COG8蛋白调控自噬溶酶体影响流感病毒感染的作用机制研究

Influenza virus infection causes serious infectious respiratory diseases, which pose a major threat to human health and public health. Therefore, studying the pathogenic mechanism of influenza virus and virus-host interaction is of great significance for the development of effective antiviral strategies. Autophagy acts as an intracellular degradation mechanism to effectively inhibit viral infection. However, influenza virus infection impedes the fusion of autophagosome with lysosome, thereby evading the autophagic antiviral mechanism. In view of the critical role of cellular autophagy in influenza virus infection, based on the previous genome-wide CRISPR/Cas9 knockout screen and identification, the aim of this project is analyzing the mechanism of host factor COG8 on influenza virus-induced autophagy. Meanwhile, From the aspect angle of both host and virus, the molecular mechanism of COG8-reuglated autolysosome affecting influenza virus infection was explored. In conclusion, the results obtained from this project will provide a theoretical basis for COG8 as a target for anti-influenza virus drugs.

流感病毒感染能引起严重的传染性呼吸道疾病，对人类健康和公共卫生存在重大威胁。因此，研究流感病毒致病机制以及病毒-宿主相互作用对开发有效的抗病毒策略具有重要意义。细胞自噬作为细胞内降解机制能有效的抑制病毒感染，然而流感病毒感染阻碍自噬溶酶体融合，从而逃避细胞自噬抗病毒机制。鉴于细胞自噬在流感病毒感染中扮演关键角色，本项目拟基于前期全基因组CRISPR/Cas9敲除筛选鉴定的宿主因子COG8为研究对象，解析COG8影响流感病毒诱导细胞自噬的作用机制；从宿主和病毒双角度挖掘出COG8调控细胞自噬溶酶体影响流感病毒感染的分子机制，为COG8作为抗流感病毒药物靶点提供理论依据。

流感病毒感染导致自噬体聚集，自噬溶酶体无法形成，促进其感染。流感病毒基质蛋白M2能通过逆转运途径从内涵体到高尔基体再到内质网，而COG8对内涵体-高尔基体逆行转运具有重要调控作用。因此，本项目预测COG8突变诱导自噬溶酶体形成抑制流感病毒感染。利用CRISPR/Cas9构建COG8敲除单克隆细胞系，发现COG8敲除后，流感病毒复制能力显著减弱；构建COG8过表达载体转入COG8敲除的单克隆细胞进行拯救实验时，流感病毒感染能力明显恢复。此外，COG8敲除能有效的减少流感病毒感染导致的自噬体的聚集，促进自噬溶酶体的形成。 COG8敲除的细胞系能够显著阻碍流感病毒从早期内涵体往晚期内涵体的迁移；同时COG8敲除影响细胞逆向转运通路，导致流感病毒基质蛋白M2不能定位到高尔基体上进行病毒组装，而是与早期内涵体一起转运到自噬体内，最后与溶酶体融合，进行降解。此外，我们利用高通量测序数据发现，多个抗病毒因子(DDX58, MDA5, TLR4, IFIT1, IFIT2, RSAD2)在COG8敲除的细胞系表达显著上调，通过qPCR进行了验证，结果一致。综上所述，COG8通过调节细胞逆向转运通路来影响自噬溶酶体形成，进而调控流感病毒感染。

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