- Bluetongue virus VP6 and genomic RNA interaction is essential for genome packaging. [Journal Article]
- JVJ Virol 2018 Dec 12
- The genomes of the Reoviridae, including the animal pathogen Bluetongue virus (BTV), are multi-segmented double-stranded (ds) RNA. During replication, single-stranded (ss) positive-sense RNA segments...
The genomes of the Reoviridae, including the animal pathogen Bluetongue virus (BTV), are multi-segmented double-stranded (ds) RNA. During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein VP6, unique for the orbiviruses, has been proposed to be involved in the RNA packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ss/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed each VP6 has multiple distinct RNA binding regions and only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay and an in vivo single cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For virus with segmented genome, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of Bluetongue virus, which is critical for genome packaging. We use multiple approaches including a robust RNA-protein finger-printing assay, which map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of the capsid-associated VP6. Together with virological and biochemical methods, within VP6, we for the first time identify the viral RNA packaging motif of a segmented dsRNA virus.
- ROTAVIRUS INFECTION ALTERS SPLICING OF THE STRESS-REGULATED TRANSCRIPTION FACTOR XBP1. [Journal Article]
- JVJ Virol 2018 Dec 12
- XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an...
XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA (XBP1es) that corresponded to exon-skipping in the XBP1 pre RNA was induced depending on the rotavirus strain used. We showed that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using mono-reassortant between ES+ ("skipping") and ES- ("nonskipping") strains of rotavirus, we showed that gene 7 encoding the viral translation enhancer NSP3 was involved in this phenomenon and that exon-skipping paralleled the nuclear relocalization of cytoplasmic PABP. We further showed, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype was linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP.IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA- translation-enhancer NSP3. XBP1 being involved in cellular stress and immune responses and the XBP1 protein made from the alternatively spliced RNA being an active transcription factor, our observations raise the question of alternative splicing being a cellular response to rotavirus infection.
- Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus PEDV Infection and the Host Innate Response. [Journal Article]
- JVJ Virol 2018 Dec 12
- Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated wi...
Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum, and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted PEDV CV777, and PEDV infection suppressed IFN production early during the infection course. IFN-lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN-α. Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN-lambda 1 over IFN-α. These events recapitulate the events that occur in vivo. This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.
- Equine-origin immunoglobulin fragments protects nonhuman primates from Ebola virus disease. [Journal Article]
- JVJ Virol 2018 Dec 12
- Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans with fatality rates reaching 90%, with no licensed, specific therapeutics to treat ill patients. Advances over the past ...
Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans with fatality rates reaching 90%, with no licensed, specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (mAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly, quantities are limited, and thus mAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal immunoglobulin fragments F(ab')2 from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100mg/kg F(ab')2 beginning at 3 or 5 days post-infection (dpi) resulted in 100% survival. Notably, NHPs that initiated treatment at 5 dpi were already highly viremic with observable signs of EBOV disease, demonstrating that F(ab')2 was still effective as a therapeutic even in symptomatic patients. These results show that F(ab')2 should be accelerated for clinical testing in preparation of future EBOV outbreaks and epidemics.IMPORTANCE Ebola is one of the deadliest viruses to humans. It has been over 40 years since Ebola was first reported, but no cure is available. Research breakthroughs over the last 5 years have shown that monoclonal antibodies (mAbs) constitute an effective therapy for Ebola. However, mAbs are expensive, difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since Ebola is endemic in several third-world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antisera fragments (F(ab')2) from horses vaccinated with an Ebola vaccine, and tested its protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after these animals were visibly ill with Ebola. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.
- A highly attenuated vesicular stomatitis virus-based vaccine platform controls HBV replication in mouse models of hepatitis B. [Journal Article]
- JVJ Virol 2018 Dec 12
- Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomati...
Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8+ T cell and antibody responses. The CD8+ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8+ T cell responses that corresponded with a reduction in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in intrahepatic HBV-specific CD8+ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multi-specific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patient's own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein, and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV.
- Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms. [Journal Article]
- JVJ Virol 2018 Dec 12
- Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus-1 (BoDV), a non-segmented, ...
Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus-1 (BoDV), a non-segmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in the N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results not only provided the mechanism of how mRNA splicing generates viral proteins that have distinct function but also a novel strategy for replication control of RNA viruses using the isoforms with different subcellular localization.IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the genus orthobornavirus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than wild-type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small sized genomes.
- A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species Specificity. [Journal Article]
- JVJ Virol 2018 Dec 12
- Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. Howev...
Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and their hosts. Here, we present evidence suggesting the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS <1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7% of NTCP amino acid (aa) residues exhibit rapid evolution under positive selection (dN/dS <1). Notably, a substitution at aa 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested a close association of the aa 158 positive selection with the pressure by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this NTCP residue. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing adaptive mutation in host receptor.IMPORTANCE Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection, while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidences supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species-specificity.
- Porcine hemagglutinating encephalomyelitis virus activation of the integrin α5β1-FAK-Cofilin pathway causes cytoskeletal rearrangement to promote its invasion of N2a cells. [Journal Article]
- JVJ Virol 2018 Dec 12
- Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dy...
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dynamics are thought to be closely related to this type of nerve damage. Currently, the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear when PHEV enters the host cells. Here, we demonstrate that entry of PHEV into N2a cells induces a biphasic remodeling of the actin cytoskeleton and a dynamic change in cofilin activity. Viral entry is affected by the disruption of actin kinetics or alteration of cofilin activity. PHEV binds to integrin α5β1 and then initiates the integrin α5β1-FAK signaling pathway, leading to virus-induced early cofilin phosphorylation and F-actin polymerization. Additionally, Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42) and downstream regulatory genes p21-activated protein kinases (PAKs) are recruited as downstream mediators of PHEV-induced dynamic changes of the cofilin activity pathway. In conclusion, we demonstrate that PHEV utilizes the integrin α5β1-FAK-Rac1/Cdc42-PAK-LIMK-Cofilin pathway to cause an actin cytoskeletal rearrangement to promote its own invasion, providing theoretical support for the development of PHEV pathogenic mechanisms and new antiviral targets.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the Coronaviridae family, is a typical neurotropic virus that primarily affects the nervous system of piglets to produce typical neurological symptoms. However, the mechanism of nerve damage caused by the virus is not fully elucidated. Actin is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells. Additionally, the morphological structure and function of nerve cells depend on the dynamic regulation of the actin skeleton. Therefore, exploring the mechanism of neuronal injury induced by PHEV from the perspective of the actin cytoskeleton not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets. This is the first report to define a mechanistic link between alterations in signaling from cytoskeleton pathways and the mechanism of PHEV invading nerve cells.
- Human Norovirus Neutralized by a Monoclonal Antibody Targeting the HBGA Pocket. [Journal Article]
- JVJ Virol 2018 Dec 12
- Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The GII.4 persistence is believed to be assoc...
Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The GII.4 persistence is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolate (1974) and a more recent epidemic GII.4 variant (2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities, yet similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivities with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus VLPs from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.IMPORTANCE The emergence of new epidemic GII.4 variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between 1974 and 2012 GII.4 variants, whereas these variants showed varying levels of reactivities against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket and blocked norovirus VLPs from binding to HBGAs and neutralized norovirus virions in the cell culture system. Raised against GII.4 2006 strain this MAb was unreactive to GII.4 1987 isolate, but was able to neutralize newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggested that the amino acid variations surrounding HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known co-factors for infection.
New Search Next
- Vaccine-induced T-cell responses do not predict the rate of acquisition after repeated intrarectal SIVmac239 challenges in Mamu-B*08+ rhesus macaques. [Journal Article]
- JVJ Virol 2018 Dec 12
- Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic phase viremia after infection with the pathog...
Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic phase viremia after infection with the pathogenic simian immunodeficiency virus (SIV)mac239 clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08+ RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T-cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsies. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges and three of them remained aviremic after 3-4 additional challenges. The rate of SIV acquisition in vaccinees was numerically lower (albeit not statistically significant) than that of controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection versus those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a slower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class-I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T-cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.