The Multi-Functional Reovirus Sigma 3 Protein Is A Virulence Factor That Suppresses Stress Granule Formation And Is Associated With Myocardial Injury

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein sigma 3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of sigma 3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of sigma 3 binding to dsRNA during viral infection is largely unknown. To identify functions of sigma 3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen sigma 3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of sigma 3. Two recombinant viruses expressing these sigma 3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that sigma 3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that sigma 3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.Author summary The sigma 3 protein of mammalian orthoreoviruses is a double-stranded RNA binding protein that has classically been thought to function by scavenging dsRNA within infected cells and thus prevents activation of cellular sensors of dsRNA such as the kinase PKR. Here we used mutagenesis to identify the region of sigma 3 responsible for binding dsRNA. Characterization of mutant viruses expressing sigma 3 proteins incapable of binding dsRNA show that contrary to expectation, dsRNA binding is not required for sigma 3-mediated inhibition of PKR. We show that one mutant virus (R296T) despite being deficient in dsRNA-binding can inhibit PKR and replicates similar to WT virus. In contrast, another mutant virus (K287T) that bears a sigma 3 protein that cannot prevent dsRNA-mediated activation of PKR induces stress granules in infected cells and replicates less efficiently than WT virus. In vivo, the K287T mutant is attenuated in its replication and unlike WT virus and the R296T mutant virus does not cause heart disease (myocarditis).