Key result
SARS-CoV-2 Nsp3 domains bind the viral RNA 5' UTR to actively load the nucleocapsid.
Why the study?
Data on SARS-CoV-2 Nsp3 molecular structure and which of its regions interact with viral RNA during replication were incomplete.
Multiple domains of SARS-CoV-2 Nsp3 interact with the 5' UTR of the viral RNA, suggesting the pore structure actively recognizes and loads viral RNA onto the nucleocapsid protein.
May guide Nsp3-targeted antivirals; leaves open translation to human infection or therapy.
Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the 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 the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein.IMPORTANCEThe largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5' UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.
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Lemak et al. (2025) studied SARS-CoV-2. Structure-function analysis of Nsp3 was evaluated on Binding of Nsp3 domains to the 5' UTR of the viral genome. Multiple domains of SARS-CoV-2 Nsp3, particularly Ubl1 and Y, interact with the 5' UTR of the viral RNA, suggesting the pore structure actively recognizes and loads viral RNA onto the nucleocapsid.
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