Amino acid T25 in the substrate-binding domain of SARS-CoV-2 nsp5 is involved in viral replication in the mouse lung

PLoS One. 2024 Dec 6;19(12):e0312800. doi: 10.1371/journal.pone.0312800. eCollection 2024.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 5 (nsp5) is a cysteine protease involved in viral replication and suppression of the host immune system. The substrate-binding domain of nsp5 is important for its protease activity. However, the relationship between nsp5 protease activity and viral replication remains unclear. We confirmed the importance of amino acid T25 in the nsp5 substrate-binding domain for viral replication using a split luciferase assay. By generating recombinant viruses using bacterial artificial chromosomes, we found that the proliferation of viruses with the T25I mutation in nsp5 was cell-dependent in culture. Furthermore, mice infected with the T25I mutant recombinant virus with a mouse acclimation backbone showed weight loss and increased lung viral load, similar to the wild-type (WT) infected group, up to 3 days after infection. However, after day 4, the lung viral load was significantly reduced in the T25I-infected group compared to that in the WT-infected group. This suggests that nsp5 T25 is involved in the pathogenesis of SARS-CoV-2.

MeSH terms

  • Animals
  • COVID-19* / metabolism
  • COVID-19* / virology
  • Chlorocebus aethiops
  • Female
  • Humans
  • Lung* / metabolism
  • Lung* / pathology
  • Lung* / virology
  • Mice
  • Mice, Inbred BALB C
  • Protein Domains
  • SARS-CoV-2* / genetics
  • SARS-CoV-2* / metabolism
  • SARS-CoV-2* / pathogenicity
  • SARS-CoV-2* / physiology
  • Vero Cells
  • Viral Load
  • Viral Nonstructural Proteins* / genetics
  • Viral Nonstructural Proteins* / metabolism
  • Virus Replication*

Substances

  • Viral Nonstructural Proteins

Grants and funding

This study was partially supported by the Japan Society for the Promotion of Science’s (JSPS) Grants-in-Aid for Scientific Research in the form of a KAKENHI grant [24K10242] to WK and the Japan Science and Technology Agency (Moonshot Research & Development) in the form of a grant [JPMJMS2025] to WK, and the Japan Agency for Medical Research and Development (Research Program on Emerging and Re-emerging Infectious Diseases) in the form of grants [JP21fk0108560h, JP20fk0108267h] to WK. SU was supported by Gunma Induction Doctoral Program for Social Innovation Relay (Support for Pioneering Research Initiated by the Next Generation). YS was supported by the Next generation-Gunma Induction Doctoral Program for Social Innovation Relay (Support for Pioneering Research Initiated by the Next Generation).