BPS2026 – Structural characterization of the SARS-CoV-2 replication and transcription complex

At around 30 kb, the SARS-CoV-2 genome is one of the largest viral genomes among RNA viruses. As such, the replication machinery must be prepared to quickly and efficiently replicate this genome in order for the viral infection to effectively persist. The replication-transcription complex (RTC) is formed by polymerase Nsp12, helicase Nsp13, and cofactors Nsp7 and Nsp8, which assemble with RNA. One of the remaining questions regards the dynamics of Nsp12 and Nsp13 with RNA, as the placement of these in complex and the directionality of their activities seem to be at odds. In this study, we used several techniques to study the SARS-CoV-2 replication and transcription complex (RTC). We assessed complex stoichiometry and binding kinetics via mass photometry (MP) and analytical ultracentrifugation (AUC). We also employed small-angle x-ray scattering (SAXS), size-exclusion chromatography-coupled small-angle x-ray scattering (SEC-SAXS), and small-angle neutron scattering (SANS) in order to characterize the structure and protein-protein interactions of the SARS-CoV-2 replication complex proteins with each other and with RNA. This data have allowed us to gain further understanding of the formation and structure of the RTC.