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Source: COVID-19 Research  Oct 16, 2020  13 days ago
BREAKING! COVID-19 Research: Coronavirus Study Reveals That SARS-CoV-2 Orf9b Protein Binds To Human Host Mitochondria Protein Tom70
BREAKING! COVID-19 Research: Coronavirus Study Reveals That SARS-CoV-2 Orf9b Protein Binds To Human Host Mitochondria Protein Tom70
Source: COVID-19 Research  Oct 16, 2020  13 days ago
COVID-19 Research: A new international comparison study of the various coronaviruses and their effects on human host proteins and various human cellular pathways have revealed among many findings that the SARS-CoV-2 coronavirus protein Orf9b actually binds with the human host mitochondria protein TOM 70.

Orf9b of SARS-CoV-2 was found to be localized to mitochondria upon overexpression as well as in SARS-CoV-2 infected cells. In line with this, the mitochondrial outer membrane protein Tom70 (encoded by TOMM70) is a high-confidence interactor of Orf9b in SARS-CoV-2 interaction maps and may act as a host dependency factor for SARS-CoV-2 .
Tom70 is one of the major import receptors in the TOM complex that recognizes and mediates the translocation of mitochondrial preproteins from the cytosol into the mitochondria in a chaperone dependent manner.
The Tom70 is normally involved in the activation of mitochondrial antiviral-signaling protein (MAVS) and is essential for an antiviral innate immune response.
Tom70 has been described as an essential import receptor for PTEN induced kinase 1 (PINK1) and therefore loss of mitochondrial import efficiency as a result of Orf9b binding to Tom70 substrate binding pocket may induce mitophagy.
The implications of this new findings are many as it suggest ways that the SARS-CoV-2 is able to disrupt certain immune responses to in via this pathway and at the same time deplete the human host ‘power house’  or ‘energy supply’ that the host is dependent on to support its defense purposes.
The mitochondria are typically the ‘powerhouses’ of the cells.

The study findings can help explain to a certain degree as to why many COVID-19 infected patients experience fatigue issues.
The many study findings from this research are published in the journal: Science but Thailand Medical News will be only focusing on this specific interaction in this article while the cover the rest in other subsequent articles. https://science.sciencemag.org/content/early/2020/10/14/science.abe9403
The SARS-CoV-2 protein Orf9b, by binding to the substrate recognition site of Tom70, inhibits Tom70's interaction with heat shock protein 90 (Hsp90), which is key for its function in the interferon pathway and induction of apoptosis upon virus infection.
In a separate international collaboration among more than 60 scientists in the QCRG led by Professor Dr Klim Verba and Professor Dr Oren Rosenberg at QBI, the structure of Orf9b bound to the active site of Tom70 was determined by cryoelectron microscopy (cryoEM) to a remarkable three-angstrom resolution.

 Interaction between Orf9b and human Tom70.

(A) Orf9b-Tom70 interaction is conserved between SARS-CoV-1 and SARS-CoV-2. (B) Viral titers in Caco-2 cells after CRISPR knockout of TOMM70 or controls. (C) Co-immunoprecipitation of endogenous Tom70 with Strep-tagged Orf9b from SARS-CoV-1 and SARS-CoV-2, Nsp2 from SARS-CoV-1, SARS-CoV-2 and MERS-CoV, or vector control in HEK293T cells. Representative blots of whole cell lysates and eluates after IP are shown. (D) Size exclusion chromatography traces (10/300 S200 Increase) of Orf9b alone, Tom70 alone and co-expressed Orf9b-Tom70 complex purified from recombinant expression in E. coli. Insert shows SDS-PAGE of the complex peak indicating presence of both proteins. (E) Immunostainings for Tom70 in HeLaM cells transfected with GFP-Strep and Orf9b from SARS-CoV-1 and SARS-CoV-2 (left) and mean fluorescence intensity ± SD values of Tom70 in GFP-Strep and Orf9b expressing cells (normalized to non-transfected cells; right). (F) Flag-Tom70 expression levels in total cell lysates of HEK293T cells upon titration of co-transfected Strep-Orf9b from SARS-CoV-1 and SARS-CoV-2. (G) Immunostaining for Orf9b and Tom70 in Caco-2 cells infected with SARS-CoV-2 (left) and mean fluorescence intensity ± SD values of Tom70 in uninfected and SARS-CoV-2 infected cells (right). SARS2 = SARS-CoV-2; SARS1 = SARS-CoV-1; MERS = MERS-CoV; IP = immunoprecipitation. **p < 0.05. B, E, G, Student’s t test. E, scale bar = 10 μm.

 A significant and rare finding showed that Orf9b, when by itself, forms a dimer and structurally a beta sheet, but exists as an alpha helix when bound to Tom70.
Utilizing the structural image of the bound proteins, the scientists were able to discover that a key residue in the interaction with Hsp90 is moved out of position, suggesting that Orf9b may modulate key aspects of the immune response, interferon and apoptosis signaling via Tom70.
The study’s functional data, however, shows that Tom70 has at least some role in promoting infection rather than inhibiting it. Utilizing cryoEM, the obtained 3 Å structure of a region of Orf9b binding to the active site of Tom70 remarkably found that Orf9b is in a drastically different conformation than previously visualized. This offers the possibility that Orf9b may partition between two distinct structural states in the cells, with each possessing a different function and possibly explaining its potential functional pleiotropy.

CryoEM structure of Orf9b-Tom70 complex reveals Orf9b adopting a helical fold and binding at the substrate recognition site of Tom70.

(A) Surface representation of the Orf9b-Tom70 structure. Tom70 is depicted as molecular surface in green, Orf9b is depicted as ribbon in orange. Region in charcoal indicates Hsp70/Hsp90 binding site on Tom70. (B) Magnified view of Orf9b-Tom70 interactions with interacting hydrophobic residues on Tom70 indicated and shown in spheres. The two phosphorylation sites on Orf9b, S50 and S53, are shown in yellow. (C) Ionic interactions between Tom70 and Orf9b are depicted as sticks. Highly conserved residues on Tom70 making hydrophobic interactions with Orf9b are depicted as spheres. (D) Diagram depicting secondary structure comparison of Orf9b as predicted by JPred server, as visualized in our structure, or as visualized in the previously-crystallized dimer structure (PDB:6Z4U) (16). Pink tubes indicate helices, charcoal arrows indicate beta strands, amino acid sequence for the region visualized in the cryoEM structure is shown on top. (E) Predicted probability of possessing an internal MTS as output by TargetP server by serially running N-terminally truncated regions of SARS-CoV-2 Orf9b. Region visualized in the cryoEM structure (amino acids 39-76) overlaps with the highest internal MTS probability region (amino acids 40-50). MTS = mitochondrial targeting signal.

The functional significance and regulation of the Orf9b-Tom70 interaction require further experimental elucidation. This interaction, however, which is conserved between SARS-CoV-1 and SARS-CoV-2, could have value as a pan-coronavirus therapeutic target.
Thailand Medical News hypothesizes that the SARS-CoV-2 Orf9b protein binding to the human host mitochondria protein TOM 70 is one way it primes a way for it to evade human host innate immune system as the mitochondrial antiviral-signaling protein (MAVS) is disrupted and certain cytokines involved with the destruction of the virus is prevented from being formed while the virus itself destroys the cellular energy supply of the host needed to launch defense cellular pathways and immune cells functions.
More research will be warranted to confirm this.

For more COVID-19 Research, keep on logging to Thailand Medical News.


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