Abstract

Ribonucleoprotein complexes (RNPs), composed of viral genomic RNA encapsidated by nucleocapsid (NP) protein and associated with the viral polymerase, are the functional units for replication and transcription of negative-sense RNA viruses (NSVs). However, how RNP assembly is regulated remains poorly understood. Here, using the plant-infecting NSV Tomato spotted wilt virus as a model, we uncover a lipid-metabolism–driven regulatory pathway linking host fatty acid synthase (FASN) activity, palmitoyltransferase, and viral protein palmitoylation to RNP assembly. The viral RNA-dependent RNA polymerase (L protein) recruits FASN and enhances its self-association, which is required for proviral activity. FASN-derived palmitate supports zDHHC14-mediated palmitoylation of NP at residue C194 in planta. Disruption of this modification selectively impairs NP-L interaction without affecting NP localization, oligomerization, or intracellular trafficking, resulting in defective RNP assembly and reduced viral replication. Together, our findings uncover a direct mechanistic link between host fatty acid synthesis and viral RNP formation, revealing an unexpected role for lipid metabolic enzymes in regulating negative-sense RNA virus replication.