Dual-barrier mechanism of superinfection exclusion in Newcastle disease virus: nucleoprotein and hemagglutinin-neuraminidase cooperate to block secondary infection

Superinfection exclusion (SIE) is a well-documented phenomenon in virology, wherein a primary viral infection prevents subsequent infection by the same or closely related viruses through various mechanisms. This phenomenon has been observed with Newcastle disease virus (NDV), a member of the Paramyxoviridae family. However, the molecular mechanisms underlying NDV-mediated SIE remain unclear. In this study, we elucidate a dual-barrier mechanism of NDV-mediated SIE, involving both the nucleoprotein (NP) and the hemagglutinin-neuraminidase (HN) glycoprotein. Our findings demonstrate that NDV SIE is initiated within 2 h of primary infection and intensifies over time, independent of viral virulence and host cell type. The NP protein forms an early cytoplasmic barrier by sequestering incoming NP subunits, while HN-neuraminidase activity at the viral membrane cleaves sialic acid receptors, preventing secondary virion adsorption. Furthermore, our data reveal that NDV-mediated SIE is specific to intraspecies interactions and does not extend to heterologous viruses, such as vesicular stomatitis virus.IMPORTANCESuperinfection exclusion (SIE) is a crucial viral strategy for limiting co-infection and ensuring the dominance of a single viral strain. The identification of the dual-barrier mechanism in Newcastle disease virus (NDV), involving both the nucleoprotein (NP) and hemagglutinin-neuraminidase (HN) proteins, significantly enhances our understanding of viral defense mechanisms. By elucidating how SIE operates at both the cytoplasmic and membrane levels, this study not only provides a mechanistic framework for NDV interference but also opens avenues for therapeutic applications. The discovery that NP and HN act synergistically to block secondary infection presents new strategies for combating viral co-infection in veterinary medicine and potentially in human viruses. Furthermore, understanding the viral determinants involved in SIE could guide the development of antiviral therapies that exploit these mechanisms to prevent superinfection and reduce viral spread.