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Proteomic Study of Nonstructural Protein 1 of SARS‐CoV to Identify Its Role in Host Shutoff
Author(s) -
Nag Anita,
Gerassimovich Yevgeniy,
MiladinovskiBengall Samantha
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.00387
Subject(s) - biotinylation , biotin , biology , ribosome , coronavirus , stress granule , ribosomal protein , hek 293 cells , microbiology and biotechnology , messenger rna , gene , rna , translation (biology) , biochemistry , covid-19 , medicine , disease , pathology , infectious disease (medical specialty)
Severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to suppress host gene expression, also known as the host shutoff. Current literature suggests that the C‐terminus of nsp1 competes for the mRNA binding site in the 40S ribosome, preventing host translation from host mRNAs. However, several mutations on surface residues of nsp1 are reported to either attenuate or augment its host shutoff function suggesting a role of these surface amino acids in dampening host gene expression. Additionally, nsp1 of SARS‐CoV and SARS‐CoV‐2 share 84% sequence identity, prompting us to believe they share a similar mechanism of host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we isolated host proteins proximal to nsp1 in human epithelial kidney (HEK) cells using the proximity‐dependent biotinylation experiment by expressing nsp1 fused to BIOID2. BioID2 is a promiscuous bacterial biotin ligase protein that attaches biotin to free lysine residues of nearby proteins. Biotin treatment of these cells incorporates biotin tag on proteins proximal to nsp1. Using streptavidin magnetic beads, we isolated these proteins and identified them using mass spectroscopy. Finally, several factors were verified by western blot. By comparing protein complexes, we identified a group of proteins including ribosomal proteins that are likely involved in host shutoff. In addition to the ribosomal proteins, factors implicated in the stress granule formation and RNA processing are identified and verified. These proteins are common in both SARS‐CoV and SARS‐CoV‐2. Currently, we are using similar methods to verify the binding of these proteins to nsp1 mutations in the N‐terminus and C‐terminus.