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.

冠状病毒的人畜共患传播仍对人类构成显著威胁。猪急性腹泻综合征冠状病毒（Swine acute diarrhea syndrome coronavirus, SADS-CoV）是一种与HKU2相关的蝙蝠冠状病毒，可给养猪业造成严重经济损失，且具备引发人类暴发疫情的潜在风险。然而，目前学界对SADS-CoV如何逃逸宿主天然免疫的机制认知仍较为有限，这阻碍了针对潜在人类暴发疫情的有效应对。
本研究证实，SADS-CoV包膜蛋白（Envelope, E）可通过巨自噬/自噬-溶酶体通路诱导信号转导与转录激活因子2（signal transducer and activator of transcription 2, STAT2）降解，从而抑制I型干扰素（type I interferon, IFN-I）信号通路。机制层面，包膜蛋白E通过由NBR1自噬货物受体（NBR1 autophagy cargo receptor, NBR1）与视神经蛋白（optineurin, OPTN）介导的自噬促进STAT2降解，以此逃逸宿主天然免疫。值得注意的是，包膜蛋白E的泛素化修饰是STAT2发生自噬降解的必要条件。此外，包膜蛋白E的赖氨酸残基K61对其稳定表达至关重要，但并不参与其泛素化修饰过程。
综上，本研究揭示了包膜蛋白E通过自噬靶向STAT2以阻断IFN-I信号通路的全新机制，加深了学界对SADS-CoV免疫逃逸策略的认知，并为防控该病毒感染提供了潜在药物靶点。

缩写说明：
3-MA：3-甲基腺嘌呤（3-methyladenine）；
ATG：自噬相关基因（autophagy related）；
BafA1：巴弗洛霉素A₁（bafilomycin A₁）；
BSA：牛血清白蛋白（bovine serum albumin）；
CALCOCO2/NDP52：钙结合卷曲螺旋结构域2（calcium binding and coiled-coil domain 2）；
CC：卷曲螺旋（coiled-coil）；
CHX：环己酰亚胺（cycloheximide）；
Co-IP：免疫共沉淀（co-immunoprecipitation）；
DAPI：4',6-二脒基-2-苯基吲哚（4’,6-diamidino-2-phenylindole）；
DBD：DNA结合结构域（DNA-binding domain）；
DMEM：达尔伯克改良伊格尔培养基（Dulbecco’s Modified Eagle’s medium）；
DMSO：二甲基亚砜（dimethyl sulfoxide）；
E：包膜蛋白（Envelope）；
FW：四色氨酸（four-tryptophan）；
GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；
HA：血凝素（hemagglutinin）；
hpt：处理后小时数（hours post-treatment）；
IF：间接免疫荧光（indirect immunofluorescence）；
IFNB/IFN-β：干扰素β（interferon beta）；
IgG：免疫球蛋白G（immunoglobulin G）；
ISG：干扰素刺激基因（IFN-stimulated genes）；
ISRE：干扰素刺激应答元件（interferon-stimulated response element）；
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；
MOI：感染复数（multiplicity of infection）；
NBR1：NBR1自噬货物受体（NBR1 autophagy cargo receptor）；
OPTN：视神经蛋白（optineurin）；
PBS：磷酸盐缓冲液（phosphate-buffered saline）；
PRRs：模式识别受体（pattern recognition receptors）；
qPCR：定量聚合酶链式反应（quantitative polymerase chain reaction）；
SAR：选择性自噬受体（selective autophagy receptor）；
SQSTM1/p62：自噬受体p62（sequestosome 1, SQSTM1/p62）；
STAT：信号转导与转录激活因子（signal transduction and activator of transcription）；
TBS-T：含吐温20的Tris缓冲盐水（Tris-buffered saline with Tween 20）；
TCID₅₀：50%组织细胞感染量（50% tissue culture infective dose）；
TOLLIP：托林相互作用蛋白（toll interacting protein）；
Ub：泛素（ubiquitin）；
UBA：C端泛素相关结构域（C-terminal ubiquitin-associated）；
VSV：水疱性口炎病毒（vesicular stomatitis virus）；
WB：蛋白质印迹（western blotting）；
WT：野生型（wild type）。