MERS冠状病毒3C样蛋白酶是一种新的细胞自噬诱导蛋白

冠状病毒（Coronavirus, CoV）3C样蛋白酶（3CLpro）在冠状病毒复制过程中起重要作用，是一种重要的潜在抗病毒药物候选靶标。细胞自噬是宿主重要抗病毒防御机制之一，但目前冠状病毒诱导细胞自噬及其机制还不很清楚。本研究以人类新发高致病性冠状病毒 --中东呼吸综合征冠状病毒（MERS CoV）为研究对象，探讨人类冠状病毒感染与细胞自噬的关系。通过免疫荧光法检测发现，MERS 3CLpro引起细胞内eGFP-LC3B绿色荧光点状聚集，同时MERS 3CLpro诱导自噬标志蛋白微管相关蛋白1-轻链3基 (LC3-II)表达增多，表明MERS 3CLpro可激活细胞自噬。进一步研究发现，MERS 3CLpro诱导细胞自噬体形成而阻断或抑制自噬溶酶体形成，即MERS 3CLpro诱导不完全细胞自噬效应，而且MERS 3CLpro诱导细胞自噬具有时间依赖性且不依赖于其蛋白酶催化活性。此外发现SARS CoV和NL63 CoV等其它人类冠状病毒3CLpro也具有诱导细胞自噬效应，表明3CLpro诱导细胞自噬可能是人类冠状病毒所具有的一种普遍生物学特性。本研究首次发现冠状病毒蛋白酶3CLpro能诱导宿主细胞自噬，是一种新型冠状病毒来源的宿主细胞自噬诱导蛋白，这一发现拓展了对人类冠状病毒蛋白酶功能的新认识，为研究冠状病毒与宿主抗病毒天然免疫以及以病毒蛋白酶为靶标的抗病毒药物研究提供了理论基础。

MERS Coronavirus 3C-like protease is an virus-encoded inducer of autophagy

Autophagy plays important roles in modulating viral replication and antiviral innate immune response. Current studies have revealed that coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to human coronavirus regulation of host innate responses. Here, we first show that the 3C-like protease (3CLpro) of human MERS coronavirus acts as a novel autophagy inducing protein. To gain insights into the relationship of between coronavirus 3C-like proteases and cells autophagy, the 3CLpro from MERS-CoV was constructed and then co-transfected with or not with eGFP-LC3B into HEK 293T cells. The immunofluorescence was performed to monitor the punctas of eGFP-LC3B, and the Western blotting was used to detect the protein level of LC3-II. The results showed that 3CLpro promoted accumulation of LC3 punctas and induced the expression of LC3-II. Moreover, 3CLpro induces autophagy formation in a time-dependent manner, which is independent on the protease activity of 3CLpro. Intriguingly, 3CLpro induces an incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes, leading to prevention of the degradation of the autophagic substrate, p62. Further study on the role of 3CLpro in autophagy indicated that autophagy was induced by various coronaviral 3CLpro, which may be a shared attribute of coronaviruses. Collectively, the results showed that, in addition to the protease activity, MERS coronavirus 3C-like protease has a new function of inducing autophagy，which may contribute to coronavirus replication and regulation of antiviral innate immunity.