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  • Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA.
    J. Virol. (IF 4.324) Pub Date : 2019-09-20
    D Sherlock,J X Leong,P C M Fogg

    Genetic exchange mediated by viruses of bacteria (bacteriophages) is the primary driver of rapid bacterial evolution. The priority of viruses is usually to propagate themselves. Most bacteriophages use the small terminase protein to identify their own genome and direct its inclusion into phage capsids. Gene transfer agents (GTAs) are descended from bacteriophages, but they instead package fragments of the entire bacterial genome without preference for their own genes. GTAs do not selectively target specific DNA, and no GTA small terminases are known. Here, we identified the small terminase from the model Rhodobacter capsulatus GTA, which then allowed prediction of analogues in other species. We examined the role of the small terminase in GTA production and propose a structural basis for random DNA packaging.IMPORTANCE Random transfer of any and all genes between bacteria could be influential in the spread of virulence or antimicrobial resistance genes. Discovery of the true prevalence of GTAs in sequenced genomes is hampered by their apparent similarity to bacteriophages. Our data allowed the prediction of small terminases in diverse GTA producer species, and defining the characteristics of a "GTA-type" terminase could be an important step toward novel GTA identification. Importantly, the GTA small terminase shares many features with its phage counterpart. We propose that the GTA terminase complex could become a streamlined model system to answer fundamental questions about double-stranded DNA (dsDNA) packaging by viruses that have not been forthcoming to date.

    更新日期:2019-11-01
  • Specific Activation In Vivo of HIV-1 by a Bromodomain Inhibitor from Monocytic Cells in Humanized Mice under Antiretroviral Therapy.
    J. Virol. (IF 4.324) Pub Date : 2019-04-12
    Guangming Li,Zheng Zhang,Natalia Reszka-Blanco,Feng Li,Liqun Chi,Jianping Ma,Jerry Jeffrey,Liang Cheng,Lishan Su

    Combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and enables HIV‑infected individuals to live long, productive lives. However, the persistence of HIV-1 reservoirs of both T and myeloid cells with latent or low-replicating HIV-1 in patients under cART makes HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies to activate and purge these reservoirs. Bromodomain and extraterminal domain proteins (BETs) are epigenetic readers involved in modulating gene expression. Several bromodomain inhibitors (BETi) are reported to activate viral transcription in vitro in HIV-1 latency cell lines in a P-TEFb (CDK9/cyclin T1)-dependent manner. Little is known about BETi efficacy in activating HIV-1 reservoir cells under cART in vivo Here we report that a BETi (I-BET151) efficiently activated HIV-1 reservoirs under effective cART in humanized mice in vivo Interestingly, I-BET151 during suppressive cART in vivo activated HIV-1 gene expression only in monocytic cells and not in CD4+ T cells. We further demonstrate that BETi preferentially enhanced HIV-1 gene expression in monocytic cells rather than in T cells and that whereas CDK9 was involved in activating HIV-1 by I-BET151 in both monocytic and T cells, CDK2 enhanced HIV-1 transcription in monocytic cells but inhibited it in T cells. Our findings reveal a role for CDK2 in differential modulation of HIV-1 gene expression in myeloid cells and in T cells and provide a novel strategy to reactivate monocytic reservoirs with BETi during cART.IMPORTANCE Bromodomain inhibitors have been reported to activate HIV-1 transcription in vitro, but their effect on activation of HIV-1 reservoirs during cART in vivo is unclear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells, in cART-treated mice. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in HIV-1-infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells and that a combination of reservoir activation agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

    更新日期:2019-11-01
  • Human Papillomavirus 11 Early Protein E6 Activates Autophagy by Repressing AKT/mTOR and Erk/mTOR.
    J. Virol. (IF 4.324) Pub Date : 2019-04-12
    Boya Zhang,Yinjing Song,Siyuan Sun,Rui Han,Chunting Hua,Stijn van der Veen,Hao Cheng

    Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which are a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 (11E6) plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-green fluorescent protein-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knock down 11E6 and showed that these effects require continued 11E6 expression. Compared with its expression in the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of the decreased phosphorylation levels of the canonical autophagy repressor mammalian target of rapamycin (mTOR) at its Ser2448 position (the mTOR complex 1 [mTORC1] phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.IMPORTANCE We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.

    更新日期:2019-11-01
  • Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Hanh T Nguyen,Nirmin Alsahafi,Andrés Finzi,Joseph G Sodroski

    Most broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.

    更新日期:2019-11-01
  • Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Suppresses Human Adenovirus Gene Expression and Replication.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Bratati Saha,Robin J Parks

    Human adenovirus (HAdV) causes minor illnesses in most patients but can lead to severe disease and death in pediatric, geriatric, and immunocompromised individuals. No approved antiviral therapy currently exists for the treatment of these severe HAdV-induced diseases. In this study, we show that the pan-histone deacetylase (HDAC) inhibitor SAHA reduces HAdV-5 gene expression and DNA replication in tissue culture, ultimately decreasing virus yield from infected cells. Importantly, SAHA also reduced gene expression from more virulent and clinically relevant serotypes, including HAdV-4 and HAdV-7. In addition to SAHA, several other HDAC inhibitors (e.g., trichostatin A, apicidin, and panobinostat) also affected HAdV gene expression. We determined that loss of class I HDAC activity, mainly HDAC2, impairs efficient expression of viral genes, and that E1A physically interacts with HDAC2. Our results suggest that HDAC activity is necessary for HAdV replication, which may represent a novel pharmacological target in HAdV-induced disease.IMPORTANCE Although human adenovirus (HAdV) can cause severe diseases that can be fatal in some populations, there are no effective treatments to combat HAdV infection. In this study, we determined that the pan-histone deacetylase (HDAC) inhibitor SAHA has inhibitory activity against several clinically relevant serotypes of HAdV. This U.S. Food and Drug Administration-approved compound affects various stages of the virus lifecycle and reduces virus yield even at low concentrations. We further report that class I HDAC activity, particularly HDAC2, is required for efficient expression of viral genes during lytic infection. Investigation of the mechanism underlying SAHA-mediated suppression of HAdV gene expression and replication will enhance current knowledge of virus-cell interaction and may aid in the development of more effective antivirals with lower toxicity for the treatment of HAdV infections.

    更新日期:2019-11-01
  • Arterivirus nsp4 Antagonizes Interferon Beta Production by Proteolytically Cleaving NEMO at Multiple Sites.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Jiyao Chen,Dang Wang,Zheng Sun,Li Gao,Xinyu Zhu,Jiahui Guo,Shangen Xu,Liurong Fang,Kui Li,Shaobo Xiao

    Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-β) production by cleaving the NF-κB essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-β production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-β production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-β transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-β-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

    更新日期:2019-11-01
  • Development of Influenza B Universal Vaccine Candidates Using the "Mosaic" Hemagglutinin Approach.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Weina Sun,Ericka Kirkpatrick,Megan Ermler,Raffael Nachbagauer,Felix Broecker,Florian Krammer,Peter Palese

    Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. However, protection by current seasonal vaccines is suboptimal due to the antigenic changes of the circulating strains. In this study, we report a novel universal influenza B virus vaccination strategy based on "mosaic" hemagglutinins. We generated mosaic B hemagglutinins by replacing the major antigenic sites of the type B hemagglutinin with corresponding sequences from exotic influenza A hemagglutinins and expressed them as soluble trimeric proteins. Sequential vaccination with recombinant mosaic B hemagglutinin proteins conferred cross-protection against both homologous and heterologous influenza B virus strains in the mouse model. Of note, we rescued recombinant influenza B viruses expressing mosaic B hemagglutinins, which could serve as the basis for a universal influenza B virus vaccine.IMPORTANCE This work reports a universal influenza B virus vaccination strategy based on focusing antibody responses to conserved head and stalk epitopes of the hemagglutinin. Recombinant mosaic influenza B hemagglutinin proteins and recombinant viruses have been generated as novel vaccine candidates. This vaccine strategy provided broad cross-protection in the mouse model. Our findings will inform and drive development toward a more effective influenza B virus vaccine.

