soluble and membrane-bound proteins involved in vesicle sorting and transport. Interestingly, many of them have been reported to interact with other viruses. Finally, thanks to the semi-quantitative nature of our data we were able to estimate the ratio among F, G and M proteins and also the ratio between cellular and viral proteins in the VLPs. We believe our data contribute to the better understanding of NiV life cycle and might facilitate future attempts for developing antiviral agents and the design of further experimental studies for this deadly infection. Traditionally viral particles have been described as pure entities carrying only viral-derived proteins. Advances in proteomics are changing this simplified view. Host proteins have been identified in many viruses (especially in enveloped viruses). These cell-derived proteins participate in multiple steps in the viral life cycle and might be as important for the survival of the virus as any other viral-encoded protein. In this work, we analyze utilizing LC-MS/MS the cellular proteins incorporated or bound to the virions of Nipah virus (NiV), an emerging, highly pathogenic, zoonotic virus from the Paramyxoviridiae family. Furthermore, we analyzed the ratio between cellular and viral proteins and among the viral F, G and M proteins in the viral particles. The characterization of the Nipah virus-human interactions occurring in the virion might facilitate the development of new therapeutic and prophylactic therapies for this viral illness. Copyright © 2017 Elsevier B.V. All rights reserved.

Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

PubMed

Sutherland, M R; Friedman, H M; Pryzdial, E L G

2007-05-01

We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host-encoded and virus-encoded procoagulant factors. These enable the virus to bypass the normal cell-regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. In the current study we investigated why these viruses may have evolved to generate thrombin. Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum-free inoculation media resulted in up to a 3-fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease-activated receptor 1 (PAR1) was supported using PAR1-activating peptides in place of thrombin and PAR1-specific antibodies to inhibit the effects of thrombin. These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1-mediated cell modulation.

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes.

PubMed

Delwart, Eric; Li, Linlin

2012-03-01

The genomes of numerous circoviruses and distantly related circular ssDNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, plants (geminivirus and nanovirus), in human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also recently identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting the very wide past host range of rep bearing viruses. An ancient origin for viruses with Rep-encoding small circular ssDNA genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular ssDNA genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular ssDNA viral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

PubMed

Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G

2014-08-29

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of Herpes Simplex Virus 1 Glycoprotein B by a Recombinant Vaccinia Virus and Protection of Mice against Lethal Herpes Simplex Virus 1 Infection

NASA Astrophysics Data System (ADS)

Cantin, Edouard M.; Eberle, Richard; Baldick, Joseph L.; Moss, Bernard; Willey, Dru E.; Notkins, Abner L.; Openshaw, Harry

1987-08-01

The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

The Interferon-Stimulated Gene Ifitm3 Restricts West Nile Virus Infection and Pathogenesis.

PubMed

Gorman, Matthew J; Poddar, Subhajit; Farzan, Michael; Diamond, Michael S

2016-09-15

The interferon-induced transmembrane protein (IFITM) family of proteins inhibit infection of several different enveloped viruses in cell culture by virtue of their ability to restrict entry and fusion from late endosomes. As few studies have evaluated the importance of Ifitm3 in vivo in restricting viral pathogenesis, we investigated its significance as an antiviral gene against West Nile virus (WNV), an encephalitic flavivirus, in cells and mice. Ifitm3(-/-) mice were more vulnerable to lethal WNV infection, and this was associated with greater virus accumulation in peripheral organs and central nervous system tissues. As no difference in viral burden in the brain or spinal cord was observed after direct intracranial inoculation, Ifitm3 likely functions as an antiviral protein in nonneuronal cells. Consistent with this, Ifitm3(-/-) fibroblasts but not dendritic cells resulted in higher yields of WNV in multistep growth analyses. Moreover, transcomplementation experiments showed that Ifitm3 inhibited WNV infection independently of Ifitm1, Ifitm2, Ifitm5, and Ifitm6. Beyond a direct effect on viral infection in cells, analysis of the immune response in WNV-infected Ifitm3(-/-) mice showed decreases in the total number of B cells, CD4(+) T cells, and antigen-specific CD8(+) T cells. Finally, bone marrow chimera experiments demonstrated that Ifitm3 functioned in both radioresistant and radiosensitive cells, as higher levels of WNV were observed in the brain only when Ifitm3 was absent from both compartments. Our analyses suggest that Ifitm3 restricts WNV pathogenesis likely through multiple mechanisms, including the direct control of infection in subsets of cells. As part of the mammalian host response to viral infections, hundreds of interferon-stimulated genes (ISGs) are induced. The inhibitory activity of individual ISGs varies depending on the specific cell type and viral pathogen. Among ISGs, the genes encoding interferon-induced transmembrane protein (IFITM) have been reported to inhibit multiple families of viruses in cell culture. However, few reports have evaluated the impact of IFITM genes on viral pathogenesis in vivo In this study, we characterized the antiviral activity of Ifitm3 against West Nile virus (WNV), an encephalitic flavivirus, using mice with a targeted gene deletion of Ifitm3 Based on extensive virological and immunological analyses, we determined that Ifitm3 protects mice from WNV-induced mortality by restricting virus accumulation in peripheral organs and, subsequently, in central nervous system tissues. Our data suggest that Ifitm3 restricts WNV pathogenesis by multiple mechanisms and functions in part by controlling infection in different cell types. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

PubMed Central

Silva, Maria C.; Yu, Qian-Chun; Enquist, Lynn; Shenk, Thomas

2003-01-01

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm. PMID:12970444

Characterization of the Rana grylio virus 3{beta}-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

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

Sun Wei; Huang Youhua; Zhao Zhe

2006-12-08

The 3{beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3{beta}-HSD gene homolog was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3{beta}-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3{beta}-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3{beta}-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3{beta}-HSDmore » in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3{beta}-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3{beta}-HSD might be a protein involved in host-virus interaction.« less

Plant rhabdoviruses.

