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

ABSTRACT 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 σ3 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 σ3 in the T1L/T3DM2 background. Our findings show that the K287T mutation in σ3 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 σ3 in the development of myocardial disease. The K287T mutation, unlike R296T, eliminates σ3’s capacity to inhibit protein kinase R (PKR) activation and to suppress NF-κB-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 σ3 protein in modulating host innate immune responses and in influencing the outcome of REOV infection and myocarditis development. IMPORTANCE Viral 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 σ3 protein to inhibit protein kinase R (PKR) and to suppress transcription of NF-κB-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.