Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease

Semliki Forest virus nonstructural protein 2 is involved in suppression of the type I interferon response

The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist

Effects of an in-frame deletion of the 6k gene locus from the genome of Ross River virus

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity