Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5′ UTR of the viral genome

Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the Ubiquitin-like 1 (Ubl1), Nucleic Acid Binding (NAB), β-coronavirus-Specific Marker (βSM) domains and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5′ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5′ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5′ UTR of viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein.