Reverse genetics reveals a role of 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 interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (OAS) and 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 an RV reversegenetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at low levels in the small intestine and were 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. IMPORTANCE Rotaviruses 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 of improving current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virusencoded 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 next-generation rotavirus vaccine design.