Residue Gln370 of Newcastle disease virus nucleoprotein mediate viral inclusion body-like structure formation via interaction with residue Ser254 of viral phosphoprotein

Abstract

Newcastle disease virus (NDV) infects more than 250 avian species and mammals. Cytoplasmic inclusion bodies (IBs) are a hallmark of paramyxovirus infection; however, the molecular mechanisms underlying NDV IB formation remain poorly understood. In this study, we investigated the interactive sites of viral protein driving IB-like structure formation via transmission electron microscopy, immunofluorescence assays (IFA), viral titration, co-immunoprecipitation (co-IP), structural modeling analysis. Both virulent and attenuated NDV strains induced IBs in chicken-, mouse-, and human-derived cells. And the virus replicated well in CEF and HeLa cells rather than MEF. Time-course analysis revealed that IBs emerged as small puncta early in infection, increased in size over time, and ultimately accumulated in the perinuclear region. Co-immunoprecipitation (co-IP) and IFA revealed that amino acid residues 367–375 of the nucleoprotein (NP) bound to the PMD of the phosphoprotein (P), and this interaction was required for IB-like structure assembly. Furthermore, structural modeling and experiments of the NP–P complex identified residues Gln370 of NP and residue Ser254 of P as the principal contact and interacting sites for IB-like structure assembly. These findings reveal a previously unrecognized NP–P binding interface that mediates NDV IB formation, providing new insights into the molecular basis of viral condensate biogenesis in paramyxoviruses and a potential target for antiviral intervention.