The alpha-coronavirus E protein inhibits the JAK-STAT pathway signaling by triggering STAT2 degradation through OPTN- and NBR1-mediated selective autophagy

The zoonotic transmission of coronaviruses continues to pose a considerable threat to humans. Swine acute diarrhea syndrome coronavirus (SADS-CoV), a bat coronavirus related to HKU2, causes severe economic losses in the pig industry and has the potential to trigger outbreaks in humans. However, our understanding of how SADS-CoV evades the host’s innate immunity remains limited, hindering effective responses to potential human outbreaks. In this study, we demonstrate that the SADS-CoV envelope protein (E) inhibits type I interferon (IFN-I) signaling by inducing the degradation of STAT2 via the macroautophagy/autophagy-lysosome pathway. Mechanistically, the E protein evades host innate immunity by promoting STAT2 degradation through autophagy, mediated by the NBR1 and OPTN receptors. Notably, ubiquitination of E protein is required for the autophagic degradation of STAT2. Additionally, lysine residue K61 of the E protein is crucial for its stable expression; however, it is not involved in its ubiquitination. In conclusion, our study reveals a novel mechanism by which the E protein disrupts IFN-I signaling by targeting STAT2 via autophagy, enhancing our understanding of SADS-CoV’s immune evasion strategies and providing potential drug targets for controlling viral infections. <b>Abbreviations</b>: 3-MA: 3-methyladenine; ATG: autophagy related; BafA1: bafilomycin A₁; BSA: bovine serum albumin; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; DBD: DNA-binding domain; DMEM: Dulbecco’s Modified Eagle’s medium; DMSO: dimethyl sulfoxide; E, Envelope. FW: four-tryptophan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HA: hemagglutinin; hpt: hours post-treatment; IF: indirect immunofluorescence; IFNB/IFN-β: interferon beta; IgG: immunoglobulin G; ISG: IFN-stimulated genes; ISRE: interferon-stimulated response element; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PBS: phosphate-buffered saline; PRRs: pattern recognition receptors; qPCR: quantitative polymerase chain reaction; SAR: selective autophagy receptor; SQSTM1/p62: sequestosome 1; STAT: signal transduction and activator of transcription; TBS-T: Tris-buffered saline with Tween 20; TCID50: 50% tissue culture infective dose; TOLLIP: toll interacting protein; Ub: ubiquitin; UBA: C-terminal ubiquitin-associated; VSV: vesicular stomatitis virus; WB: western blotting. WT: wild type.