- Downregulation of eca-mir-128 in seminal exosomes and enhanced expression of CXCL16 in the stallion reproductive tract are associated with long-term persistence of equine arteritis virus. [Journal Article]
- JVJ Virol 2018 Feb 14
- Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associ...
Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of theCXCL16gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. Here, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of miRNAs. Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented herein suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.IMPORTANCEEquine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.
- The Superimposed Deubiquitination Effect of OTULIN and PRRSV Nsp11 Promoted the Multiplication of PRRSV. [Journal Article]
- JVJ Virol 2018 Feb 14
- Linear ubiquitination plays an important role in the regulation of the immune response by regulating the nuclear factor κB (NF-κB). The linear ubiquitin-specific deubiquitinase OTULIN can control imm...
Linear ubiquitination plays an important role in the regulation of the immune response by regulating the nuclear factor κB (NF-κB). The linear ubiquitin-specific deubiquitinase OTULIN can control immune signaling transduction pathway by restricting Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in PRRSV-infected cell model. PRRSV infection promotes the expression of OTULIN gene, in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is a negative regulation of innate immunity with OTULIN during viral infection. The cooperative effect of swine OTULIN and PRRSV Nsp11 potentiates the ability to reduce cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a non-enzymatic combination to enhance its ability of removing linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs. Our study presented a new model of virus utilize ubiquitin-protease system in vivo from the perspective of the viral proteins interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV.ImportanceDeubiquitination effect of swine OTULIN were identified.The interaction between porcine OTULIN and PRRSV Nsp11 dependent on OUT domain.PRRSV Nsp11 recruits OTULIN through a non-enzymatic combination to promote removing linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs.
- Hepatitis C virus core+1/ARFP modulates Cyclin D1/pRb pathway and promotes carcinogenesis. [Journal Article]
- JVJ Virol 2018 Feb 14
- Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate timely their gene expression. HCV...
Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate timely their gene expression. HCV possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein designated initially as ARFP or F or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L initiating from codon 26 and core+1/S initiating from codons 85/87 of the polyprotein coding region, respectively. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell combiningin vitroandin vivoapproaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCEThis study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery leading to acceleration of cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as biomarker of disease progression.
- Karyopherin alpha6 Is Required for the Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus. [Journal Article]
- JVJ Virol 2018 Feb 14
- Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating nucleocytoplas...
Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating nucleocytoplasmic translocation of proteins through the NPC. In this study, we discovered that karyopherin alpha6 (KPNA6, also known as importin alpha7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that of mock-infected controls, whereas KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of KPNA6 half-life and the elevation of KPNA6 level in comparison with mock-infected cells. PRRSV nsp12 protein induced the KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6 knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCEPositive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin-alpha6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced elevation of the KPNA6 protein level due to an extension of KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, restitution of KPNA6 expression in the KPNA6 knockout cells led to the restoration of nsp1β nuclear translocation and the ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.
- Interaction of human cytomegalovirus tegument proteins ppUL35 and ppUL35A with Sorting Nexin 5 regulates glycoprotein B (gpUL55) localization. [Journal Article]
- JVJ Virol 2018 Feb 14
- Human cytomegalovirus (HCMV) is a wide-spread human pathogen that causes asymptomatic infection in healthy individuals, but poses a serious threat to immunocompromised patients. During the late phase...
Human cytomegalovirus (HCMV) is a wide-spread human pathogen that causes asymptomatic infection in healthy individuals, but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC) where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wildtype proteins but not mutants defective in SNX5 binding resulted in cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB, gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.ImportanceHuman cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic, cycle human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.
- The 38K-mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus. [Journal Article]
- JVJ Virol 2018 Feb 14
- Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a co...
Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene,38K(ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes under unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrated that 38K mediates the dephosphorylation of specific sites at the C-terminus of P6.9, which is essential for viral genome encapsidation.IMPORTANCEGenome packaging is a fundamental process in virus life cycle, and viruses have different strategies to perform this step. For several dsDNA viruses, the procapsid is formed before genome encapsidation, and the latter may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene38Kis involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C-terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly.
