Negative-sense RNA virus nucleocapsid as a versatile platform for gene delivery, vaccine development, and antiviral screening.

Viruses typically infiltrate host cells via specialized cellular receptors, a pivotal step that is challenging when suitable permissive cell lines or hosts are unavailable. Leveraging the unique internal replication machinery of negative-stranded (ns) RNA viruses, we demonstrate that purified nucleocapsid (NCs), their functional genomic templates, can directly initiate a complete viral life cycle upon intracellular delivery, bypassing conventional cell surface interaction. We successfully purified vesicular stomatitis virus (VSV) NCs, including those from a G-deleted pseudotyped variant (rVSV-ΔG-TFP). Exogenous delivery of these NCs into cells stimulated gene expression and generated infectious progeny virions (from full-length NCs). Critically, VSV-ΔG-TFP NCs, though self-amplifying, are inherently non-infectious, offering a safer, potentially more effective alternative for a vaccine development platform compared to live/attenuated viruses or mRNA-based systems. Furthermore, these NCs provide a secure method for transporting components of highly pathogenic nsRNA viruses (e.g., Nipah virus), acting as an inherent self-deactivating feature against accidental exposure. This study establishes NCs as a novel, intrinsically safer, and self-amplifying platform for antiviral screening, vaccine development, gene delivery, and biosafety in pathogen research.