    更新日期:2019-11-01
  • C19ORF66 Broadly Escapes Virus-Induced Endonuclease Cleavage and Restricts Kaposi's Sarcoma-Associated Herpesvirus.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    William Rodriguez,Kumaraman Srivastav,Mandy Muller

    One striking characteristic of certain herpesviruses is their ability to induce rapid and widespread RNA decay in order to gain access to host resources. This phenotype is induced by viral endoribonucleases, including SOX in Kaposi's sarcoma-associated herpesvirus (KSHV), muSOX in murine gammaherpesvirus 68 (MHV68), BGLF5 in Epstein-Barr virus (EBV), and vhs in herpes simplex virus 1 (HSV-1). Here, we performed comparative transcriptome sequencing (RNA-seq) upon expression of these herpesviral endonucleases in order to characterize their effect on the host transcriptome. Consistent with previous reports, we found that approximately two-thirds of transcripts were downregulated in cells expressing any of these viral endonucleases. Among the transcripts spared from degradation, we uncovered a cluster of transcripts that systematically escaped degradation from all tested endonucleases. Among these escapees, we identified C19ORF66 and reveal that this transcript is protected from degradation by its 3' untranslated region (UTR). We then show that C19ORF66 is a potent KSHV restriction factor by impeding early viral gene expression, suggesting that its ability to escape viral cleavage may be an important component of the host response to viral infection. Collectively, our comparative approach is a powerful tool to pinpoint key regulators of the viral-host interplay and led us to uncover a novel KSHV regulator.IMPORTANCE Viruses are master regulators of the host gene expression machinery. This is crucial to promote viral infection and to dampen host immune responses. Many viruses, including herpesviruses, express RNases that reduce host gene expression through widespread mRNA decay. However, it emerged that some mRNAs escape this fate, although it has been difficult to determine whether these escaping transcripts benefit viral infection or instead participate in an antiviral mechanism. To tackle this question, we compared the effect of the herpesviral RNases on the human transcriptome and identified a cluster of transcripts consistently escaping degradation from all tested endonucleases. Among the protected mRNAs, we identified the transcript C19ORF66 and showed that it restricts Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Collectively, these results provide a framework to explore how the control of RNA fate in the context of viral-induced widespread mRNA degradation may influence the outcome of viral infection.

    更新日期:2019-11-01
  • Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Derek L Carbaugh,Ralph S Baric,Helen M Lazear

    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice.IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

    更新日期:2019-11-01
  • Reconstruction of the Genetic History and the Current Spread of HIV-1 Subtype A in Germany.
    J. Virol. (IF 4.324) Pub Date : 2019-04-05
    Kirsten Hanke,Nuno Rodrigues Faria,Denise Kühnert,Kaveh Pouran Yousef,Andrea Hauser,Karolin Meixenberger,Alexandra Hofmann,Viviane Bremer,Barbara Bartmeyer,Oliver Pybus,Claudia Kücherer,Max von Kleist,Norbert Bannert

    HIV-1 non-B infections have been increasing in Europe for several years. In Germany, subtype A belongs to the most abundant non-B subtypes showing an increasing prevalence of 8.3% among new infections in 2016. Here we trace the origin and examine the current spread of the German HIV-1 subtype A epidemic. Bayesian coalescence and birth-death analyses were performed with 180 German HIV-1 pol sequences and 528 related and publicly available sequences to reconstruct the population dynamics and fluctuations for each of the transmission groups. Our reconstructions indicate two distinct sources of the German subtype A epidemic, with an Eastern European and an Eastern African lineage both cocirculating in the country. A total of 13 German-origin clusters were identified; among these, 6 clusters showed recent activity. Introductions leading to further countrywide spread originated predominantly from Eastern Africa when introduced before 2005. Since 2005, however, spreading introductions have occurred exclusively within the Eastern European clade. Moreover, we observed changes in the main route of subtype A transmission. The beginning of the German epidemic (1985 to 1995) was dominated by heterosexual transmission of the Eastern African lineage. Since 2005, transmissions among German men who have sex with men (MSM) have been increasing and have been associated with the Eastern European lineage. Infections among people who inject drugs dominated between 1998 and 2005. Our findings on HIV-1 subtype A infections provide new insights into the spread of this virus and extend the understanding of the HIV epidemic in Germany.IMPORTANCE HIV-1 subtype A is the second most prevalent subtype worldwide, with a high prevalence in Eastern Africa and Eastern Europe. However, an increase of non-B infections, including subtype A infections, has been observed in Germany and other European countries. There has simultaneously been an increased flow of refugees into Europe and especially into Germany, raising the question of whether the surge in non-B infections resulted from this increased immigration or whether German transmission chains are mainly involved. This study is the first comprehensive subtype A study from a western European country analyzing in detail its phylogenetic origin, the impact of various transmission routes, and its current spread. The results and conclusions presented provide new and substantial insights for virologists, epidemiologists, and the general public health sector. In this regard, they should be useful to those authorities responsible for developing public health intervention strategies to combat the further spread of HIV/AIDS.

    更新日期:2019-11-01
  • Voltage-Dependent Anion Channel Protein 2 (VDAC2) and Receptor of Activated Protein C Kinase 1 (RACK1) Act as Functional Receptors for Lymphocystis Disease Virus Infection.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Ying Zhong,Xiaoqian Tang,Xiuzhen Sheng,Jing Xing,Wenbin Zhan

    In previous research, a 27.8-kDa protein in flounder Paralichthys olivaceus gill (FG) cells was identified as a putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32-kDa viral attachment protein (VAP) of LCDV, and monoclonal antibodies (MAbs) against 27.8R and 32-kDa VAP were developed. In this study, the 27.8R was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder. Recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1, and 27.8-kDa proteins in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Preincubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies significantly decreased the percentages of LCDV-infected cells and LCDV copy numbers, blocked virus infection, and delayed the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were upregulated to maximum levels 12 h and 48 h after LCDV infection, respectively. VDAC2/RACK1 knockdown through short interfering RNA (siRNA) significantly reduced VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls, while VDAC2/RACK1 expression on LCDV-nonpermissive epithelial papillosum cells (EPCs) conferred susceptibility to LCDV infection, indicating the VDAC2 and RACK1 were sufficient to allow LCDV entry and infection. All these results collectively showed that VDAC2 and RACK1 function as receptors for LCDV entry and infection.IMPORTANCE Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying the LCDV-host interaction remains unclear. Here, the 27.8-kDa putative cellular receptor for LCDV was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1), and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and that they are functional receptors that initiate LCDV infection for the first time, which leads to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interactions.

    更新日期:2019-11-01
  • Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Fushun Zhang,Xiangzhi Meng,Michael B Townsend,Panayampalli Subbian Satheshkumar,Yan Xiang

    Orthopoxviruses (OPXVs) have a broad host range in mammalian cells, but Chinese hamster ovary (CHO) cells are nonpermissive for vaccinia virus (VACV). Here, we revealed a species-specific difference in host restriction factor SAMD9L as the cause for the restriction and identified orthopoxvirus CP77 as a unique inhibitor capable of antagonizing Chinese hamster SAMD9L (chSAMD9L). Two known VACV inhibitors of SAMD9 and SAMD9L (SAMD9&L), K1 and C7, can bind human and mouse SAMD9&L, but neither can bind chSAMD9L. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 knockout of chSAMD9L from CHO cells removed the restriction for VACV, while ectopic expression of chSAMD9L imposed the restriction for VACV in a human cell line, demonstrating that chSAMD9L is a potent restriction factor for VACV. In contrast to K1 and C7, cowpox virus CP77 can bind chSAMD9L and rescue VACV replication in cells expressing chSAMD9L, indicating that CP77 is yet another SAMD9L inhibitor but has a unique specificity for chSAMD9L. Binding studies showed that the N-terminal 382 amino acids of CP77 were sufficient for binding chSAMD9L and that both K1 and CP77 target a common internal region of SAMD9L. Growth studies with nearly all OPXV species showed that the ability of OPXVs to antagonize chSAMD9L correlates with CP77 gene status and that a functional CP77 ortholog was maintained in many OPXVs, including monkeypox virus. Our data suggest that a species-specific difference in rodent SAMD9L poses a barrier for cross-species OPXV infection and that OPXVs have evolved three SAMD9&L inhibitors with different specificities to overcome this barrier.IMPORTANCE Several OPXV species, including monkeypox virus and cowpox virus, cause zoonotic infection in humans. They are believed to use wild rodents as the reservoir or intermediate hosts, but the host or viral factors that are important for OPXV host range in rodents are unknown. Here, we showed that the abortive replication of several OPXV species in a Chinese hamster cell line was caused by a species-specific difference in the host antiviral factor SAMD9L, suggesting that SAMD9L divergence in different rodent species poses a barrier for cross-species OPXV infection. While the Chinese hamster SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human and mouse SAMD9&L, it can be inhibited by cowpox virus CP77, indicating that OPXVs encode three SAMD9&L inhibitors with different specificities. Our data suggest that OPXV host range in broad rodent species depends on three SAMD9&L inhibitors with different specificities.