- Vaccinia virus C9 ankyrin-repeat/F-box protein is a newly identified antagonist of the type I interferon-induced antiviral state. [Journal Article]
- JVJ Virol 2018 Feb 14
- Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN resp...
Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells co-evolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pre-treated with IFNβ, suggesting that one or more of the missing 17 open reading frames (ORFs) encodes an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN-resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pre-treated with IFNβ. Expression of viral early genes occurred but subsequent events including genome uncoating, genome replication and post-replicative gene expression were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 was predicted to have six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that co-purified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins.IMPORTANCEPoxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds and mammals all produce type I interferon homologs. In humans, interferon stimulates the synthesis of more than 300 proteins thought to have roles in host defense. Conversely, viruses have evolved means to thwart the host defenses. We are attempting to deconstruct the established virus-host relationship in order to better understand the molecular mechanisms involved. In the present study, we identified a vaccinia virus gene that prevents interferon-mediated inhibition of very early stages of viral replication and is conserved in orthopoxviruses. The viral protein was shown to interact with host proteins involved in proteolysis, suggesting that vaccinia virus may subvert the cellular apparatus for its own defense.
- A functional link between RNA replication and virion assembly in the potyvirusPlum pox virus. [Journal Article]
- JVJ Virol 2018 Feb 14
- Accurate assembly of viral particles in the potyvirusPlum pox virus(PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other vir...
Accurate assembly of viral particles in the potyvirusPlum pox virus(PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced inNicotiana benthamianaleaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro suppliedin trans, was assembled into virus like particles and remained stable afterin vitroincubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential co-agroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads inN. benthamianaleaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a non-replicating viral RNA can be stably assembled into virions as long as it is co-expressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection.IMPORTANCEViruses of the familyPotyviridaeare among the most dangerous threats for basically every important crop, and such socio-economical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case ofPlum pox virus(genusPotyvirus), for instance, we have previously shown that the multitasking viral factor HCPro plays a role during the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unravelling a previously unknown connection of these two key events of the potyviral infection cycle.
- Lactic acid downregulates viral microRNA to promote Epstein-Barr Virus-immortalized B lymphoblastic cell adhesion and growth. [Journal Article]
- JVJ Virol 2018 Feb 14
- High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EB...
High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B cell lymphoma. Here, we report that lactate dehydrogenase (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA, acidic lactate) at low concentration triggers EBV-infected B cell adhesion, morphological changes, and proliferationin vitroandin vivoMoreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression ofSMAD3,JUN, andCOL1Agenes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel potential mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment.IMPORTANCEThe tumor microenvironment is complicated, and lactate, which is created by cell metabolism, contributes to an acidic microenvironment that facilitates cancer progression. However, how lactic acid (LA) operates in virus-associated cancers is unclear. Thus, we studied how the Epstein-Barr virus (EBV, first tumor virus identified in humans; it is associated with many cancers) upregulates the expression of lactate dehydrogenase (LDH-A) and lactate production in B-lymphoma cells. Elevated LA induces adhesion and the growth of EBV-infected B cells by inhibiting viral microRNA transcription. Thus, we offer a novel understanding of how EBV utilizes an acidic microenvironment to promote cancer development.
New Search Next
- Dynamics of Evolution of Poliovirus Neutralizing Antigenic Sites and Other Capsid Functional Domains during a Large and Prolonged Outbreak. [Journal Article]
- JVJ Virol 2018 Feb 14
- We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functio...
We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: 1) neutralizing antigenic (NAg) sites, 2) residues binding the poliovirus receptor (PVR), 3) VP1 residues 1-32, and 4) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites, and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0-2 replacements/isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1-32 of VP1 were highly variable (133 different variants; 0-6 replacements/isolate; 5/32 invariant positions), with residues 1-18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements into the structural core were the most conservative and were fixed at an overall rate ∼20-fold lower than rates for the NAg sites and VP1 1-32, and ∼5-fold lower than the rate for the PVR-binding sites. Only VP1-143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection.IMPORTANCEThe high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During seven years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.