The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected more than 29 million individuals worldwide with nearly ~1 million fatalities. Understanding how SARS-CoV-2 initiates viral pathogenesis is of the utmost importance for development of antiviral drugs. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks underline the urgent need for better understanding the mechanism of viral subversion of autophagy. Using murine hepatitis virus-A59 (MHV-A59) as a model betacoronavirus, time-course infections revealed a significant loss in the protein level of ULK1, a canonical autophagy regulating serine-threonine kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target this protein, we conducted in vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PL^pro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PL^pro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from the C-terminal substrate recognition region. Consistent with this, over-expression of SARS-CoV-2 PL^pro is sufficient to impair starvation-induced canonical autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PL^pro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagic pathway.

中文翻译：

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冠状病毒的木瓜蛋白酶样蛋白酶切割ULK1破坏宿主自噬

持续的COVID-19大流行以及2003年SARS和2012年MERS的爆发凸显了将β冠状病毒理解为全球健康挑战的重要性。SAV-CoV-2是COVID-19的病原体，已感染了全球2900万人，死亡人数接近100万人。理解SARS-CoV-2如何启动病毒发病机制，对于开发抗病毒药物至关重要。自噬调节剂已成为对抗SARS-CoV-2的潜在治疗候选药物，但最近的临床挫折突显了对更好地了解自噬病毒颠覆机制的迫切需求。使用鼠肝炎病毒A59（MHV-A59）作为模型冠状病毒，时程感染显示ULK1的蛋白质水平显着下降，规范的自噬调节丝氨酸-苏氨酸激酶，并伴随可能的裂解片段的出现。为了研究病毒编码的蛋白酶是否靶向该蛋白，我们进行了体外和细胞裂解实验，并将ULK1鉴定为SARS-CoV-2木瓜蛋白酶样蛋白酶（PL^亲）。诱变研究发现，ULK1在G499之后以保守的PL ^pro识别序列（LGGG）裂解，将其N端激酶结构域与C端底物识别区分开。与此相一致，SARS-CoV-2 PL ^pro的过度表达足以削弱饥饿诱导的规范自噬并破坏ULK1-ATG13复合物的形成。最后，我们展示了ULK1在MHV-A59复制中的双重作用，在早期复制过程中发挥了亲病毒功能，该功能在感染后期被灭活。总之，我们的研究确定了一种新的机制，β冠状病毒的PL ^pro通过靶向细胞自噬途径来诱导病毒发病。