Structural studies of the full-length nucleocapsid protein of SARS-CoV-2

Project Summary The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The nucleocapsid (N) protein of SARS-CoV-2 plays several functionally critical roles in the life cycle of the virus, similar to those in other coronaviruses. It assembles with the genomic RNA into an RNA- protein complex, which is packaged into virions. The protein also forms a replication-transcriptional complex with the RNA synthesis machinery to create replicating organelles for efficient transcription and replication. It protects viral double-stranded RNA (dsRNA), which would otherwise be degraded by the RNA interference (RNAi)-based antiviral immune defense mechanism in the host cells. N protein adopts a modular architecture with two well- folded domains, including N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three intrinsically disordered regions. Crystal structures of the NTD and CTD have been determined, but high- resolution information of the full-length N protein, in particular, in complex with RNA is still lacking. We hypothesize that intrinsically disordered regions of the N protein from SARS-CoV-2 adopt defined structures when bound to RNA that are critical for viral assembly and replication. In this project, we plan to determine the high-resolution structure of the full-length N protein in complex with the viral RNA by cryogenic electron microscopy (cryo-EM). The goal is to visualize detailed structural features of the full-length N protein in the context of RNA to advance our understanding of its function and to inform development of intervention strategies. Our specific aim is to obtain a high-resolution structure of intact SARS-CoV-2 N protein in complex of RNA.