The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region

This study investigated the antiviral activity of mammalian Mx1 proteins against Thogoto virus, focusing on the molecular determinants responsible for viral restriction. Using polymerase minireplicon assays, infection experiments, mutational analyses, and protein interaction studies, the authors compared Mx1 proteins from different mammalian species. Among them, equine Mx1 (eqMx1) exhibited strong antiviral activity comparable to human MxA, whereas most other mammalian Mx1 proteins were inactive. In contrast, none of the tested Mx1 proteins inhibited the related Dhori virus, indicating virus-specific differences in susceptibility to Mx-mediated restriction. Detailed mutational analyses identified the flexible loop L4 in the stalk domain of eqMx1 as the key determinant of antiviral specificity. In particular, two adjacent residues, tryptophan (W562) and glycine (G563), were essential for THOV inhibition. Substitution of either residue abolished antiviral activity, while maintaining a bulky aromatic residue at position 562 together with glycine at position 563 preserved restriction. Importantly, introducing this motif into otherwise inactive bovine Mx1 conferred antiviral activity, demonstrating its functional sufficiency. Overall, the findings reveal that a minimal structural motif within loop L4 governs the antiviral activity of eqMx1 and highlight this region as an evolutionary hotspot shaping host–virus interactions in thogotoviruses.
(TMR)