Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor

Abstract

Background SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology. Methods We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction. Results We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells. Conclusions Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.

Crosstalk between nucleocytoplasmic trafficking and the innate immune response to viral infection

“…Other factors bind to the nucleocytoplasmic transport machinery, including NSP4 (which interacts with GP210), NSP9 (which associates with Nup54, Nup58, Nup62, Nup88, and Nup214), NSP15 (which binds to NTF2), and ORF6 (which interacts with the Nup98–RAE1 complex) ( 179 ). Indeed, this last interaction has been confirmed by several other groups ( 180 , 181 , 182 , 183 ) and was shown to disrupt bidirectional nucleocytoplasmic transport, likely inhibiting host response to viral infection ( 183 ). This interaction also blocks nuclear translocation of STAT1 to antagonize IFN signaling ( 181 ).…”

“…Verdinexor was demonstrated to inhibit the infection of respiratory syncytial virus, VEEV, IAV, EBV, KSHV, AdV-5, as well as HPV-11 ( 297 , 298 , 299 , 300 ). More recently, studies revealed that selinexor, another nuclear export inhibitor that selectively binds CRM1, could be repurposed to directly target the interaction between the SARS-CoV-2 protein ORF6 and other host proteins such as Nup98-RAE1 ( 179 , 182 ). Although none of the drugs has been approved in clinical trials for treating viral infections, these studies show that the comprehensive understanding of the crosstalk between nucleocytoplasmic transport and host antiviral immune responses to viral infection, together with high-throughput drug screening, will contribute to the development of novel antiviral therapies in the future.…”

“…other groups (180)(181)(182)(183) and was shown to disrupt bidirectional nucleocytoplasmic transport, likely inhibiting host response to viral infection (183). This interaction also blocks nuclear translocation of STAT1 to antagonize IFN signaling (181).…”

“…Since RAE1 plays a role in mRNA export, ORF6 also alters the mRNA export and expression of innate immunity proteins. Aside from Nup98–RAE1 complex, ORF6 also interacts with many other key members of the nuclear pore machinery, including Nups, such as RanBP2/Nup358, Nup160, Nup188, Nup210, Nup37, and Nup93, importins, such as importin-5 (also known as importin-β3), importin-8, RanBP6, and importin-β1, exportins, such as CRM1 and exportin-T (also known as XPO3), as well as spliceosome components, such as THO complex subunit 3, a member of TREX complex known for splicing-coupled mRNA export ( 182 ).…”

SARS-CoV-2 Accessory Proteins in Viral Pathogenesis: Knowns and Unknowns

“…The ORF6 gene is common to all Sarbecoviruses , including SARS-CoV and SARS-CoV-2, and no orthologues have been found in other Betacoronaviruses , such as MERS-CoV. The protein encoded by SARS-CoV-2 ORF6 gene is 61 aa residues and has been localized to the endoplasmic reticulum and membrane of vesicles such as autophagosomes and lysosomes ( 34 ). SARS-CoV-2 ORF6 protein, is a potent IFN antagonist, a role that was previously described by Kopecky-Bromberg and coworkers in SARS-CoV ( 35 ).…”