丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus，HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein，MAVS)来逃逸天然免疫识别，但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程，探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein，GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462)，用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示，病毒复制早期MAVS切割效率较低；NS3/4A高效切割MAVS发生于HCV复制晚期，且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞，在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况，发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现，不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示，HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积，对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。

Kinetics of hepatitis C virus NS3/4A-mediated MAVS cleavage during hepatitis C virus infection

Hepatitis C virus(HCV)protease NS3/4A cleaves mitochondrial antiviral signaling protein (MAVS)to evade host innate recognition and interferon production.However,HCV infection induces interferon production in certain cell types and HCV could replicate in cells expressing NS 3/4A cleavage-resistant MAVS.The roles of NS3/4A-mediated MAVS cleavage in host innate evasion and restriction of viral replication need to be clarified.In this study,by using an HCV NS3/4A-mediated MAVS cleavage reporter system,in which Huh7.5 cells express GFP-NLS-MAVS-TM462,we dissected the kinetics of MAVS cleavage during HCV infection.It was found that MAVS was barely cleaved during the early time but was efficiently cleaved only in the late time during viral infection.Using ypet-tagged HCV virus and Huh7.5 cells expressing RFP-NLS-MAVS-TM462,we dissected the MAVS cleavage in the HCV-positive (ypet-positive)cells on a single-cell manner.It was found that MAVS cleavage occurred only in part of the HCV-infected cells.We also assessed the MAVS cleavage by HCV NS3/4A from different genotypes,and found the cleavage efficiency was correlated with the protein level of HCV NS3/4A.Taken together,we found NS3/4A-mediated MAVS cleavage occurs in the late stage during viral replication and is correlated with NS3 protein level.We proposed HCV NS3/4A-mediated MAVS cleavage does not contribute to host innate evasion during early viral replication step.