Abstract
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.
Keywords:
B.1.1.7; COVID19; Hsp90; Orf9b; SARS-CoV-2; TOM70; interferon; mitochondria; variant of concern.
MeSH terms
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Animals
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Binding Sites / genetics
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COVID-19 / immunology
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COVID-19 / transmission*
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COVID-19 / virology
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Chlorocebus aethiops
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Coronavirus Nucleocapsid Proteins / genetics
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Coronavirus Nucleocapsid Proteins / immunology
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Coronavirus Nucleocapsid Proteins / isolation & purification
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Coronavirus Nucleocapsid Proteins / metabolism*
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HSP90 Heat-Shock Proteins / metabolism*
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Humans
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Interferon Type I / immunology
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Interferon Type I / metabolism
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Mitochondrial Membrane Transport Proteins / genetics
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Mitochondrial Membrane Transport Proteins / isolation & purification
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Mitochondrial Membrane Transport Proteins / metabolism*
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Mitochondrial Precursor Protein Import Complex Proteins
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Mutation
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Phosphoproteins / genetics
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Phosphoproteins / immunology
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Phosphoproteins / isolation & purification
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Domains / genetics
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Recombinant Proteins / genetics
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Recombinant Proteins / immunology
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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SARS-CoV-2 / genetics
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SARS-CoV-2 / metabolism
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SARS-CoV-2 / pathogenicity*
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Vero Cells
Substances
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Coronavirus Nucleocapsid Proteins
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HSP90 Heat-Shock Proteins
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Interferon Type I
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Mitochondrial Membrane Transport Proteins
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Mitochondrial Precursor Protein Import Complex Proteins
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Phosphoproteins
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Recombinant Proteins
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TOMM70 protein, human
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nucleocapsid phosphoprotein, SARS-CoV-2