Structural and evolutionary insights into deleterious nsSNPs in bovine MDA5 suggest their role in viral dsRNA recognition

Abstract

The bovine IFIH1 gene, which encodes melanoma differentiation-associated protein 5 (MDA5), is a major part of antiviral innate immunity in cattle and mediates recognition of long, double-stranded viral RNA. To elucidate the functional significance of naturally occurring IFIH1 variants, we performed a comprehensive survey of SNPs in the core MDA5 and identified 53 missense variants through database mining and in silico prediction tools. Eighteen of those variants were predicted to be deleterious and eight of those were consistently classified as destabilizing the MDA5 structure. Furthermore, four of those eight variants (G490D, I507V, I717M, and L729H) were found to be highly conserved and functionally essential residues and classified as high-risk nsSNPs. MD simulations of wild-type and mutant dsRNA-bound MDA5 high-risk variants, particularly I717M and G490D, induced increased backbone fluctuations and reduced structural adaptability based on essential dynamics analysis. Moreover, the domain-based RMSF profiling highlighted enhanced local flexibility in key helicase and C-terminal regions, correlating with weakening of dsRNA contacts in MDA5 variants. Although the dsRNA itself remained stably accommodated in its binding pocket, the mutant proteins consistently showed compromised domain closure and altered residue interactions that would facilitate RNA sensing oligomeric filament formation. These results reveal the molecular basis by which deleterious IFIH1 nsSNPs disrupt innate immune signaling in cattle and emphasize their potential role in disease resistance and livestock health. Furthermore, this study aids rational genetic selection and provides structural insight that may support future studies on antiviral responses in agriculture and livestock industries.