    更新日期:2019-11-01
  • Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Bora Nam,Zelalem Mekuria,Mariano Carossino,Ganwu Li,Ying Zheng,Jianqiang Zhang,R Frank Cook,Kathleen M Shuck,Juliana R Campos,Edward L Squires,Mats H T Troedsson,Peter J Timoney,Udeni B R Balasuriya

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

    更新日期:2019-11-01
  • Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Xufang Deng,Robert C Mettelman,Amornrat O'Brien,John A Thompson,Timothy E O'Brien,Susan C Baker

    Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

    更新日期:2019-11-01
  • Interferon Gamma Inhibits Varicella-Zoster Virus Replication in a Cell Line-Dependent Manner.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Akhalesh K Shakya,Dennis J O'Callaghan,Seong K Kim

    The major immediate early 62 (IE62) protein of varicella-zoster virus (VZV) is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate early (IE), early (E), and late (L) genes. Interferon gamma (IFN-γ) is a potent cytokine produced following primary VZV infection. Furthermore, VZV reactivation correlates with a decline in IFN-γ-producing immune cells. Our results showed that treatment with 20 ng/ml of IFN-γ completely reduced intracellular VZV yield in A549 lung epithelial cells, MRC-5 lung fibroblasts, and ARPE-19 retinal epithelial cells at 4 days post-VZV infection. However, IFN-γ reduced virus yield only 2-fold in MeWo melanoma cells compared to that of untreated cells. IFN-β significantly inhibited VZV replication in both ARPE-19 and MeWo cells. In luciferase assays with VZV open reading frame 61 (ORF61) promoter reporter plasmid, IFN-γ abrogated the transactivation activity of IE62 by 95%, 97%, and 89% in A549, ARPE-19, and MRC-5 cells, respectively. However, IFN-γ abrogated IE62's transactivation activity by 16% in MeWo cells, indicating that IFN-γ inhibits VZV replication as well as IE62-mediated transactivation in a cell line-dependent manner. The expression of VZV IE62 and ORF63 suppressed by IFN-γ was restored by JAK1 inhibitor treatment, indicating that the inhibition of VZV replication is mediated by JAK/STAT1 signaling. In the presence of IFN-γ, knockdown of interferon response factor 1 (IRF1) increased VZV replication. Ectopic expression of IRF1 reduced VZV yields 4,000-fold in MRC-5 and ARPE-19 cells but 3-fold in MeWo cells. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner.IMPORTANCE Our results showed that IFN-γ significantly inhibited VZV replication in a cell line-dependent manner. IFN-γ inhibited VZV gene expression after the immediate early stage of infection and abrogated IE62-mediated transactivation. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner. Understanding the mechanisms by which IFN-γ plays a role in VZV gene programming may be important in determining the tissue restriction of VZV.

    更新日期:2019-11-01
  • High-Throughput Screening and Identification of Potent Broad-Spectrum Inhibitors of Coronaviruses.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Liang Shen,Junwei Niu,Chunhua Wang,Baoying Huang,Wenling Wang,Na Zhu,Yao Deng,Huijuan Wang,Fei Ye,Shan Cen,Wenjie Tan

    Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

    更新日期:2019-11-01
  • Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Daniel J Rawle,Dongsheng Li,Zhonglan Wu,Lu Wang,Marcus Choong,Mary Lor,Robert C Reid,David P Fairlie,Jonathan Harris,Gilda Tachedjian,Sally-Ann Poulsen,David Harrich

    HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

    更新日期:2019-11-01
  • Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.
    J. Virol. (IF 4.324) Pub Date : 2019-03-29
    Jana Tvarogová,Ramakanth Madhugiri,Ganesh Bylapudi,Lyndsey J Ferguson,Nadja Karl,John Ziebuhr

    Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3' polyadenylation of viral plus-strand RNAs.IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

    更新日期:2019-11-01
  • Astrovirus Replication Is Inhibited by Nitazoxanide In Vitro and In Vivo.
    J. Virol. (IF 4.324) Pub Date : null
    Virginia Hargest,Bridgett Sharp,Brandi Livingston,Valerie Cortez,Stacey Schultz-Cherry

    Astroviruses (AstV) are a leading cause of diarrhea especially in the very young, the elderly, and immunocompromised populations. Despite their significant impact on public health, no drug therapies for astrovirus have been identified. In this study we fill this gap in knowledge and demonstrate that the FDA-approved broad-spectrum anti-infective drug nitazoxanide (NTZ) blocks astrovirus replication in vitro with a 50% effective concentration (EC50) of approximately 1.47 μM. It can be administered up to 8 hours post-infection and is effective against multiple human astrovirus serotypes including clinical isolates. Most importantly, NTZ reduces viral shed in vivo, exhibiting its potential as a future clinical therapeutic.Importance Human astroviruses (HAstV) are thought to cause between 2 and 9% of acute, non-bacterial diarrhea cases in children worldwide. HAstV infection can be especially problematic in immunocompromised people and infants where the virus has been associated with necrotizing enterocolitis, severe and persistent diarrhea, as well as rare instances of systemic and fatal disease. Yet no antivirals have been identified to treat astrovirus infection. Our study provides the first evidence that nitazoxanide may be an effective therapeutic strategy against astrovirus disease.

    更新日期:2019-11-01
  • A MUC16 IgG binding activity selects for a restricted subset of IgG enriched for certain SIV epitope specificities.
    J. Virol. (IF 4.324) Pub Date : null
    Jeffrey R Schneider,Xiaoying Shen,Chiara Orlandi,Tinashe Nyanhete,Sheetal Sawant,Ann M Carias,Archer D Smith,Neil L Kelleher,Ronald S Veazey,George K Lewis,Georgia D Tomaras,Thomas J Hope

    We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from chronically infected HIV patients, who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV infected macaques revealed the MUC16-eluted antibodies were enriched for certain specific epitopes including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and subsequent association with MUC16 suggests the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.Importance Understanding how antibodies are distributed in the mucosal environment is valuable for development of a vaccine to block HIV infection. Here we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16 and interestingly these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model which is shown here to have MUC16 targeted antigen responses.

    更新日期:2019-11-01
  • The IFNL4 gene is a non-canonical interferon gene with a unique but evolutionarily conserved regulation.
    J. Virol. (IF 4.324) Pub Date : null
    Hao Zhou,Michelle Møhlenberg,Ewa Terczyńska-Dyla,Kasper Grønbjerg Winther,Nanna Hougaard Hansen,Johan Vad-Nielsen,Laura Laloli,Ronald Dijkman,Anders Lade Nielsen,Hans Henrik Gad,Rune Hartmann

    Interferon lambda 4 (IFNL4) is a recently identified enigmatic member of the interferon lambda family. Genetic data suggest that the IFNL4 gene acts in a pro-viral and anti-inflammatory manner in patients. However, the protein is in vitro indistinguishable from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4 This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.Importance Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner as patients with a non-functional IFNL4 gene exhibit increased clearance of hepatitis C virus but also increased liver inflammation. This suggests that the IFNL4 gene acts in a pro-viral and anti-inflammatory manner. Those surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product IFN-λ4. We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of IFNs, i.e. the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its non-canonical transcriptional regulation.

