Reverse genetics reveals a role of the rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication in vivo.

Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo Here, we present both in vitro and in vivo evidence that the host IFN-inducible 2'-5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2'-5'-phosphodiesterase (PDE) domain to counteract RNase L mediated antiviral signaling. Using a RV reverse genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at a lower level in the small intestine and shed less in the feces of wild-type mice and such defects were rescued in Rnasel^-/- suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well characterized function as the viral RNA capping enzyme.ImportanceRotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low to middle income countries, where the burden of the diseases is the most severe. With the ultimate goal to improve current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that the interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus encoded VP3 antagonizes RNase L activity both in vitro and in vivo These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for the next-generation rotavirus vaccine design.