SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70.
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ABSTRACT:
SUBMITTER: Jiang HW
PROVIDER: S-EPMC7387808 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC8430508 | biostudies-literature
Project description:The emergence of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a great threat to global health. ORF9b, an important accessory protein of SARS-CoV-2, plays a critical role in the viral host interaction, targeting TOM70, a member of the mitochondrial translocase of the outer membrane complex. The assembly between ORF9b and TOM70 is implicated in disrupting mitochondrial antiviral signaling, leading to immune evasion. We describe the expression, purification, and characterization of ORF9b alone or coexpressed with the cytosolic domain of human TOM70 in E. coli. ORF9b has 97 residues and was purified as a homodimer with an molecular mass of 22 kDa as determined by SEC-MALS. Circular dichroism experiments showed that Orf9b alone exhibits a random conformation. The ORF9b-TOM70 complex characterized by CD and differential scanning calorimetry showed that the complex is folded and more thermally stable than free TOM70, indicating strong binding. Importantly, protein-protein interaction assays demonstrated that full-length human Hsp90 is capable of binding to free TOM70 but not to the ORF9b-TOM70 complex. To narrow down the nature of this inhibition, the isolated C-terminal domain of Hsp90 was also tested. These results were used to build a model of the mechanism of inhibition, in which ORF9b efficiently targets two sites of interaction between TOM70 and Hsp90. The findings showed that ORF9b complexed with TOM70 prevents the interaction with Hsp90, and this is one major explanation for SARS-CoV-2 evasion of host innate immunity via the inhibition of the interferon activation pathway.
| S-EPMC9132681 | biostudies-literature
Project description:Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the β-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar KD. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.
| S-EPMC8121815 | biostudies-literature
Project description:Type I interferons (IFN-? and IFN-?) are important for protection against many viral infections, whereas type II interferon (IFN-?) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-? and IFN-? gene expression programs. IFN-? and its downstream vitamin D-dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-? and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-?-induced macrophage vitamin D-dependent antimicrobial peptide response was inhibited by IFN-? and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.
| S-EPMC3653587 | biostudies-literature
Project description:The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-β promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-β treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.
| S-EPMC7392898 | biostudies-literature
Project description:SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.
| S-EPMC7654262 | biostudies-literature
Project description:Extant fold-switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer's, and autoimmune disorders. Thus, predictive methods are needed to identify more fold-switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high-throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS-CoV-1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high-throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes 2019 coronavirus disease (COVID-19), poses a significant threat to global public health security. Like other coronaviruses, SARS-CoV-2 has developed various strategies to inhibit the production of interferon (IFN). Here, we have discovered that SARS-CoV-2 Nsp15 obviously reduces the expression of IFN-β and IFN-stimulated genes (ISG56, CXCL10), and also inhibits IRF3 phosphorylation and nuclear translocation by antagonizing the RLR-mediated antiviral signaling pathway. Mechanically, we found that the poly-U-specific endonuclease domain (EndoU) of Nsp15 directly associates with the kinase domain (KD) of TBK1 to interfere TBK1 interacting with IRF3 and the flowing TBK1-mediated IRF3 phosphorylation. Furthermore, Nsp15 also prevented nuclear translocation of phosphorylated IRF3 via binding to the nuclear import adaptor karyopherin α1 (KPNA1) and promoting it autophagy-dependent degradation. These findings collectively reveal a novel mechanism by which Nsp15 antagonizes host's innate immune response.
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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.
| S-EPMC10491932 | biostudies-literature
Project description:The Kallikrein-Kinin System (KKS), comprised of kallikreins (klks), bradykinins (BKs) angiotensin-converting enzyme (ACE), and many other molecules, regulates a number of physiological processes, including inflammation, coagulation, angiogenesis, and control of blood pressure. In this report, we show that KKS regulates Type I IFN responses, thought to be important in lupus pathogenesis. We used CpG (TLR9 ligand), R848 (TLR7 ligand), or recombinant IFN-? to induce interferon-stimulated genes (ISGs) and proteins, and observed that this response was markedly diminished by BKs, klk1 (tissue kallikrein), or captopril (an ACE inhibitor). BKs significantly decreased the ISGs induced by TLRs in vitro and in vivo (in normal and lupus-prone mice), and in human PBMCs, especially the induction of Irf7 gene (p?<?0.05), the master regulator of Type I IFNs. ISGs induced by IFN-? were also suppressed by the KKS. MHC Class I upregulation, a classic response to Type I IFNs, was reduced by BKs in murine dendritic cells (DCs). BKs decreased phosphorylation of STAT2 molecules that mediate IFN signaling. Among the secreted pro-inflammatory cytokines/chemokines analyzed (IL-6, IL12p70, and CXCL10), the strongest suppressive effect was on CXCL10, a highly Type I IFN-dependent cytokine, upon CpG stimulation, both in normal and lupus-prone DCs. klks that break down into BKs, also suppressed CpG-induced ISGs in murine DCs. Captopril, a drug that inhibits ACE and increases BK, suppressed ISGs, both in mouse DCs and human PBMCs. The effects of BK were reversed with indomethacin (compound that inhibits production of PGE2), suggesting that BK suppression of IFN responses may be mediated via prostaglandins. These results highlight a novel regulatory mechanism in which members of the KKS control the Type I IFN response and suggest a role for modulators of IFNs in the pathogenesis of lupus and interferonopathies.
| S-EPMC5801412 | biostudies-literature