    更新日期:2019-11-01
  • Chronic Lymphocytic Choriomeningitis infection causes susceptibility to mousepox and impairs Natural Killer cell maturation and function.
    J. Virol. (IF 4.324) Pub Date : null
    Pedro Alves-Peixoto,Maria Férez,Cory Knudson,Colby Stotesbury,Carolina R Melo-Silva,Eric B Wong,Margarida Correia-Neves,Luis J Sigal

    Chronic viral infections like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus; or of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) result in immune dysfunction that predispose to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV) and that this resistance requires Natural Killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox while most of those that recovered from acute infection with the LCMV Armstrong strain (Arm) survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm have reduced frequency and number of NK cells. However, at steady state, NK cells in Arm-recovered mice mature normally and, in response to ECTV, get activated, become more mature, proliferate and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated and, following ECTV infection, they do not mature, proliferate or increase their cytotoxicity. Given the well established importance of NK cells in resistance to mousepox, these data suggests that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.Importance Infection of adult mice with the Clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, Alves-Peixoto et al. show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV, the agent of mousepox), and that Natural Killer (NK) cells in CL13 infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues for why humans chronically infected with certain pathogens are less resistant to viral diseases.

    更新日期:2019-11-01
  • A murine model for the study of influenza D virus.
    J. Virol. (IF 4.324) Pub Date : null
    J Oliva,J Mettier,L Sedano,M Delverdier,N Bourgès-Abella,B Hause,J Loupias,I Pardo,C Bleuart,P J Bordignon,E Meunier,R Le Goffic,G Meyer,M F Ducatez

    A novel genus within the Orthomyxoviridae family was identified in the USA and named Influenza D virus (IDV). Bovine have been proposed to be the primary host and three main viral lineages (D/OK-like, D/660-like and D/Japan-like) have been described. Experimental infections were so far performed in swine, ferret, calf and guinea pig, in order to study IDV pathogenesis.We developed a murine experimental model to ease the study of IDV pathogenesis and immune response. DBA/2 mice were inoculated with 105 TCID50 of D/bovine/France/5920/2014 (D/OK-like). No clinical signs and weight loss were observed. Viral replication was observed mainly in the upper respiratory tract (nasal turbinates) but also in lower respiratory tract of infected mice, with a peak at 4 days post-infection. Moreover, the virus was also detected in the intestines. All infected mice seroconverted by 14 days post infection. Transcriptomic analyses demonstrated that IDV induced an activation of pro-inflammatory genes such as IFN-γ and CCL2. Inoculation of NFκB-luciferase and Ifnar1-/- mice demonstrated that IDV induced mild inflammation and that type I interferons response was not necessary in IDV clearance. Adaptation of IDV by serial passages in mice was not sufficient to induce disease or increased pathogenesis.Taken together, present data and comparisons with the calf model show that our mouse model allows for the study of IDV replication and fitness (before selected viruses may be inoculated on calves) and also of the immune response.ImportanceInfluenza D virus (IDV), a new genus of Orthomyxoviridae family, presents a large host range and a worldwide circulation. The pathogenicity of this virus has been studied in the calf model. The mouse model is frequently used to enable a first assessment of a pathogen's fitness, replication and pathogenesis for influenza A and B viruses. We showed that DBA/2 mice are a relevant in vivo model for the study of IDV replication. This model will allow for rapid IDV fitness and replication evaluation and will enable phenotypic comparisons between isolated viruses. It will also allow for a better understanding of the immune response induced after IDV infection.

    更新日期:2019-11-01
  • Breaking the convention: Sialoglycan variants, Co-receptors and Alternative Receptors for Influenza A Virus Entry.
    J. Virol. (IF 4.324) Pub Date : null
    Umut Karakus,Marie O Pohl,Silke Stertz

    The influenza A virus (IAV) envelope protein haemagglutinin binds α2,6- or α2,3-linked sialic acid as host cell receptor. Bat IAV subtypes H17N10 and H18N11 form an exception to this rule and do not bind sialic acid but enter cells via MHC class II complexes. Here, we review current knowledge on IAV receptors with a focus on sialoglycan variants, protein co-receptors and alternative receptors that impact IAV attachment and internalization beyond the well-described sialic acid binding.

    更新日期:2019-11-01
  • Boosting with AIDSVAX B/E enhances Env constant region 1 and 2 antibody-dependent cellular cytotoxicity breadth and potency.
    J. Virol. (IF 4.324) Pub Date : null
    David Easterhoff,Justin Pollara,Kan Luo,William D Tolbert,Brianna Young,Dieter Mielke,Shalini Jha,Robert J O'Connell,Sandhya Vasan,Jerome Kim,Nelson L Michael,Jean-Louis Excler,Merlin L Robb,Supachai Rerks-Ngarm,Jaranit Kaewkungwal,Punnee Pitisuttithum,Sorachai Nitayaphan,Faruk Sinangil,James Tartaglia,Sanjay Phogat,Thomas B Kepler,S Munir Alam,Kevin Wiehe,Kevin O Saunders,David C Montefiori,Georgia D Tomaras,M Anthony Moody,Marzena Pazgier,Barton F Haynes,Guido Ferrari

    Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce non-neutralizing antibodies (NNAbs) that kill virus-infected cells as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) monoclonal antibodies (mAbs) frequently mediate potent antibody dependent cellular cytotoxicity (ADCC) making them an important vaccine target. Here we explore the effect of delayed and repetitive boosting of RV144 vaccine-recipients with AIDSVAX B/E on the C1C2-specific mAb repertoire. It was found that boosting increased clonal lineage specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific mAb showed that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity mature these type of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than seen in the RV144 trial.SIGNIFICANCE Over one million people become infected with HIV-1 each year making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine-regimen is the only HIV-1 clinical trial to date to demonstrate vaccine-efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine-efficacy. The RV305 HIV-1 vaccine-regimen was a follow-up boost of RV144 vaccine-recipients that occurred 6-8 years after the conclusion of RV144. Our study focused on the effect of delayed boosting in humans on the vaccine-induced Env constant region 1 and 2 (C1C2) - specific antibody repertoire. It was found that boosting with a HIV-1 Env vaccine increased C1C2-specific antibody dependent cellular cytotoxicity potency and breadth.

    更新日期:2019-11-01
  • Specific Anchoring and Local Translation of Poxviral ATI mRNA at Cytoplasmic Inclusion Bodies.
    J. Virol. (IF 4.324) Pub Date : null
    George C Katsafanas,Bernard Moss

    On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membrane-less sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA-binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membrane-less protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, re-localization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N-terminus of the ATI or large truncations are made at the N- or C-termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation.IMPORTANCE Poxvirus genome replication, transcription, translation and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.

    更新日期:2019-11-01
  • Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival.
    J. Virol. (IF 4.324) Pub Date : null
    Kai-Min Lin,Sue-Jane Lin,Juin-Han Lin,Pei-Yi Lin,Pu-Lin Teng,Hsueh-Erh Wu,Te-Huei Yeh,Ying-Piao Wang,Mei-Ru Chen,Ching-Hwa Tsai

    The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet, the underlying mechanisms of how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of mitogen-activated protein kinases (MAPKs), during EBV infection and immortalization. Thirteen DUSPs, including ten typical and three atypical types of DUSPs, were tested. Most of them were down-regulated post EBV infection. Herein, a role of the viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates ERK or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this study provides the first description of the interplay between EB viral genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.Importance The infection of ubiquitous Epstein-Barr virus (EBV) is associated with a wide spectrum of lymphomas and carcinomas. It has been well-documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual specificity phosphates (DUSPs) exert great ability in regulating MAPK activities for their abilities to dephosphorylate MAPKs. In this study, we found that DUSPs generally are down-regulated post EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppresses DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation could be continuously activated. Furthermore, DUSP down-regulation contributes greatly to prevent apoptosis of EBV infected cells. To sum up, this study sheds light on a novel molecular mechanism on how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.

    更新日期:2019-11-01
  • Viral determinants in H5N1 influenza A virus enable productive infection of HeLa cells.
    J. Virol. (IF 4.324) Pub Date : null
    Ariel Rodriguez-Frandsen,Laura Martin-Sancho,Anshu P Gounder,Max W Chang,Wen-Chun Liu,Paul D De Jesus,Jessica von Recum-Knepper,Miriam S Dutra,Nicholas J Huffmaster,Monica Chavarria,Ignacio Mena,Laura Riva,Courtney B Nguyen,Saunil Dobariya,Kristina M Herbert,Christopher Benner,Randy A Albrecht,Adolfo García-Sastre,Sumit K Chanda

    Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, and sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to growth of human H1N1 and H3N2, and low pathogenic avian influenza (LPAIs) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) virus H5N1 successfully propagated in HeLa cells to levels comparable to a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 growth, suggesting the absence of a host factor(s) required for replication of H1N1, but not H5N1, in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, and deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from H5N1 was necessary and sufficient to enable H1N1 growth. Overall, this study suggests the absence of one or more cellular factors in HeLa cells that results in abortive replication of H1N1, H3N2, and LPAI viruses, but can be circumvented upon introduction of H5N1 NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into virus-host interactions that underlie IAV pathogenesis and tropism.IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk for these zoonotic viruses to acquire adaptive mutations to efficiently propagate and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon introduction of H5N1 HA and NP proteins. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 pathogenesis and tropism.

