Genome-scale eCLIP mapping of SARS-CoV-2 RNA interactions with NSP8, NSP12, and N reveals regulatory sites

The global impact of the COVID-19 pandemic highlights the need for a deeper understanding of SARS-CoV-2 replication. Although the essential roles of the RNA-dependent RNA polymerase (NSP12), primase (NSP8), and nucleocapsid (N) proteins are established, their contact points with the full-length viral RNA genome during active infection require further characterization. Here, we used enhanced crosslinking and immunoprecipitation (eCLIP) to map direct interactions of NSP8, NSP12, and N across the SARS-CoV-2 genome during live virus infection at early and late stages of replication. We mapped NSP8 and NSP12 binding to the 5′ and 3′ ends of positive- and negative-sense RNA, consistent with their roles in viral replication, transcription, and translation. We also observed an NSP12 binding site within the conserved Y1 domain of NSP3 that modulates RNA and protein abundance. In addition, N binds the 5′ untranslated region (UTR), supporting a role in translational regulation early in infection, and associates with the frameshift element, where it modulates ribosomal frameshifting. Together, these findings map SARS-CoV-2 RNA–protein interactions across the viral genome and delineate specific regulatory regions for potential antiviral intervention.