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SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response.

ABSTRACT: In the past year and a half, SARS-CoV-2 has caused 240 million confirmed cases and 5 million deaths worldwide. Autophagy is a conserved process that either promotes or inhibits viral infections. Although coronaviruses are known to utilize the transport of autophagy-dependent vesicles for the viral life cycle, the underlying autophagy-inducing mechanisms remain largely unexplored. Using several autophagy-deficient cell lines and autophagy inhibitors, we demonstrated that SARS-CoV-2 ORF3a was able to induce incomplete autophagy in a FIP200/Beclin-1-dependent manner. Moreover, ORF3a was involved in the induction of the UPR (unfolded protein response), while the IRE1 and ATF6 pathways, but not the PERK pathway, were responsible for mediating the ORF3a-induced autophagy. These results identify the role of the UPR pathway in the ORF3a-induced classical autophagy process, which may provide us with a better understanding of SARS-CoV-2 and suggest new therapeutic modalities in the treatment of COVID-19.

SUBMITTER: Su WQ

PROVIDER: S-EPMC8706696 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SARS-CoV-2 ORF3a accessory protein was found to be involved in virus release, immunomodulation and exhibited a pro-apoptotic character. In order to unravel a potential ORF3a-induced apoptotic and inflammatory death mechanism, lung epithelial cells (A549) were transfected with in vitro synthesized ORF3a mRNA. The protein’s dynamic involvement as “stress factor” for the endoplasmic reticulum, causing the activation of PERK kinase and other UPR-involved proteins and therefore the upregulation of their signaling pathway executioners (ATF6, XBP-1s, PERK, phospho eIF2a, ATF4, CHOP, GADD34), has been clearly demonstrated. Furthermore, the overexpression of BAX and BH3-only pro-apoptotic protein PUMA, the upregulation of Bcl-2 family genes (BAX, BAK, BID, BAD), the reduced expression of Bcl-2 in mRNA and protein levels, and lastly, the cleavage of PARP-1 and caspase family members (caspase-3,-8 and -9) indicate that ORF3a displays its apoptotic character through the mitochondrial pathway of apoptosis. Moreover, the upregulation of NFκB, phosphorylation of p65 and IκΒα and the elevated expression of pro-inflammatory cytokines (IL-1b, IL-6, IL-8 and IL-18) in transfected cells with ORF3a mRNA indicate that this protein causes the inflammatory response through NFκB activation and therefore triggers lung injury. An intriguing finding of our study is that upon treatment of the ORF3a-transfected cells with GSK2606414, a selective PERK inhibitor, both complications (apoptosis and inflammatory response) were neutralized, and cell survival was favored, whereas treatment of transfected cells with z-VAD (a pan-caspase inhibitor) despite inhibiting cell death, could not ameliorate the inflammatory response of transfected A549 cells. Given the above, we point out that PERK kinase is a “master tactician” and its activation constitutes the main stimulus for the emergence of ORF3a apoptotic and inflammatory nature and therefore could serve as potential target for developing novel therapeutic approaches against COVID-19.

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SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response.

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