    更新日期:2019-11-01
  • Herpes simplex virus latency is noisier the closer we look.
    J. Virol. (IF 4.324) Pub Date : null
    Navneet Singh,David C Tscharke

    During herpes simplex virus (HSV) latency the viral genome is harboured in peripheral neurons in the absence of infectious virus, but with the potential to restart infection. Advances in epigenetics have helped explain how viral gene expression is largely inhibited during latency. Paradoxically at the same time, the view that latency is entirely silent has been eroding. This low-level noise has implications for our understanding of HSV latency and should not be ignored.

    更新日期:2019-11-01
  • Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle.
    J. Virol. (IF 4.324) Pub Date : null
    Yingying Cong,Mustafa Ulasli,Hein Schepers,Mario Mauthe,Philip V'kovski,Franziska Kriegenburg,Volker Thiel,Cornelis A M de Haan,Fulvio Reggiori

    Coronaviruses (CoV) nucleocapsid N proteins are key in incorporating the genomic RNA into the progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used CoV model virus, interacts with non-structural protein 3 (nsp3), a component of the RTCs. These two aspects of CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 but not with other RTCs components using a systematic yeast two-hybrid approach, and identified two distinct regions in the N protein that redundantly mediates this interaction. A selective N protein variant carrying point mutations in these two regions fail to bind nsp3 in vitro, resulting in an inhibition of its recruitment to the RTCs in vivo Furthermore, opposite to the wild type N protein, this N protein variant impairs the stimulation of gRNA and viral mRNA transcription in vivo and in vitro, which in turn leads to an impairment in MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in CoV life cycle as it is critical for optimal viral RNA synthesis.IMPORTANCE Coronaviruses (CoV) have been regarded for a long time as a relatively harmless pathogen for humans. Two severe respiratory tract infection outbreaks caused by SARS-CoV and MERS-CoV, however, have caused high pathogenicity and mortality rates in humans. This highlighted the relevance of being able to control CoV infections. We used a CoV model virus, mouse hepatitis virus (MHV), to investigate the importance of the recruitment of nucleocapsid (N) protein, a central component of CoV virions, to intracellular platforms where CoV replicate, transcribe and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial in promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain both an important aspect of CoV life cycle and reveal an interaction between viral proteins that could be targeted in antiviral therapies.

    更新日期:2019-11-01
  • Genetic diversity, compartmentalization and age of HIV proviruses persisting in CD4+ T cell subsets during long-term combination antiretroviral therapy.
    J. Virol. (IF 4.324) Pub Date : null
    Bradley R Jones,Rachel L Miller,Natalie N Kinloch,Olivia Tsai,Hawley Rigsby,Hanwei Sudderuddin,Aniqa Shahid,Bruce Ganase,Chanson J Brumme,Marianne Harris,Art F Y Poon,Mark A Brockman,Rémi Fromentin,Nicolas Chomont,Jeffrey B Joy,Zabrina L Brumme

    The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4+ T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4+ T cell subsets however remains incomplete. Using single-genome amplification we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory and effector memory CD4+ T cells from five HIV-infected individuals on long-term cART, and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV datasets spanning a median 19.5 (range 10-20) years per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity and genetic compartmentalization between CD4+ T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4+ T cell subsets, combined analyses revealed no evidence that any particular CD4+ T cell subset harbored the longest-persisting, most genetically diverse and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three others we observed no significant differences in proviral ages between subsets. In one participant, "old" proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) of their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches.ImportanceThe main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4+ T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4+ T cells however constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic "landscapes" of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4+ T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4+ T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity, and support personalized strategies for HIV eradication.

    更新日期:2019-11-01
  • HIV-1 accessory protein Vpr interacts with REAF/RPRD2 to mitigate its antiviral activity.
    J. Virol. (IF 4.324) Pub Date : null
    Joseph M Gibbons,Kelly M Marno,Rebecca Pike,Wing-Yiu Jason Lee,Christopher E Jones,Babatunji W Ogunkolade,Claire Pardieu,Alexander Bryan,Rebecca Menhua Fu,Gary Warnes,Paul A Rowley,Richard D Sloan,Áine McKnight

    The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intact vpr is more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.Importance For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

    更新日期:2019-11-01
  • Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5.
    J. Virol. (IF 4.324) Pub Date : null
    Guoqing Chen,Qing Yan,Haoran Wang,Shufen Chao,Lijuan Wu,Peter J Krell,Guozhong Feng

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL) from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleolus of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleolus of transfected cells. Multiple point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed, several NoLS-mutated LEF5s were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production and occlusion body production.IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleolus of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

    更新日期:2019-11-01
  • Two Pioneer Transcription Factors, Krüppel-Like Transcription Factor 4 and Glucocorticoid Receptor, Cooperatively Transactivate the Bovine Herpesvirus 1 ICP0 Early Promoter and Stimulate Productive Infection.
    J. Virol. (IF 4.324) Pub Date : null
    Fouad S El-Mayet,Laximan Sawant,Prasanth Thunuguntla,Jing Zhao,Clinton Jones

    An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR+KLF family members transactivate the bICP0 E promoter. GR+KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity via a ligand independent manner in mouse neuroblastoma cells (Neuro-2A): furthermore, GR+KLF4 stimulated productive infection. Mutating both ½ GR response elements did not significantly reduce GR+KLF4 mediated transactivation of the bICP0 E promoter suggesting a novel mechanism exists for transactivation. GR+KLF15 cooperatively stimulated bICP0 activity less efficiently than GR+KL4: however, KLF6 and PLZF plus GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes life-long latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict increased corticosteroid levels activate the glucocorticoid receptor (GR): consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Krüppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR+KLF15 cooperate to stimulate bICP0 E promoter activity, but significantly less relative to GR+KLF4. The bICP0 E promoter contain distinct enhancer-like domains necessary for GR+KLF4 mediated transactivation relative to GR+KLF15. Stress induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.

    更新日期:2019-11-01
  • Molecular basis of binding between MERS-CoV and CD26s from seven bat species.
    J. Virol. (IF 4.324) Pub Date : null
    Yuan Yuan,Jianxun Qi,Ruchao Peng,Chunrui Li,Guangwen Lu,Jinghua Yan,Qihui Wang,George Fu Gao

    Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting human have occurred since its identification in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozens of spill-over infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats, and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with K D values ranging from several to hundreds of micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.Importance In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.

