Multiple Capsid Protein Binding Sites Mediate Selective Packaging of the Alphavirus Genomic RNA

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, we comprehensively defined binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrated Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments showed that PS was not required for production of infectious SFV or Chikungunya virus. Instead, we identified multiple novel Cp binding sites that were enriched on gRNA-specific regions and promoted infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrated that budding caused discrete changes in Cp-gRNA interactions. Notably, Cps top binding site was maintained throughout virus assembly, and specifically bound and assembled with Cp into core-like particles in vitro. Together our data suggest a new model for selective alphavirus genome recognition and assembly.