Grass Carp Reovirus VP56 Allies VP4, Recruits, Blocks, and Degrades RIG-I to More Effectively Attenuate IFN Responses and Facilitate Viral Evasion

Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection.

Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction

ABSTRACT Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic double-stranded RNA (dsRNA). The reovirus outer capsid protein μ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with μ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of μ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity. IMPORTANCE We use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here, we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

Reovirus σ3 protein limits interferon expression and cell death induction

ABSTRACTInduction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic dsRNA. The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating cell death, we used siRNA-mediated knockdown. Similar to that observed by diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. σ3 knockdown does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or cell death. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and cell death following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCEWe use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. σ3 limits the induction of necroptosis by preventing excessive production of interferon following infection.

MODELACIÓN POR COMPARACIÓN Y ACOPLAMIENTO MULTIMÉRICO DE LA PROTEÍNA EXTERIOR PEQUEÑA DE LA CÁPSIDE DEL BACTERIÓFAGO IME08

The small outer capsid protein plays a stabilizing role in the viral assembly, adhering to the<br />capsid during the later stages of maturation. This protein acts as glue among adjacent<br />capsomers, protecting the virus against extreme changes. The small outer capsid protein of the<br />bacteriophage IME08 was modelled using structural protein homology. A trimeric protein<br />docking was developed with the best-scored model and important sites of the molecules<br />interfaces were identified. It was used the Swiss Model platform for developing the protein<br />structure. Reliability was assessed by the QMEAN, Verify3D and ERRAT indices. The quality of<br />the whole model was verified by Ramachandran plot and the trimerization model was<br />performed on the platform ClusPro 2.0 Protein-Protein Docking. The structure obtained has a<br />reliability estimator QMEANscore4 of 0.769, rating it as a suitable model. The Z-Score QMEAN<br />value was 0.133, showing that the obtained model is not different from the experimental<br />structures stored in PDB database. The estimators and the Ramachandran plot evaluated<br />positively the model. Finally we identified a loop between two secondary structures as an<br />important site of the interaction of small outer capsid proteins, indicating that from residues 35<br />to 41 are relevant in the trimerization process.

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response

Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/MS, and then verified by co-IP and an advanced far-red fluorescence complementation system. VP4 was proved to bind to the CARD and RD domains of RIG-I and promoted K48-linked ubiquitination of RIG-I to degrade RIG-I. VP4 reduced mRNA and promoter activities of key genes of RLR pathway and sequential IFN production. As a consequence, antiviral effectors were suppressed and GCRV replication increased, resulting in intensified cytopathic effect. Furthermore, results of transcriptome sequencing of VP4 stably expressed CIK (C. idella kidney) cells indicated that VP4 activated the MyD88-dependent TLR pathway. Knockdown of VP4 obtained opposite effects. These results collectively revealed that VP4 interacts with RIG-I to restrain interferon response and assist GCRV invasion. This study lays the foundation for anti-dsRNA virus molecular function research in teleost and provides a novel insight into the strategy of immune evasion for aquatic virus.

Generation of simian rotavirus reassortants with diverse VP4 genes using reverse genetics

Species A rotaviruses (RVAs) are a major cause of gastroenteritis in animals and humans. Their genome consists of 11 segments of dsRNA, and reassortment events between animal and human strains can contribute to the high genetic diversity of RVAs. We used a plasmid-based reverse genetics system to investigate the reassortment potential of the genome segment encoding the viral outer capsid protein VP4, which is a major antigenic determinant, mediates viral entry and plays an important role in host cell tropism. We rescued reassortant viruses containing VP4 from porcine, bovine, bat, pheasant or chicken RVA strains in the backbone of simian strain SA11. The VP4 reassortants could be stably passaged in MA-104 cells and induced cytopathic effects. However, analysis of growth kinetics revealed marked differences in replication efficiency. Our results show that the VP4-encoding genome segment has a high reassortment potential, even between virus strains from highly divergent species. This can result in replication-competent reassortants with new genomic, growth and antigenic features.

Cell Entry-Independent Role for the Reovirus μ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products

ABSTRACTThe reovirus outer capsid protein μ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized μ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized μ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of μ1 also affects aspects of viral replication. We found that, while μ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the μ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when μ1 is knocked down. Following μ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of μ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to μ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCEWe used mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for the reovirus outer capsid protein μ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection; μ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.