PubMed

Redinbaugh, M G; Hogenhout, S A

2005-01-01

This chapter provides an overview of plant rhabdovirus structure and taxonomy, genome structure, protein function, and insect and plant infection. It is focused on recent research and unique aspects of rhabdovirus biology. Plant rhabdoviruses are transmitted by aphid, leafhopper or planthopper vectors, and the viruses replicate in both their insect and plant hosts. The two plant rhabdovirus genera, Nucleorhabdovirus and Cytorhabdovirus, can be distinguished on the basis of their intracellular site of morphogenesis in plant cells. All plant rhabdoviruses carry analogs of the five core genes: the nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large or polymerase (L). However, compared to vesiculoviruses that are composed of the five core genes, all plant rhabdoviruses encode more than these five genes, at least one of which is inserted between the P and M genes in the rhabdoviral genome. Interestingly, while these extra genes are not similar among plant rhabdoviruses, two encode proteins with similarity to the 30K superfamily of plant virus movement proteins. Analysis of nucleorhabdoviral protein sequences revealed nuclear localization signals for the N, P, M and L proteins, consistent with virus replication and morphogenesis of these viruses in the nucleus. Plant and insect factors that limit virus infection and transmission are discussed.

Molecular cloning, overproduction, purification, and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses

PubMed Central

Zhang, Di; Tözsér, József; Waugh, David S.

2009-01-01

Alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. The nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. Its 39-kDa C-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. In the present study, we evaluated nsp2pro domains from the following three sources as reagents for site-specific cleavage of fusion proteins: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV) and Sindbis Virus (SIN). All three alphavirus proteases cleaved model fusion protein substrates with high specificity but they were much less efficient enzymes than potyviral proteases from tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Oligopeptide substrates were also cleaved with very low efficiency by the alphavirus proteases. We conclude that, in general, alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases. PMID:19013248

A novel monopartite dsRNA virus isolated from the phytopathogenic fungus Ustilaginoidea virens and ancestrally related to a mitochondria-associated dsRNA in the green alga Bryopsis.

PubMed

Zhang, Tingting; Jiang, Yinhui; Dong, Wubei

2014-08-01

In this study, we describe a novel mycovirus isolated from Ustilaginoidea virens, which was designated Ustilaginoidea virens nonsegmented virus 1 (UvNV-1). The sequence analysis revealed that UvNV-1 has two open reading frames (ORFs). ORF1 encodes an unknown protein, which is similar to the hypothetical protein BN7_5177 of Wickerhamomyces ciferrii. ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp), which is most closely related to Bryopsis mitochondria-associated dsRNA (BDRM) and is likely expressed by a +1 ribosomal frameshift within the sequence CCC_UUU_CGA. The phylogenetic analysis of the RdRp of UvNV-1 showed that UvNV-1 represents a new virus taxon of mycoviruses with a partitivirus-like lineage that is classified into the family of picorna-like viruses. Based on northern hybridization, UvNV-1 was found to be common to U. virens from different geographic locations in China. The biological comparison of virus-free and infected fungal strains revealed that UvNV-1 is likely to be cryptic to its host. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Creation of Rift Valley Fever Viruses with Four-Segmented Genomes Reveals Flexibility in Bunyavirus Genome Packaging

PubMed Central

Oreshkova, Nadia; Moormann, Rob J. M.; Kortekaas, Jeroen

2014-01-01

ABSTRACT Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. IMPORTANCE Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines. PMID:25008937

Creation of Rift Valley fever viruses with four-segmented genomes reveals flexibility in bunyavirus genome packaging.

PubMed

Wichgers Schreur, Paul J; Oreshkova, Nadia; Moormann, Rob J M; Kortekaas, Jeroen

2014-09-01

Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

PubMed Central

Vogler, Hannes; Kwon, Myoung-Ok; Dang, Vy; Sambade, Adrian; Fasler, Monika; Ashby, Jamie; Heinlein, Manfred

2008-01-01

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA. PMID:18389061

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.

Pathobiologic Roles of Epstein–Barr Virus-Encoded MicroRNAs in Human Lymphomas

PubMed Central

Navari, Mohsen; Etebari, Maryam; Ibrahimi, Mostafa; Leoncini, Lorenzo

2018-01-01

Epstein–Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs—small RNAs with a posttranscriptional regulating role—are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas. PMID:29649101

The Envelope Glycoprotein of Friend Spleen Focus-Forming Virus Covalently Interacts with and Constitutively Activates a Truncated Form of the Receptor Tyrosine Kinase Stk

PubMed Central

Nishigaki, Kazuo; Thompson, Delores; Hanson, Charlotte; Yugawa, Takashi; Ruscetti, Sandra

2001-01-01

The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene Fv-2, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55P, the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk. PMID:11483734

Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodeling complex.