    更新日期:2019-11-01
  • HPV E7 Oncoprotein Subverts Host Innate Immunity Via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes.
    J. Virol. (IF 4.324) Pub Date : null
    Irene Lo Cigno,Federica Calati,Cinzia Borgogna,Alessandra Zevini,Silvia Albertini,Licia Martuscelli,Marco De Andrea,John Hiscott,Santo Landolfo,Marisa Gariglio

    Subversion of innate immunity by oncoviruses, such as human papillomavirus (HPV), favors carcinogenesis because the mechanism(s) of viral immune evasion can also hamper cancer immunosurveillance. Previously, we demonstrated that high-risk (hr) HPVs trigger simultaneous epigenetic silencing of multiple effectors of innate immunity to promote viral persistence. Here, we expand on those observations and show that the HPV E7 oncoprotein upregulates the H3K9-specific methyltransferase, whose action shuts down the host innate immune response. Specifically, we demonstrate that SUV39H1 contributes to chromatin repression at the promoter regions of the viral nucleic acid sensors RIG-I, cGAS and the adaptor molecule STING in HPV-transformed cells. Inhibition of SUV39H1 leads to transcriptional activation of these genes, especially RIG-I, followed by increased IFNβ and λ1 production after poly(dA:dT) or RIG-I agonist M8 transfection, Collectively, our findings provide new evidence that the E7 oncoprotein plays a central role in dampening host innate immunity and raise the possibility that targeting the downstream effector SUV39H1 or the RIG-I pathway may be a viable strategy to treat viral and neoplastic disease.IMPORTANCE High-risk HPVs are major viral human carcinogens responsible for approximately 5% of all human cancers. The growth of HPV-transformed cells depends on the ability of viral oncoproteins to manipulate a variety of cellular circuits, including those involved in innate immunity. Here, we show that one of these strategies relies on E7-mediated transcriptional activation of the chromatin repressor SUV39H1, which then promotes epigenetic silencing of RIG-I, cGAS and STING genes, thereby shutting down interferon secretion in HPV-transformed cells. Pharmacological or genetic inhibition of SUV39H1 restored the innate response in HPV-transformed cells, mostly through activation of RIG-I signaling. We also show that IFN production upon transfection of poly(dA:dT) or the RIG-I agonist M8 predominantly occurs through RIG-I signaling. Altogether, the reversible nature of the modifications associated with E7-mediated SUV39H1 upregulation provides a rationale for the design of novel anticancer and antiviral therapies targeting these molecules.

    更新日期:2019-11-01
  • Polymorphisms in the most oncolytic reovirus strain confer enhanced cell attachment, transcription and single-step replication kinetics.
    J. Virol. (IF 4.324) Pub Date : null
    Adil Mohamed,James R Smiley,Maya Shmulevitz

    Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strain of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo This study aimed to determine why the most-oncolytic T3DPL lab strain of reovirus replicates more efficiently in cancer cells than other commonly used laboratory strains, T3DKC and T3DTD In single step growth curves, T3DPL titers increased at faster rates and produced ∼9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3DPL than T3DTD In conclusion, the most-oncolytic T3DPL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3DPL were identified. First, T3DPL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (≤3) σ1 cell attachment proteins. Second, T3DPL transcribed RNA at superior rates to the less-oncolytic T3D strains, attributed to polymorphisms in M1-encoding μ2 protein, as confirmed in an in vitro transcription assay, demonstrating that T3DPL has an inherent transcription advantage that is cell-independent. Accordingly, T3DPL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains, and mechanistically, reveal the importance of rapid onset of infection for reovirus oncolysis.Importance: Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently it was discovered that highly related laboratory strains of T3D exhibit large differences in their ability to replicate in cancer cells in vitro, which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3DPL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3DPL binds to cells more efficiency and more-rapidly produces viral RNAs; this increased rate of infection relative to the less-oncolytic strains gives T3DPL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread.

    更新日期:2019-11-01
  • An Alu element insertion into intron 1 results in the aberrant alternative splicing of APOBEC3G pre-mRNA in northern pig-tailed macaques (Macaca leonina) may reduce APOBEC3G-mediated hypermutation pressure on HIV-1.
    J. Virol. (IF 4.324) Pub Date : null
    Xiao-Liang Zhang,Meng-Ting Luo,Jia-Hao Song,Wei Pang,Yong-Tang Zheng

    APOBEC3 family members, particularly APOBEC3F and APOBEC3G, inhibit the replication and spread of various retroviruses by inducing hypermutation in newly synthesized viral DNA. Viral hypermutation by APOBEC3 is associated with viral evolution, viral transmission and disease progression. In recent years, increasing attention has been paid to targeting APOBEC3G for AIDS therapy. Thus, a controllable model system using species such as macaques, which provide a relatively ideal in vivo system, is needed for the study of APOBEC3-related issues. To appropriately utilize this animal model for biomedical research, the important differences between human and macaque APOBEC3s must be considered. In this study, we found that the ratio of APOBEC3G-mediated/APOBEC3s-mediated HIV-1 hypermutation footprints was much lower in peripheral blood mononuclear cells (PBMCs) from northern pig-tailed macaques than that in PBMCs from humans. Then we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and have resulted from Alu element insertion into the macaque APOBEC3G gene intron 1. This alternative splicing pattern generating an aberrant APOBEC3G mRNA isoform may significantly dilute the full length APOBEC3G and reduce APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques, which was supported by the elimination of other possibilities accounting for this hypermutation difference between the two hosts.IMPORTANCE APOBEC3 family members, particularly APOBEC3F and APOBEC3G, are important cellular antiviral factors. Recently, more attention has been paid to targeting APOBEC3G for AIDS therapy. To appropriately utilize macaque animal models for the study of APOBEC3-related issues, it is important that the differences between human and macaque APOBEC3s are clarified. In this study, we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and which may reduce the APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques (NPMs). Our work provides important information for the proper application of macaque animal models for APOBEC3-related issues in AIDS research and a better understanding of the biological functions of APOBEC3 proteins.

    更新日期:2019-11-01
  • Intact HIV proviruses persist in children 7-9 years after initiation of ART in the first year of life.
    J. Virol. (IF 4.324) Pub Date : null
    Mary Grace Katusiime,Elias K Halvas,Imogen Wright,Kevin Joseph,Michael J Bale,Bronwyn Kirby-McCullough,Susan Engelbrecht,Wei Shao,Wei-Shau Hu,Mark F Cotton,John W Mellors,Mary F Kearney,Gert U van Zyl

    In adults starting ART during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. By contrast, a recent report in early treated children failed to detect sequence intact proviruses. We sought to detect and characterize proviral sequences in another cohort of early treated children after 6-9 years on suppressive ART. PBMCs from perinatally-infected children from the CHER study were analysed. Near full-length proviral amplification and sequencing (NFL-PAS) was performed at one time point after 6-9 years on ART. Amplicons with large internal deletions were excluded (<9kb). All amplicons ≥9kb were sequenced and analysed through a bio-informatic pipeline to detect indels, frameshifts or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range: 2.0 - 11.1), 733 single NFL-PAS amplicons were generated. Of these, 534(72.9%) had large internal deletions, 174(23.7%) had hypermutation, 15(1.4%) had small internal deletions, 3(1.0%) had deletions in the packaging signal/major splice donor site and 7(1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age; of whom one had two identical intact sequences, suggestive of a cell clone harbouring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably just as in adults, maintained by clonal expansion of cells infected before ART initiation.Importance There is limited data on the proviral landscape in early treated, long-term suppressed children, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through near full-length proviral amplification and sequencing (NFL-PAS) we generated 733 NFL-PAS amplicons from 8 children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower than (p<0.05) reported for HIV subtype B infected adults treated during early HIV infection. We show that cells harbouring genetically intact HIV proviruses are rare in early treated long-term suppressed children and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs particularly in paediatric studies where sample availability is limited.

    更新日期:2019-11-01
  • Bromo- and extraterminal domain chromatin regulators serve as cofactors for murine leukemia virus integration.
    J. Virol. (IF 4.324) Pub Date : 2013-09-21
    Saumya Shree Gupta,Tobias Maetzig,Goedele N Maertens,Azar Sharif,Michael Rothe,Magdalena Weidner-Glunde,Melanie Galla,Axel Schambach,Peter Cherepanov,Thomas F Schulz

    Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV IN in vitro. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integration in vitro and, in the case of BRD2, also in vivo. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.

