Inhibition of PERK Kinase, an Orchestrator of the Unfolded Protein Response (UPR), Significantly Reduces Apoptosis and Inflammation of Lung Epithelial Cells Triggered by SARS-CoV-2 ORF3a Protein

Abstract

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.

Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation

“…While both L-ORF3a and E-ORF3a proteins have been shown to induce cytopathic effects by increasing NF-kB-mediated cytokine levels ( 14 , 15 , 17 , 27 ), the precise molecular mechanism responsible for NF-kB activation by ORF3a remains elusive. Initial reports suggested that the interaction of TRAF3 with ORF3a is necessary for ORF3a-induced NF-κB and NLRP3 inflammasome activation ( 10 , 39 ).…”

“…Furthermore, our results have revealed a significant crosstalk between ER stress and autophagy, a process referred to as reticulophagy or ER-phagy ( 31 ). We compared the induction of autophagy and ER stress, as assessed by the accumulation of LC3-II and the levels of XBP1s ( 27 , 43 , 44 ). As anticipated, ORF3a proteins primarily localized to lysosomes not only induce autophagy but also trigger ER stress ( Fig.…”

“…While present at ER, ORF3a causes cellular ER stress response ( 25 – 27 ). Once ORF3a arrives at lysosomes, it triggers a host cellular antiviral autophagy response, resulting in the activation of the HOPS complex.…”

“…Additionally, there is a crosstalk between ER stress and autophagy, a process that is known as reticulophagy or ER-phagy where ER stress elicits a selective form of autophagy, aka ER-phagy, that transpires ER-associated degradation (ERAD) leading to degradation of ER-residing proteins or ER destruction ( 31 ). Early studies showed that induction of reticulophagy by SARS-CoV-2 is essential for viral infection and replication, and ORF3a elicits reticulophagy response and then disrupts ER homeostasis to induce ER stress and inflammatory responses during SARS-CoV-2 infection ( 25 – 27 ).…”

Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells

“…By contrast, OC43 infection caused a detectable activation of PERK, consistent with its ability to trigger ER stress in infected cells [ 33 , 46 , 47 ]. Interestingly, PERK activation was previously shown to either inhibit (transmissible gastroenteritis virus, [ 28 ]) or promote (porcine epidemic diarrhea virus, [ 44 ]) coronavirus replication. In our study, we demonstrate that PERK is an antiviral factor for OC43, as the silencing of PERK expression in 293A cells enhanced viral protein accumulation and infectious virion release.…”