PubMed

Joubert, D Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J; Paradkar, Prasad N; Moseley, Gregory W; Walker, Peter J

2015-01-15

Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Wongabel Rhabdovirus Accessory Protein U3 Targets the SWI/SNF Chromatin Remodeling Complex

PubMed Central

Joubert, D. Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J.; Paradkar, Prasad N.; Moseley, Gregory W.

2014-01-01

ABSTRACT Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. PMID:25392228

Structural insights into the multifunctional protein VP3 of birnaviruses.

PubMed

Casañas, Arnau; Navarro, Aitor; Ferrer-Orta, Cristina; González, Dolores; Rodríguez, José F; Verdaguer, Núria

2008-01-01

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two alpha-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

PubMed Central

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei

2015-01-01

ABSTRACT Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. PMID:26559827

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

PubMed

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei; Wang, Hualin; Lou, Zhiyong

2016-01-15

Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

An Overview of Live Attenuated Recombinant Pseudorabies Viruses for Use as Novel Vaccines

PubMed Central

Dong, Bo; Zarlenga, Dante S.; Ren, Xiaofeng

2014-01-01

Pseudorabies virus (PRV) is a double-stranded, DNA-based swine virus with a genome approximating 150 kb in size. PRV has many nonessential genes which can be replaced with genes encoding heterologous antigens but without deleterious effects on virus propagation. Recombinant PRVs expressing both native and foreign antigens are able to stimulate immune responses. In this paper, we review the current status of live attenuated recombinant PRVs and live PRV-based vector vaccines with potential for controlling viral infections in animals. PMID:24995348

Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

PubMed Central

Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Kekilli, Demet; Samso, Monserrat

2013-01-01

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle. PMID:23152528

Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

PubMed

Kim, Saet-Byul; Kang, Won-Hee; Huy, Hoang Ngoc; Yeom, Seon-In; An, Jeong-Tak; Kim, Seungill; Kang, Min-Young; Kim, Hyun Jung; Jo, Yeong Deuk; Ha, Yeaseong; Choi, Doil; Kang, Byoung-Cheorl

2017-01-01

Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

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

Johnson, Karen E.; Knipe, David M., E-mail: david_knipe@hms.harvard.ed

2010-01-05

Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expressionmore » is induced after IFNalpha treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27{sup -} HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.« less

Isolation and characterization of a novel mycovirus from Penicillium digitatum.

PubMed

Niu, Yuhui; Zhang, Tingfu; Zhu, Ying; Yuan, Yongze; Wang, Shengqiang; Liu, Jing; Liu, Deli

2016-07-01

A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed spherical particles ~35nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. Copyright © 2016 Elsevier Inc. All rights reserved.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

PubMed Central

Russo, James J.; Bohenzky, Roy A.; Chien, Ming-Cheng; Chen, Jing; Yan, Ming; Maddalena, Dawn; Parry, J. Preston; Peruzzi, Daniela; Edelman, Isidore S.; Chang, Yuan; Moore, Patrick S.

1996-01-01

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. PMID:8962146

Protective immunity and lack of histopathological damage two years after DNA vaccination against infectious hematopoietic necrosis virus in trout

USGS Publications Warehouse

Kurath, Gael; Garver, Kyle A.; Corbeil, Serge; Elliott, Diane G.; Anderson, Eric D.; LaPatra, Scott E.

2006-01-01

The DNA vaccine pIHNw-G encodes the glycoprotein of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination. At three months all fish vaccinated with 0.1 μg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47–69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after two years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination.

Recurrent Loss of APOBEC3H Activity during Primate Evolution.

PubMed

Garcia, Erin I; Emerman, Michael

2018-06-20

Genes in the APOBEC3 family encode cytidine deaminases that provide a barrier against viral infection and retrotransposition. Of all APOBEC3 genes in humans, APOBEC3H ( A3H ) is the most polymorphic: some haplotypes encode stable and active A3H proteins, while others are unstable and poorly antiviral. Such variation in human A3H affects interactions with the lentiviral antagonist Vif, which counteracts A3H via proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions, as well as its evolution in Old World monkeys, we characterized A3H variation within four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of function was partially related to protein expression levels but was also influenced by amino acid mutations in the N-terminus. Moreover, we demonstrate that the evolution of A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests that activity of A3H is evolutionarily dynamic and may have a negative effect on host fitness, resulting in its recurrent loss in primates. IMPORTANCE Adaptation of viruses to their hosts is critical for transmission of viruses between different species. Previous studies had identified changes in a protein from the APOBEC3 family that influenced species-specificity of simian immunodeficiency viruses (SIVs) in African green monkeys. We studied the evolution of a related protein in the same system, APOBEC3H, which has experienced a loss of function in humans. This evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken place during primate evolution suggesting that APOBEC3H places a fitness cost on hosts. The variability of APOBEC3H activity between different primates highlights the differential selective pressures on the APOBEC3 gene family. Copyright © 2018 American Society for Microbiology.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

PubMed

Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

2016-04-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

PubMed Central

Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

2016-01-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses.

PubMed

Tse, Longping V; Moller-Tank, Sven; Meganck, Rita M; Asokan, Aravind

2018-04-25

Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly. Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a distinct role and can be engineered for carrying out new functions. Copyright © 2018 American Society for Microbiology.

Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells

PubMed Central

Shi, Stephanie T.; Schiller, Jennifer J.; Kanjanahaluethai, Amornrat; Baker, Susan C.; Oh, Jong-Won; Lai, Michael M. C.

1999-01-01

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines. PMID:10364348

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

PubMed

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-06-26

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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

2016-12-13

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

Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae

PubMed Central

Arslan, Defne; Legendre, Matthieu; Seltzer, Virginie; Abergel, Chantal; Claverie, Jean-Michel

2011-01-01

Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein). PMID:21987820

Bovine Herpes Virus 1 (BHV-1) and Herpes Simplex Virus Type 1 (HSV-1) Promote Survival of Latently Infected Sensory Neurons, in Part by Inhibiting Apoptosis

PubMed Central

Jones, Clinton

2013-01-01

α-Herpesvirinae subfamily members, including herpes simplex virus type 1 (HSV-1) and bovine herpes virus 1 (BHV-1), initiate infection in mucosal surfaces. BHV-1 and HSV-1 enter sensory neurons by cell-cell spread where a burst of viral gene expression occurs. When compared to non-neuronal cells, viral gene expression is quickly extinguished in sensory neurons resulting in neuronal survival and latency. The HSV-1 latency associated transcript (LAT), which is abundantly expressed in latently infected neurons, inhibits apoptosis, viral transcription, and productive infection, and directly or indirectly enhances reactivation from latency in small animal models. Three anti-apoptosis genes can be substituted for LAT, which will restore wild type levels of reactivation from latency to a LAT null mutant virus. Two small non-coding RNAs encoded by LAT possess anti-apoptosis functions in transfected cells. The BHV-1 latency related RNA (LR-RNA), like LAT, is abundantly expressed during latency. The LR-RNA encodes a protein (ORF2) and two microRNAs that are expressed in certain latently infected neurons. Wild-type expression of LR gene products is required for stress-induced reactivation from latency in cattle. ORF2 has anti-apoptosis functions and interacts with certain cellular transcription factors that stimulate viral transcription and productive infection. ORF2 is predicted to promote survival of infected neurons by inhibiting apoptosis and sequestering cellular transcription factors which stimulate productive infection. In addition, the LR encoded microRNAs inhibit viral transcription and apoptosis. In summary, the ability of BHV-1 and HSV-1 to interfere with apoptosis and productive infection in sensory neurons is crucial for the life-long latency-reactivation cycle in their respective hosts. PMID:25278776

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

PubMed Central

Boccara, Martine; Hamilton, William D. O.; Baulcombe, David C.

1986-01-01

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor. ImagesFig. 2.Fig. 3. PMID:16453668

Different haplotypes encode the same protein for independent sources of zucchini yellow mosaic virus resistance in cucumber

USDA-ARS?s Scientific Manuscript database

Cucumber (Cucumis sativus) production is negatively affected by zucchini yellow mosaic virus (ZYMV). Three sources of ZYMV resistance have been commercially deployed and all three resistances are conditioned by a single recessive gene. A vacuolar protein sorting-associated protein 4-like (VPS4-like)...

Identification of a maize chlorotic dwarf virus silencing suppressor protein

USDA-ARS?s Scientific Manuscript database

Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389 kDa proteolytically processed polyprotein. We show that an N-terminal 78kDa polyprotein (R78) has silencing suppressor activity, that it is cleaved by the viral...

Complete genome sequence of a divergent strain of Japanese yam mosaic virus from China

USDA-ARS?s Scientific Manuscript database

A novel strain of Japanese yam mosaic virus (JYMV-CN) was identified in a yam plant with foliar mottle symptoms in China. The complete genomic sequence of JYMV-CN was determined. Its genomic sequence of 9701 nucleotides encodes a polyprotein of 3247 amino acids. Its organization was virtually identi...

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

PubMed

Koehler, Heather; Cotsmire, Samantha; Langland, Jeffrey; Kibler, Karen V; Kalman, Daniel; Upton, Jason W; Mocarski, Edward S; Jacobs, Bertram L

2017-10-24

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species.

PubMed

Martins, Mathias; Joshi, Lok R; Rodrigues, Fernando S; Anziliero, Deniz; Frandoloso, Rafael; Kutish, Gerald F; Rock, Daniel L; Weiblen, Rudi; Flores, Eduardo F; Diel, Diego G

2017-11-01

The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV ∆024 RABV-G or ORFV ∆121 RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV ∆024 RABV-G and ORFV ∆121 RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV ∆121 RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV ∆024 RABV-G-immunized animals, indicating a higher immunogenicity of ORFV Δ121 -based vectors in these animal species. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of giant Mimivirus protein functions using RNA interference

PubMed Central

Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

2015-01-01

Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases. PMID:25972846

Identification of Strawberry vein banding virus encoded P6 as an RNA silencing suppressor.