    更新日期:2019-11-01
  • Kaposi's sarcoma-associated herpesvirus LANA-1 interacts with the short variant of BRD4 and releases cells from a BRD4- and BRD2/RING3-induced G1 cell cycle arrest.
    J. Virol. (IF 4.324) Pub Date : 2006-08-25
    Matthias Ottinger,Thomas Christalla,Kavita Nathan,Melanie M Brinkmann,Abel Viejo-Borbolla,Thomas F Schulz

    The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA-1) is required for the replication of episomal viral genomes. Regions in its N-terminal and C-terminal domains mediate the interaction with host cell chromatin. Several cellular nuclear proteins, e.g., BRD2/RING3, histones H2A and H2B, MeCP2, DEK, and HP1alpha, have been suggested to mediate this interaction. In this work, we identify the double-bromodomain proteins BRD4 and BRD3/ORFX as additional LANA-1 interaction partners. The carboxy-terminal region of the short variant of BRD4 (BRD4S) containing the highly conserved extraterminal domain directly interacts with an element in the LANA-1 carboxy-terminal domain. We show that ectopically expressed BRD4S and BRD2/RING3 delay progression into the S phase of the cell cycle in epithelial and B-cell lines and increase cyclin E promoter activity. LANA-1 partly releases epithelial and B cells from a BRD4S- and BRD2/RING3-induced G1 cell cycle arrest and also promotes S-phase entry in the presence of BRD4S and BRD2/RING3. This is accompanied by a reduction in BRD4S-mediated cyclin E promoter activity. Our data are in keeping with the notion that the direct interaction of KSHV LANA-1 with BRD4 and other BRD proteins could play a role in the G1/S phase-promoting functions of KSHV LANA-1. Further, our data support a model in which the LANA-1 C terminus contributes to a functional attachment to acetylated histones H3 and H4 via BRD4 and BRD2, in addition to the recently demonstrated direct interaction (A. J. Barbera, J. V. Chodaparambil, B. Kelley-Clarke, V. Joukov, J. C. Walter, K. Luger, and K. M. Kaye, Science 311:856-861, 2006) of the LANA-1 N terminus with histones H2A and H2B.

    更新日期:2019-11-01
  • Brd2/RING3 interacts with a chromatin-binding domain in the Kaposi's Sarcoma-associated herpesvirus latency-associated nuclear antigen 1 (LANA-1) that is required for multiple functions of LANA-1.
    J. Virol. (IF 4.324) Pub Date : 2005-10-18
    Abel Viejo-Borbolla,Matthias Ottinger,Eva Brüning,Antje Bürger,Regina König,Emrah Kati,Julie A Sheldon,Thomas F Schulz

    Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episomal DNA, to modulate transcription, and to interact with Brd2/RING3. Similar to the recently described tethering of bovine papillomavirus E2 protein to host chromatin via Brd4/MCAP, Brd2/RING3, another member of the Brd family of chromatin-binding proteins, therefore interacts with a chromatin-binding region of another viral latent nuclear protein and could play a role in its multiple functions.

    更新日期:2019-11-01
  • Latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene.
    J. Virol. (IF 4.324) Pub Date : 1999-11-13
    G M Platt,G R Simpson,S Mittnacht,T F Schulz

    Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is the likely infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and some cases of multicentric Castleman's disease. Its latent nuclear antigen (LANA) is expressed in the nuclei of latently infected cells and may play a role in the persistence of episomal viral DNA in dividing cells. Here we report that LANA interacts with RING3, a nuclear protein and member of the Drosophila fsh (female sterile homeotic) family of proteins, some of which have previously been implicated in controlling gene expression. Binding of RING3 to LANA involves the ET domain, characteristic of fsh-related proteins, suggesting that this highly conserved region is involved in protein-protein interactions. The interaction between RING3 and LANA results in phosphorylation of serine and threonine residues located between amino acids 951 and 1107 in the carboxy-terminal region of LANA. However, RING3 is not itself a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into the complex which it forms with LANA.

    更新日期:2019-11-01
  • Acknowledgment of Ad Hoc Reviewers.
    J. Virol. (IF 4.324) Pub Date : null
    Rozanne M Sandri-Goldin

    更新日期:2019-11-01
  • Recognizing the Top 25 Peer Reviewers for the Journal of Virology.
    J. Virol. (IF 4.324) Pub Date : null
    Rozanne M Sandri-Goldin

    更新日期:2019-11-01
  • Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor.
    J. Virol. (IF 4.324) Pub Date : 2019-10-11
    Vânia Passos,Thomas Zillinger,Nicoletta Casartelli,Amelie S Wachs,Shuting Xu,Angelina Malassa,Katja Steppich,Hildegard Schilling,Sergej Franz,Daniel Todt,Eike Steinmann,Kathrin Sutter,Ulf Dittmer,Jens Bohne,Olivier Schwartz,Winfried Barchet,Christine Goffinet

    When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

    更新日期:2019-11-01
  • Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System.
    J. Virol. (IF 4.324) Pub Date : 2019-10-11
    Asha A Philip,Jacob L Perry,Heather E Eaton,Maya Shmulevitz,Joseph M Hyser,John T Patton

    Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

    更新日期:2019-11-01
  • Epstein-Barr Virus-Specific CD8 T Cells Selectively Infiltrate the Brain in Multiple Sclerosis and Interact Locally with Virus-Infected Cells: Clue for a Virus-Driven Immunopathological Mechanism.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Barbara Serafini,Barbara Rosicarelli,Caterina Veroni,Gina Adriana Mazzola,Francesca Aloisi

    Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (≤0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury.IMPORTANCE EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.

    更新日期:2019-11-01
  • Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Jennifer A Bohn,Justin DaSilva,Siarhei Kharytonchyk,Maria Mercedes,Jennifer Vosters,Alice Telesnitsky,Theodora Hatziioannou,Janet L Smith

    APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination.IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

    更新日期:2019-11-01
  • HIV-2 Depletes CD4 T Cells through Pyroptosis despite Vpx-Dependent Degradation of SAMHD1.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Xiaoyu Luo,Eytan Herzig,Gilad Doitsh,Zachary W Grimmett,Isa Muñoz-Arias,Warner C Greene

    Human immunodeficiency virus type 2 (HIV-2) infection results in a milder course of disease and slower progression to AIDS than does HIV-1. We hypothesized that this difference may be due to degradation of the sterile alpha motif and HD domain 1 (SAMHD1) host restriction factor by the HIV-2 Vpx gene product, thereby diminishing abortive infection and pyroptotic cell death within bystander CD4 T cells. We have compared CD4 T cell death in tonsil-derived human lymphoid aggregate cultures (HLACs) infected with wild-type HIV-2, HIV-2 ΔVpx, or HIV-1. In contrast to our hypothesis, HIV-2, HIV-2 ΔVpx, and HIV-1 induced similar levels of bystander CD4 T cell death. In all cases, cell death was blocked by AMD3100, a CXCR4 entry inhibitor, but not by raltegravir, an integrase, indicating that only early life cycle events were required. Cell death was also blocked by a caspase-1 inhibitor, a key enzyme promoting pyroptosis, but not by a caspase-3 inhibitor, an important enzyme in apoptosis. HIV-1-induced abortive infection and pyroptotic cell death were also not reduced by forced encapsidation of HIV-2 Vpx into HIV-1 virions. Together, these findings indicate that HIV-2 and HIV-1 support similar levels of CD4 T cell depletion in vitro despite HIV-2 Vpx-mediated degradation of the SAMHD1 transcription factor. The milder disease course observed with HIV-2 infection likely stems from factors other than abortive infection and caspase-1-dependent pyroptosis in bystander CD4 T cells.IMPORTANCE CD4 T cell depletion during HIV-1 infection involves the demise of bystander CD4 T cells due to abortive infection, viral DNA sensing, inflammasome assembly, and death by caspase-1-dependent pyroptosis. HIV-2 infection is associated with milder disease and lower rates of CD4 T cell loss. We hypothesized that HIV-2 infection produces lower levels of pyroptosis due to the action of its Vpx gene product. Vpx degrades the SAMHD1 restriction factor, potentially reducing abortive forms of infection. However, in tonsil cell cultures, HIV-2, HIV-2 ΔVpx, and HIV-1 induced indistinguishable levels of pyroptosis. Forced encapsidation of Vpx into HIV-1 virions also did not reduce pyroptosis. Thus, SAMHD1 does not appear to play a key role in the induction of bystander cell pyroptosis. Additionally, the milder clinical course of HIV-2-induced disease is apparently not explained by a decrease in this inflammatory form of programmed cell death.

    更新日期:2019-11-01
  • Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Tonya Kueck,Louis-Marie Bloyet,Elena Cassella,Trinity Zang,Fabian Schmidt,Vesna Brusic,Gergely Tekes,Owen Pornillos,Sean P J Whelan,Paul D Bieniasz

    Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis.IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.

    更新日期:2019-11-01
  • How HIV Nef Proteins Hijack Membrane Traffic To Promote Infection.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Cosmo Z Buffalo,Yuichiro Iwamoto,James H Hurley,Xuefeng Ren

    The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.

