Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction.

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.

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

The 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.IMPORTANCE We 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.