GTPase domain of porcine Mx1 protein interacts with the N protein of porcine deltacoronavirus to inhibit viral replication

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that poses significant economic threats to the swine industry and carries potential zoonotic risk. However, the interactions between PDCoV and host innate immunity, particularly those involving interferon-stimulated genes (ISGs), remain poorly understood. Porcine myxovirus resistance protein 1 (pMx1), a well-characterized ISG with broad-spectrum antiviral activity, has not been investigated in the context of PDCoV infection. This study demonstrates that PDCoV infection upregulates endogenous pMx1 expression in porcine intestinal epithelial cells (IPEC-J2 and IPI-2I) through a type I interferon alpha-dependent pathway. Functional analyses further revealed that pMx1 exerts potent antiviral activity against PDCoV. Mechanistically, we identified a direct interaction between the pMx1 protein and the PDCoV nucleocapsid (N) protein and found the GTPase domain of pMx1 as the critical binding region. This finding was strongly supported by a predicted interaction complex structural model generated using AlphaFold 3, and the GTPase domain was shown to be indispensable for the antiviral function of pMx1. Furthermore, we identified a critical synergistic interaction between the PDCoV N protein and the viral RNA-dependent RNA polymerase – nonstructural protein 12, a core component of the replication–transcription complex. Co-immunoprecipitation assays demonstrated that pMx1 effectively disrupts this interaction, suggesting that pMx1 may inhibit viral genome replication by impairing the formation or stability of the viral replication complex. Collectively, our study elucidates the mechanism by which the pMx1 protein, as a host antiviral factor, inhibits PDCoV infection. These findings provide novel insights into the innate immune defence against coronaviruses and highlight the pMx1 gene as a promising host-derived antiviral factor with potential applications in antiviral therapeutics and genetic improvement strategies for PDCoV control.