    更新日期:2019-11-01
  • Macromolecular Synthesis Shutoff Resistance by Myeloid Cells Is Critical to IRF7-Dependent Systemic Interferon Alpha/Beta Induction after Alphavirus Infection.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Nishank Bhalla,Christina L Gardner,Sierra N Downs,Matthew Dunn,Chengqun Sun,William B Klimstra

    Alphavirus infection of fibroblastic cell types in vitro inhibits host cell translation and transcription, leading to suppression of interferon alpha/beta (IFN-α/β) production. However, the effect of infection upon myeloid cells, which are often the first cells encountered by alphaviruses in vivo, is unclear. Previous studies demonstrated an association of systemic IFN-α/β production with myeloid cell infection efficiency. Murine infection with wild-type Venezuelan equine encephalitis virus (VEEV), a highly myeloid-cell-tropic alphavirus, results in secretion of very high systemic levels of IFN-α/β, suggesting that stress responses in responding cells are active. Here, we infected myeloid cell cultures with VEEV to identify the cellular source of IFN-α/β, the timing and extent of translation and/or transcription inhibition in infected cells, and the transcription factors responsible for IFN-α/β induction. In contrast to fibroblast infection, myeloid cell cultures infected with VEEV secreted IFN-α/β that increased until cell death was observed. VEEV inhibited translation in most cells early after infection (<6 h postinfection [p.i.]), while transcription inhibition occurred later (>6 h p.i.). Furthermore, the interferon regulatory factor 7 (IRF7), but not IRF3, transcription factor was critical for IFN-α/β induction in vitro and in sera of mice. We identified a subset of infected Raw 264.7 myeloid cells that resisted VEEV-induced translation inhibition and secreted IFN-α/β despite virus infection. However, in the absence of IFN receptor signaling, the size of this cell population was diminished. These results indicate that IFN-α/β induction in vivo is IRF7 dependent and arises in part from a subset of myeloid cells that are resistant, in an IFN-α/β-dependent manner, to VEEV-induced macromolecular synthesis inhibition.IMPORTANCE Most previous research exploring the interaction of alphaviruses with host cell antiviral responses has been conducted using fibroblast lineage cell lines. Previous studies have led to the discovery of virus-mediated activities that antagonize host cell antiviral defense pathways, such as host cell translation and transcription inhibition and suppression of STAT1 signaling. However, their relevance and impact upon myeloid lineage cell types, which are key responders during the initial stages of alphavirus infection in vivo, have not been well studied. Here, we demonstrate the different abilities of myeloid cells to resist VEEV infection compared to nonmyeloid cell types and begin to elucidate the mechanisms by which host antiviral responses are upregulated in myeloid cells despite the actions of virus-encoded antagonists.

    更新日期:2019-11-01
  • Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Deanna A Kulpa,Aarthi Talla,Jessica H Brehm,Susan Pereira Ribeiro,Sally Yuan,Anne-Gaelle Bebin-Blackwell,Michael Miller,Richard Barnard,Steven G Deeks,Daria Hazuda,Nicolas Chomont,Rafick-Pierre Sékaly

    During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal.IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

    更新日期:2019-11-01
  • Small-Molecule Antiviral β-d-N 4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Maria L Agostini,Andrea J Pruijssers,James D Chappell,Jennifer Gribble,Xiaotao Lu,Erica L Andres,Gregory R Bluemling,Mark A Lockwood,Timothy P Sheahan,Amy C Sims,Michael G Natchus,Manohar Saindane,Alexander A Kolykhalov,George R Painter,Ralph S Baric,Mark R Denison

    Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). β-d-N 4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N 4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

    更新日期:2019-11-01
  • Clearance of Chikungunya Virus Infection in Lymphoid Tissues Is Promoted by Treatment with an Agonistic Anti-CD137 Antibody.
    J. Virol. (IF 4.324) Pub Date : 2019-10-04
    Jun P Hong,Mary K McCarthy,Bennett J Davenport,Thomas E Morrison,Michael S Diamond

    CD137, a member of the tumor necrosis factor receptor superfamily of cell surface proteins, acts as a costimulatory receptor on T cells, natural killer cells, B cell subsets, and some dendritic cells. Agonistic anti-CD137 monoclonal antibody (MAb) therapy has been combined with other chemotherapeutic agents in human cancer trials. Based on its ability to promote tumor clearance, we hypothesized that anti-CD137 MAb might activate immune responses and resolve chronic viral infections. We evaluated anti-CD137 MAb therapy in a mouse infection model of chikungunya virus (CHIKV), an alphavirus that causes chronic polyarthritis in humans and is associated with reservoirs of CHIKV RNA that are not cleared efficiently by adaptive immune responses. Analysis of viral tropism revealed that CHIKV RNA was present preferentially in splenic B cells and follicular dendritic cells during the persistent phase of infection, and animals lacking B cells did not develop persistent CHIKV infection in lymphoid tissue. Anti-CD137 MAb treatment resulted in T cell-dependent clearance of CHIKV RNA in lymphoid tissue, although this effect was not observed in musculoskeletal tissue. The clearance of CHIKV RNA from lymphoid tissue by anti-CD137 MAb was associated with reductions in the numbers of germinal center B cells and follicular dendritic cells. Similar results were observed with anti-CD137 MAb treatment of mice infected with Mayaro virus, a related arthritogenic alphavirus. Thus, anti-CD137 MAb treatment promotes resolution of chronic alphavirus infection in lymphoid tissues by reducing the numbers of target cells for infection and persistence.IMPORTANCE Although CHIKV causes persistent infection in lymphoid and musculoskeletal tissues in multiple animals, the basis for this is poorly understood, which has hampered pharmacological efforts to promote viral clearance. Here, we evaluated the therapeutic effects on persistent CHIKV infection of an agonistic anti-CD137 MAb that can activate T cell and natural killer cell responses to clear tumors. We show that treatment with anti-CD137 MAb promotes the clearance of persistent alphavirus RNA from lymphoid but not musculoskeletal tissues. This occurs because anti-CD137 MAb-triggered T cells reduce the numbers of target germinal center B cells and follicular dendritic cells, which are the primary reservoirs for CHIKV in the spleen and lymph nodes. Our studies help to elucidate the basis for CHIKV persistence and begin to provide strategies that can clear long-term cellular reservoirs of infection.

    更新日期:2019-11-01
  • NF-κB Activation Promotes Alphavirus Replication in Mature Neurons.
    J. Virol. (IF 4.324) Pub Date : 2019-09-27
    Jane X Yeh,Eunhye Park,Kimberly L W Schultz,Diane E Griffin

    Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-κB pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-κB during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-κB activation, as evidenced by the degradation of IκBα and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream IκB kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-κB inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) was decreased, and host protein synthesis was maintained, suggesting that NF-κB activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2α phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-κB activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation.IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-κB in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-κB activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-κB is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

    更新日期:2019-11-01
  • Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication.
    J. Virol. (IF 4.324) Pub Date : 2019-09-27
    Hongyun Hao,Tian Han,Baoqin Xuan,Yamei Sun,Shubing Tang,Nan Yue,Zhikang Qian

    DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

    更新日期:2019-11-01
  • Authentication Analysis of MT-4 Cells Distributed by the National Institutes of Health AIDS Reagent Program.
    J. Virol. (IF 4.324) Pub Date : 2019-09-27
    Melissa V Fernandez,Krista A Delviks-Frankenberry,David A Scheiblin,Christine Happel,Vinay K Pathak,Eric O Freed

    The MT-4 human T-cell line expresses HTLV-1 Tax and is permissive for replication of an HIV-1 gp41 mutant lacking the cytoplasmic tail. MT-4 cells (lot 150048), distributed by the NIH AIDS Reagent Program (NIH-ARP), were found to be Tax deficient and unable to host replication of the gp41-truncated HIV-1 mutant. These findings, together with short tandem repeat profiling, established that lot 150048 are not bona fide MT-4 cells.

    更新日期:2019-11-01
  • Activity-Based Protein Profiling Identifies ATG4B as a Key Host Factor for Enterovirus 71 Proliferation.
    J. Virol. (IF 4.324) Pub Date : 2019-09-27
    Yang Sun,Qizhen Zheng,Yaxin Wang,Zhengyuan Pang,Jingwei Liu,Zheng Yin,Zhiyong Lou

    Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3Cpro), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3Cpro and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation in vivo Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.IMPORTANCE Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.

    更新日期:2019-11-01
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