PubMed

Feng, Mingfeng; Zuo, Dengpan; Jiang, Xizi; Li, Shuai; Chen, Jing; Jiang, Lei; Zhou, Xueping; Jiang, Tong

2018-07-01

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Therapeutic implications of Epstein–Barr virus infection for the treatment of nasopharyngeal carcinoma

PubMed Central

Hutajulu, Susanna Hilda; Kurnianda, Johan; Tan, I Bing; Middeldorp, Jaap M

2014-01-01

Nasopharyngeal carcinoma (NPC) is highly endemic in certain regions including the People’s Republic of China and Southeast Asia. Its etiology is unique and multifactorial, involving genetic background, epigenetic, and environment factors, including Epstein–Barr virus (EBV) infection. The presence of EBV in all tumor cells, aberrant pattern of antibodies against EBV antigens in patient sera, and elevated viral DNA in patient circulation as well as nasopharyngeal site underline the role of EBV during NPC development. In NPC tumors, EBV expresses latency type II, where three EBV-encoded proteins, Epstein–Barr nuclear antigen 1, latent membrane protein 1 and 2 (LMP1, 2), are expressed along with BamH1-A rightward reading frame 1, Epstein–Barr virus-encoded small nuclear RNAs, and BamH1-A rightward transcripts. Among all encoded proteins, LMP1 plays a central role in the propagation of NPC. Standard treatment of NPC consists of radiotherapy with or without chemotherapy for early stage, concurrent chemoradiotherapy in locally advanced tumors, and palliative systemic chemotherapy in metastatic disease. However, this standard care has limitations, allowing recurrences and disease progression in a certain proportion of cases. Although the pathophysiological link and molecular process of EBV-induced oncogenesis are not fully understood, therapeutic approaches targeting the virus may increase the cure rate and add clinical benefit. The promising results of early phase clinical trials on EBV-specific immunotherapy, epigenetic therapy, and treatment with viral lytic induction offer new options for treating NPC. PMID:25228810

Molecular Determinants of Human T-lymphotropic Virus Type 1 Transmission and Spread

PubMed Central

Lairmore, Michael D.; Anupam, Rajaneesh; Bowden, Nadine; Haines, Robyn; Haynes, Rashade A. H.; Ratner, Lee; Green, Patrick L.

2011-01-01

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1. PMID:21994774

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

PubMed Central

Huang, M; Jolicoeur, P

1990-01-01

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus. Images PMID:2243376

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

PubMed Central

Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

2010-01-01

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

Identification of VP1/2A and 2C as Virulence Genes of Hepatitis A Virus and Demonstration of Genetic Instability of 2C

PubMed Central

Emerson, Suzanne U.; Huang, Ying K.; Nguyen, Hanh; Brockington, Alicia; Govindarajan, Sugantha; St. Claire, Marisa; Shapiro, Max; Purcell, Robert H.

2002-01-01

Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins. PMID:12163575

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.

Comparison of immune responses to different foot-and-mouth disease genetically engineered vaccines in guinea pigs.

PubMed

Yao, Qingxia; Qian, Ping; Huang, Qinfeng; Cao, Yi; Chen, Huanchun

2008-01-01

The P12A3C gene from FMDV (serotype O) encoding the capsid precursor protein, and the highly immunogenic gene FHG, which encodes multiple epitopes of FMDV capsid proteins, were inserted into eukaryotic expression vectors to compare different candidate genetically engineered vaccines for foot-and-mouth disease (FMD). A modified live pseudorabies virus (MLPRV) was also used to deliver P12A3C. Guinea pigs were inoculated intramuscularly with the candidate vaccines to compare the ability to elicit immunity of the DNA vector and a live viral vector. An indirect enzyme-linked immunosorbent assay (iELISA), virus-neutralization test and lymphoproliferation assay were used to detect antibody and cellular responses. The group immunized with P12A3C delivered by MLPRV produced significantly greater antibody and cellular responses indicating that MLPRV has a greater ability to mediate exogenous gene delivery than the plasmid DNA vector. Comparison of the immune responses induced by P12A3C and FHG, which were both mediated by DNA plasmids, showed that FHG and P12A3C elicited similar cellular responses, while P12A3C induced higher antibody levels, suggesting that P12A3C is a more powerful immunogen than FHG. In challenge experiments, guinea pigs vaccinated with P12A3C delivered by MLPRV were protected fully from FMDV challenge, whereas guinea pigs vaccinated with P12A3C or FHG delivered by DNA plasmid were only protected partially. This study provides a basis for future construction of a genetically engineered vaccine for FMDV.

Non-Structural Proteins of Arthropod-Borne Bunyaviruses: Roles and Functions

PubMed Central

Eifan, Saleh; Schnettler, Esther; Dietrich, Isabelle; Kohl, Alain; Blomström, Anne-Lie

2013-01-01

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod. PMID:24100888

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Phylogenetic relationships and the occurrence of interspecific recombination between beet chlorosis virus (BChV) and Beet mild yellowing virus (BMYV).

PubMed

Kozlowska-Makulska, Anna; Hasiow-Jaroszewska, Beata; Szyndel, Marek S; Herrbach, Etienne; Bouzoubaa, Salah; Lemaire, Olivier; Beuve, Monique

2015-02-01

Samples containing two viruses belonging to the genus Polerovirus, beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV), were collected from French and Polish sugar beet fields. The molecular properties of 24 isolates of BChV and BMYV were investigated, and their genetic diversity was examined in the coat protein (CP)- and P0-encoding genes. For the first time, we have demonstrated that beet polerovirus populations include recombinants between BChV and BMYV containing breakpoints within the CP gene. Moreover, a partial correlation between geographic origin and phylogenetic clustering was observed for BMYV isolates.

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

PubMed Central

Mundt, E; Vakharia, V N

1996-01-01

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines. Images Fig. 2 Fig. 3 Fig. 4 PMID:8855321

Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein.

