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PreprintCited by 8SARS-CoV-2 and COVID-19 ResearchEnzyme Structure and FunctionAnimal Virus Infections Studies

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

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Lemak, Sofia, Skarina, Tatiana, Flick, Robert, Patel, Deepak T., Stogios, Peter J., Savchenko, Alexei·2024·Microbiology Spectrum
DOI 10.1101/2024.05.09.593331PMC PMC12323374

Preprint — published as 10.1128/spectrum.02871-24

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Abstract

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.

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Metadata

Authors
Lemak, S., Skarina, T., Flick, R., Patel, D. T., Stogios, P. J., & Savchenko, A.
Affiliations
  • University of Toronto (CA)
  • University of Calgary (CA)
Corresponding author
  • Stogios, Peter J.
  • Savchenko, Alexei
ORCID
Venue
Microbiology Spectrum
Publisher
openRxiv
Published
2024-05-09
Volume
13
Issue
8
Pages
e0287124-e0287124
ISSN
2165-0497 (ISSN-L)
DOI
10.1101/2024.05.09.593331
PMC
PMC12323374
OpenAlex
W4412090233
Cited by
8
References
1
Citations by year
2026: 7 · 2025: 1
FWCI
6.15
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbiology Spectrum (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Temerty Faculty of Medicine, University of Toronto
  • National Institute of Allergy and Infectious Diseases (#75N93022C00035, HHSN272201700060C)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
Keywords
SARS-CoV-2, NSP3, 5' untranslated region, double-membrane vesicles, viral RNA replication, nucleocapsid protein, crystal structure, nucleic acid-binding domain, RNA translocation, structure-function analysis
MeSH terms
Protein Domains, COVID-19, SARS-CoV-2, Humans, Protein Binding, RNA, Viral, Virus Replication, Genome, Viral, Viral Nonstructural Proteins, Crystallography, X-Ray, 5' Untranslated Regions

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