Identification of Cleavage Sites Proteolytically Processed by NS2B-NS3 Protease in Polyprotein of Japanese Encephalitis Virus

文献类型： 外文期刊

第一作者： Wahaab, Abdul

作者： Wahaab, Abdul;Liu, Ke;Hameed, Muddassar;Anwar, Muhammad Naveed;Kang, Lei;Li, Chenxi;Ma, Xiaochun;Wajid, Abdul;Yang, Yi;Khan, Umair Hassan;Wei, Jianchao;Li, Beibei;Shao, Donghua;Qiu, Yafeng;Ma, Zhiyong

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关键词： Japanese encephalitis virus; NS2B-NS3 protease; cleavage site; polyprotein; substrate; pathogenesis

期刊名称：PATHOGENS （ 影响因子：3.492； 五年影响因子：4.066 ）

ISSN：

年卷期： 2021 年 10 卷 2 期

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收录情况： SCI

摘要： Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.

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