A single point mutation in the σ3 protein encoded by the reovirus S4 gene substantially attenuates the virulence of a highly myocarditic reassortant virus in neonatal mice

Viral myocarditis is a major cause of heart damage, sudden death, and heart failure. Some strains of Mammalian Orthoreoviruses (REOV) cause myocarditis in newborn mice. This study examines the role of the REOV {sigma}3 protein, encoded by the S4 gene, in modulating the virulence and myocarditic potential of a highly virulent T1L/T3DM2 reassortant virus that contains the M2 gene from the Type 3 Dearing (T3D) strain in the Type 1 Lang background. We introduced single-point mutations in the double-stranded RNA-binding region of {sigma}3 in the T1L/T3DM2 background. Our findings show that the K287T mutation in {sigma}3 prevents the myocarditic phenotype and significantly reduces the virulence of T1L/T3DM2. Unlike the parental reassortant virus and a control reassortant mutant S4(R296T), infection of neonatal mice with the T1L-S4(K287T)/T3DM2 virus resulted in 100% survival, lower viral titers, particularly in the heart and spleen, and no gross or severe histological signs of myocarditis. This attenuation, despite similar in vitro growth and in vivo dissemination, indicates a tissue-specific replication deficit and highlights a key role for {sigma}3 in the development of myocardial disease. The K287T mutation, unlike R296T, eliminates {sigma}3s capacity to inhibit protein kinase R (PKR) activation and to suppress NF-{kappa}B-driven transcription, leading to a strong innate immune response that likely controls viral replication and reduces cardiac damage. These results underscore the crucial role of the {sigma}3 protein in modulating host innate immune responses and in influencing the outcome of REOV infection and myocarditis development. IMPORTANCEViral myocarditis is a serious disease with life-threatening consequences, particularly in neonates and young animals. Infection of neonatal mice with certain strains of mammalian reovirus induces myocarditis. A reassortant virus containing the M2 gene from the Type 3 Dearing Strain in an otherwise Type 1 Lang background causes severe myocarditis in 100% of neonatal mice. Here, we show that a single point mutation in the S4 gene, which abolishes the capacity of the encoded {sigma}3 protein to inhibit protein kinase R (PKR) and to suppress transcription of NF-{kappa}B-dependent genes, significantly attenuates the lethality of the T1L/T3DM2 reassortant and significantly reduces the severity of myocarditis. These findings highlight the importance of viral innate immune suppression in the induction of myocarditis.