PubMed

Allison, A B; Palacios, G; Travassos da Rosa, A; Popov, V L; Lu, L; Xiao, S Y; DeToy, K; Briese, T; Lipkin, W I; Keel, M K; Stallknecht, D E; Bishop, G R; Tesh, R B

2011-01-01

The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. Copyright © 2010 Elsevier B.V. All rights reserved.

Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein

PubMed Central

Allison, A. B.; Palacios, G.; Rosa, A. Travassos da; Popov, V. L.; Lu, L.; Xiao, S. Y.; DeToy, K.; Briese, T.; Lipkin, W. Ian; Keel, M. K.; Stallknecht, D. E.; Bishop, G. R.; Tesh, R. B.

2010-01-01

The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase proteins indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. PMID:20863863

Epstein–Barr virus latent genes

PubMed Central

Kang, Myung-Soo; Kieff, Elliott

2015-01-01

Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized. PMID:25613728

The open reading frames in the 3' long terminal repeats of several mouse mammary tumor virus integrants encode V beta 3-specific superantigens

PubMed Central

1992-01-01

Mice expressing the minor lymphocyte stimulation antigens, Mls-1a, -2a, or -3a, singly on the B10.BR background have been generated. Mls phenotypes correlate with the integration of mouse mammary tumor viruses (MTV) in the mouse genome. The open reading frames within the 3' long terminal repeats of the integrated MTVs 1, 3, 6, and 13 encode V beta 3-specific superantigens. Sequence data for these viral superantigens is presented, indicating that it is the COOH-terminal portion of the viral superantigen that interacts with the T cell receptor V beta element. PMID:1309854

Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

PubMed Central

Pruss, G; Ge, X; Shi, X M; Carrington, J C; Bowman Vance, V

1997-01-01

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication. PMID:9212462

Epstein–Barr virus in the multiple sclerosis brain: a controversial issue—report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria

PubMed Central

Niedobitek, Gerald; Aloisi, Francesca; Middeldorp, Jaap M.

2011-01-01

Recent epidemiological and immunological studies provide evidence for an association between Epstein–Barr virus infection and multiple sclerosis, suggesting a role of Epstein–Barr virus infection in disease induction and pathogenesis. A key question in this context is whether Epstein–Barr virus-infected B lymphocytes are present within the central nervous system and the lesions of patients with multiple sclerosis. Previous studies on this topic provided highly controversial results, showing Epstein–Barr virus reactivity in B cells in the vast majority of multiple sclerosis cases and lesions, or only exceptional Epstein–Barr virus-positive B cells in rare cases. In an attempt to explain the reasons for these divergent results, a workshop was organized under the umbrella of the European Union FP6 NeuroproMiSe project, the outcome of which is presented here. This report summarizes the current knowledge of Epstein–Barr virus biology and shows that Epstein–Barr virus infection is highly complex. There are still major controversies, how to unequivocally identify Epstein–Barr virus infection in pathological tissues, particularly in situations other than Epstein–Barr virus-driven lymphomas or acute Epstein–Barr virus infections. It further highlights that unequivocal proof of Epstein–Barr virus infection in multiple sclerosis lesions is still lacking, due to issues related to the sensitivity and specificity of the detection methods. PMID:21846731

In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

PubMed Central

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T.; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-01-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/). PMID:12634390

In silico pattern-based analysis of the human cytomegalovirus genome.

PubMed

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-04-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

The Influence of Multiple Host Contacts on the Acquisition and Transmission of Dengue-2 Virus

DTIC Science & Technology

1993-01-01

5500 Stanoard Form 298 (Rev 2-89) PIs~~ bv AtI %t .34-| II Best Available Copy ABSTRACT Title of Dissertation: THE INFLUENCE OF MULTIPLE HOST CONTACTS...ABSTRACT Title of the Dissertaton: THE INFLUENCE OF MULTIPLE HOST CONTACTS ON THE ACQUISITION AND TRANSMISSION OF DENGUE-2 VIRUS BY A=DISAEX2 . John L...virus does not alter the feeding behavior of An. n•ngy~i. THE INFLUENCE OF MULTIPLE HOST CONTACTS ON THE ACQUISITION AND TRANSMISSION OF DENGUE-2 VIRUS BY

A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus.

PubMed

Sela, Noa; Lachman, Oded; Reingold, Victoria; Dombrovsky, Aviv

2013-10-01

A novel virus was detected in watermelon plants (Citrullus lanatus Thunb.) infected with Melon necrotic spot virus (MNSV) using SOLiD next-generation sequence analysis. In addition to the expected MSNV genome, two double-stranded RNA (dsRNA) segments of 1,312 and 1,118 bp were also identified and sequenced from the purified virus preparations. These two dsRNA segments encode two putative partitivirus-related proteins, an RNA-dependent RNA polymerase (RdRP) and a capsid protein, which were sequenced. Genomic-sequence analysis and analysis of phylogenetic relationships indicate that these two dsRNAs together make up the genome of a novel Partitivirus. This virus was found to be closely related to the Pepper cryptic virus 1 and Raphanus sativus cryptic virus. It is suggested that this novel virus putatively named Citrullus lanatus cryptic virus be considered as a new member of the family Partitiviridae.

The enigmatic archaeal virosphere.

