Identification of the SARS-CoV-2 genome packaging signal in the nsp12-coding region

Selective genome packaging is a critical step for RNA viruses, which must distinguish genomic RNA from other abundant transcripts. For SARS-CoV-2, the cis-acting packaging signal is thought to be recognized by the nucleocapsid (N) protein, but its identity and mechanistic basis for selective recognition remain undefined. Here we identify the packaging signal within the nsp12 polymerase-coding region. CLIP-seq maps N-bound sites and, together with virus-like particle assays, pinpoints a conserved segment with strong packaging activity. An orthogonal defective-interfering RNA approach confirms its role in genome selection. We further delineate two critical subregions, α and β, that engage the N C-terminal domain. Synonymous mutations in either subregion selectively disrupt packaging and reduce viral fitness. Notably, the α subregion encompasses the ribosomal frameshifting element, revealing its dual role in viral translation and assembly. These findings establish the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target.