A feline coronavirus nucleocapsid protein disrupts ZC3HAV1–viral RNA association to counteract host RNA-level restriction

BACKGROUND: Coronaviruses have evolved intricate mechanisms to evade host RNA surveillance systems and sustain efficient replication. The feline infectious peritonitis virus (FIPV), a highly lethal feline coronavirus that causes systemic vasculitis and fatal peritonitis, represents a major challenge in veterinary medicine and serves as a valuable model for studying conserved coronavirus-host interactions. Among coronavirus structural proteins, the nucleocapsid (N) protein not only packages the viral genome but also modulates host antiviral responses, yet its role in counteracting RNA restriction remains incompletely understood. RESULTS: Using co-immunoprecipitation coupled with mass spectrometry screening, we identified the host RNA-binding antiviral factor ZC3HAV1 (also known as ZAP or PARP13) as a prominent N-associated host protein. ZC3HAV1 restricted FIPV replication by associating with viral RNA and reducing viral RNA accumulation and persistence. We show that the FIPV N protein associates with the RBD-containing region of ZC3HAV1 and that the ZC3HAV1-associated region of N contributes to efficient viral counter-restriction activity. Through coordinated protein-protein and protein-RNA interactions, FIPV N interferes with ZC3HAV1-viral RNA association, thereby supporting viral RNA accumulation and replication. Expression of FIPV N in a heterologous Japanese encephalitis virus system also alleviated ZC3HAV1-mediated RNA-level restriction, indicating that this antagonism activity is functionally transferable and not entirely dependent on FIPV-specific replication machinery. Despite the low CpG content of FIPV RNA, ZC3HAV1 efficiently associated with multiple viral transcripts, suggesting that ZC3HAV1-FIPV RNA recognition is not fully explained by overall CpG abundance and may also be influenced by subgenomic RNA context or structural features. CONCLUSIONS: These findings reveal a previously unrecognized mechanism by which a highly pathogenic feline coronavirus circumvents host RNA restriction. By engaging the RBD-containing region of ZC3HAV1 and disrupting ZC3HAV1-viral RNA association, the FIPV N protein effectively attenuates ZC3HAV1-associated antiviral restriction and helps preserve viral RNA accumulation and persistence. This work expands the functional understanding of coronavirus N proteins beyond genome packaging and immune modulation, and provides new insight into FIPV-host interactions that may inform future studies of RNA-level counter-restriction mechanisms in other animal and human coronaviruses.