PubMed

Prangishvili, David; Bamford, Dennis H; Forterre, Patrick; Iranzo, Jaime; Koonin, Eugene V; Krupovic, Mart

2017-11-10

One of the most prominent features of archaea is the extraordinary diversity of their DNA viruses. Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic viruses and represent unique virus families. The distinct nature of archaeal viruses also extends to the gene composition and architectures of their genomes and the properties of the proteins that they encode. Environmental research has revealed prominent roles of archaeal viruses in influencing microbial communities in ocean ecosystems, and recent metagenomic studies have uncovered new groups of archaeal viruses that infect extremophiles and mesophiles in diverse habitats. In this Review, we summarize recent advances in our understanding of the genomic and morphological diversity of archaeal viruses and the molecular biology of their life cycles and virus-host interactions, including interactions with archaeal CRISPR-Cas systems. We also examine the potential origins and evolution of archaeal viruses and discuss their place in the global virosphere.

Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes.

PubMed

Colson, Philippe; La Scola, Bernard; Raoult, Didier

2017-09-29

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Cardiomyopathy Syndrome of Atlantic Salmon (Salmo salar L.) Is Caused by a Double-Stranded RNA Virus of the Totiviridae Family▿

PubMed Central

Haugland, Øyvind; Mikalsen, Aase B.; Nilsen, Pål; Lindmo, Karine; Thu, Beate J.; Eliassen, Trygve M.; Roos, Norbert; Rode, Marit; Evensen, Øystein

2011-01-01

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease. PMID:21411528

Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile.

PubMed

Kibenge, F S; Gárate, O N; Johnson, G; Arriagada, R; Kibenge, M J; Wadowska, D

2001-05-04

The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV.

The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

PubMed

Gammon, Don B; Evans, David H

2009-05-01

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

PubMed Central

Tugume, Arthur K.; Amayo, Robert; Weinheimer, Isabel; Mukasa, Settumba B.; Rubaihayo, Patrick R.; Valkonen, Jari P. T.

2013-01-01

Background The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. Methodology/Principal Findings Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis. Conclusions/Significance SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV. PMID:24278443

Evidence for positive selection and recombination hotspots in Deformed wing virus (DWV).

PubMed

Dalmon, A; Desbiez, C; Coulon, M; Thomasson, M; Le Conte, Y; Alaux, C; Vallon, J; Moury, B

2017-01-25

Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.

Construction and characterization of a recombinant invertebrate iridovirus.

PubMed

Ozgen, Arzu; Muratoglu, Hacer; Demirbag, Zihni; Vlak, Just M; van Oers, Monique M; Nalcacioglu, Remziye

2014-08-30

Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol. Copyright © 2014 Elsevier B.V. All rights reserved.

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

PubMed

Kines, Kristine J; Mann, Victoria H; Morales, Maria E; Shelby, Bryan D; Kalinna, Bernd H; Gobert, Geoffrey N; Chirgwin, Sharon R; Brindley, Paul J

2006-04-01

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

The Ep152R ORF of African swine fever virus strain Georgia encodes for an essential gene that interacts with host protein BAG6.

PubMed

Borca, Manuel V; O'Donnell, Vivian; Holinka, Lauren G; Rai, Devendra K; Sanford, Brenton; Alfano, Marialexia; Carlson, Jolene; Azzinaro, Paul A; Alonso, Covadonga; Gladue, Douglas P

2016-09-02

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few of these genes have been studied in some detail. Here we report the characterization of open reading frame Ep152R that has a predicted complement control module/SCR domain. This domain is found in Vaccinia virus proteins that are involved in blocking the immune response during viral infection. A recombinant ASFV harboring a HA tagged version of the Ep152R protein was developed (ASFV-G-Ep152R-HA) and used to demonstrate that Ep152R is an early virus protein. Attempts to construct recombinant viruses having a deleted Ep152R gene were consistently unsuccessful indicating that Ep152R is an essential gene. Interestingly, analysis of host-protein interactions for Ep152R using a yeast two-hybrid screen, identified BAG6, a protein previously identified as being required for ASFV replication. Furthermore, fluorescent microscopy analysis confirms that Ep152R-BAG6 interaction actually occurs in cells infected with ASFV. Published by Elsevier B.V.

Functional characterization of the triple gene block 1 (TGB1) gene of Pepino mosaic virus in tomato

USDA-ARS?s Scientific Manuscript database

Pepino mosaic virus (PepMV) has caused serious economic losses to many greenhouse tomato productions around the world. This potexvirus genome contains five major open reading frames (ORFs) encoding for a 164-kDa RNA-dependent RNA polymerase (RdRp), three triple gene block (TGB) proteins of 26, 14 an...

Complete genome sequence of Paris mosaic necrosis virus, a distinct member of the genus Potyvirus

USDA-ARS?s Scientific Manuscript database

The complete genomic sequence of a novel potyvirus was determined from Paris polyphylla var. yunnanensis. Its genomic RNA consists of 9,660 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing a single open reading frame (ORF) encoding a large polyprotein. The virus shares 52.1-69.7%...

Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus

Treesearch

David S. Bischoff; James M. Slavicek

1997-01-01

A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear...

Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

Treesearch

David S. Bischoff; James M. Slavicek

1995-01-01

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted Mr of 22000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M...

