The Capsid Protein of Semliki Forest Virus Antagonizes RNA Interference in Mammalian Cells

Abstract

RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus. IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

Mechanism and Function of Antiviral RNA Interference in Mice

“…Thus, when VSR is rendered nonexpressing or nonfunctional, the resulting mutant viruses induce abundant vsiRNAs, replicate less efficiently than parental viruses in mESCs, mature murine, monkey, and human cells, and/or newborn mice and are efficiently rescued by knocking out all four Ago genes in mESCs or Dicer gene in human 293T cells ( 10 , 12 – 15 ). Moreover, ebolavirus VP35 and the nucleocapsid protein of yellow fever virus ( Flaviviridae ), Semliki Forest virus ( Togaviridae ), and severe acute respiratory syndrome coronavirus (SARS CoV) and SARS CoV-2 also display activities of dsRNA-binding VSRs ( 15 , 17 – 19 , 64 , 65 ). Together, these findings reveal a new mammalian antiviral response mediated by the RNAi pathway with striking similarities to the siRNA-directed antiviral response characterized extensively in plants and invertebrates ( 7 – 9 , 20 ).…”

The Capsid Protein of Rubella Virus Antagonizes RNA Interference in Mammalian Cells

“…Previous studies show that the RuV capsid forms a disulfide-linked dimer [ 47 ]. Moreover, previous studies showed that homodimerization is critical for VSR’s activity [ 18 , 19 , 20 , 29 , 43 ]; we focused on the key region or residues responsible for the dimerization of the RuV capsid.…”

“…The full-length cDNA of the FHV RNA1 and FHV RNA1 ΔB2 was described previously [ 12 ]. For the purification of the MBP fusion capsid protein, its ORF was inserted into the pFastBac-MBP vector [ 43 ]. The EGFP-siRNA (siEGFP) was chemically synthesized by Rui Bo, Guangzhou, China.…”

“…SFV (strain SFV4) and VSR-deficient SFV were from our laboratory; both were amplified in HEK293T cells [ 43 ]. EV-A71 (VR-1432, strain H) and VSR-deficient EV-A71 were from our laboratory; both were amplified in RD cells [ 18 ].…”

“…The RuV capsid protein is the sole virus-encoded protein component of the nucleocapsid that participates in many processes critical for the viral life cycle, including membrane association, RNA-binding [ 37 , 38 ], modulating viral RNA replication [ 39 , 40 ], mitochondrial rearrangements [ 41 ], and inhibiting host protein translation by a nonenzymatic mechanism [ 42 ]. Our previous study demonstrated that the capsid protein encoded by the Semliki Forest virus (SFV), an important virus belonging to the Alphavirus genus in the family of Togaviridae , is a bona fide VSR that antagonizes antiviral RNAi in the context of SFV infection in cells [ 43 ]. Because the genomic structure and expression pattern of RuV and alphaviruses are similar, it would be intriguing to exploit whether the strategy of using capsid protein to suppress RNAi by SFV is also shared by RuV.…”