Homodimerization of Marek's disease virus encoded Meq protein is not sufficient for transformation of lymphocytes in chicken

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), the etiologic agent of Marek’s disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV codes for an oncoprotein, Meq, which shares resemblance wit...

Complete genomic sequence of a tobacco rattle virus isolate from Michigan-grown potatoes

USDA-ARS?s Scientific Manuscript database

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16 kDa gene of the...

Rift Valley fever virus structural and non-structural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep

USDA-ARS?s Scientific Manuscript database

The Rift Valley fever virus (RVFV) encodes structural proteins, nucleoprotein (N), N-terminus glycoprotein (Gn), C-terminus glycoprotein (Gc) and L protein, 78-kDa and non-structural proteins NSm and NSs. Using the baculovirus system we expressed the full-length coding sequence of N, NSs, NSm, Gc an...

Genetic characterization of novel putative rhabdovirus and dsRNA virus from Japanese persimmon.

PubMed

Ito, Takao; Suzaki, Koichi; Nakano, Masaaki

2013-08-01

Deep-sequencing analysis of nucleic acids from leaf tissue of Japanese persimmon trees exhibiting fruit apex disorder in some fruits detected two molecules that were graft transmitted to healthy seedlings. One of the complete genomes consisted of 13 467 nt and encoded six genes similar to those of plant rhabdoviruses. The virus formed a distinct cluster in the genus Cytorhabdovirus with lettuce necrotic yellows virus, lettuce yellow mottle virus and strawberry crinkle virus in a phylogenetic tree based on the L protein (RNA-dependent RNA polymerase, RdRp). The other consisted of 7475 nt and shared a genome organization similar to those of some insect and fungal viruses having dsRNA genomes. In a phylogenetic tree using the RdRp sequence of several unassigned dsRNA viruses, the virus formed a possible new genus cluster with two insect viruses, Circulifer tenellus virus 1 and Spissistilus festinus virus 1, and one plant virus, cucurbit yellows-associated virus.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

PubMed

Luu, Van-Trinh; Moon, Hye Yun; Hwang, Jee Youn; Kang, Bo-Kyu; Kang, Hyun Ah

2017-08-01

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC.

PubMed

Jarosinski, Keith W; Margulis, Neil G; Kamil, Jeremy P; Spatz, Stephen J; Nair, Venugopal K; Osterrieder, Nikolaus

2007-10-01

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

CARD games between virus and host get a new player.

PubMed

Johnson, Cynthia L; Gale, Michael

2006-01-01

A growing family of cellular proteins encoding the caspase activation and recruitment domain (CARD) has a crucial role in immunity by sensing virus infection and signaling antiviral immune defenses. Four independent studies have identified a novel CARD-containing protein, variously called IPS-1, MAVS, VISA and Cardif, which is an essential signaling adaptor of the host defense mediating CARD-CARD interactions with retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDAS), sensors of virus infection. Disruption of this novel signaling pathway by hepatitis C virus (HCV) might provide a foundation for viral persistence.

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

PubMed Central

Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

2013-01-01

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

Respiratory viruses among children with non-severe community-acquired pneumonia: A prospective cohort study.

PubMed

Nascimento-Carvalho, Amanda C; Vilas-Boas, Ana-Luisa; Fontoura, Maria-Socorro H; Vuorinen, Tytti; Nascimento-Carvalho, Cristiana M

2018-06-06

Community-acquired pneumonia (CAP) causes a major burden to the health care system among children under-5 years worldwide. Information on respiratory viruses in non-severe CAP cases is scarce. To estimate the frequency of respiratory viruses among non-severe CAP cases. Prospective study conducted in Salvador, Brazil. Out of 820 children aged 2-59 months with non-severe CAP diagnosed by pediatricians (respiratory complaints and radiographic pulmonary infiltrate/consolidation), recruited in a clinical trial (ClinicalTrials.gov Identifier NCT01200706), nasopharyngeal aspirate samples were obtained from 774 (94.4%) patients and tested for 16 respiratory viruses by PCRs. Viruses were detected in 708 (91.5%; 95%CI: 89.3-93.3) cases, out of which 491 (69.4%; 95%CI: 65.9-72.7) harbored multiple viruses. Rhinovirus (46.1%; 95%CI: 42.6-49.6), adenovirus (38.4%; 95%CI: 35.0-41.8), and enterovirus (26.5%; 95%CI: 23.5-29.7) were the most commonly found viruses. The most frequent combination comprised rhinovirus plus adenovirus. No difference was found in the frequency of RSVA (16.1% vs. 14.6%; P = 0.6), RSVB (10.9% vs. 13.2%; P = 0.4) influenza (Flu) A (6.3% vs. 5.1%; P = 0.5), FluB (4.5% vs. 1.8%; P = 0.09), parainfluenza virus (PIV) 1 (5.1% vs. 2.8%; P = 0.2), or PIV4 (7.7% vs. 4.1%; P = 0.08), when children with multiple or sole virus detection were compared. Conversely, rhinovirus, adenovirus, enterovirus, bocavirus, PIV2, PIV3, metapneumovirus, coronavirus OC43, NL63, 229E were significantly more frequent among cases with multiple virus detection. Respiratory viruses were detected in over 90% of the cases, out of which 70% had multiple viruses. Several viruses are more commonly found in multiple virus detection whereas other viruses are similarly found in sole and in multiple virus detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.