应用携带TC标签的猪瘟病毒研究Npro蛋白核定位分子机制

Npro蛋白是瘟病毒属成员特有的自体蛋白酶，具有拮抗宿主Ⅰ型干扰素产生的功能。此前我们通过猪瘟病毒标记和活细胞成像技术首次发现，Npro蛋白具有核定位现象，但Npro蛋白的核定位分子机制及其生物学功能目前尚不清楚。本项目拟利用插入TC分子标签的猪瘟病毒实时示踪Npro蛋白的入核过程；通过被动扩散、选择性易位试验确定Npro蛋白的入核方式；利用绿色荧光蛋白标记和截短表达界定Npro蛋白的核定位信号；通过 GST pull-down、免疫共沉淀、哺乳动物细胞双杂交、激光共聚焦、RNA干扰等技术筛选和鉴定与Npro蛋白相互作用的细胞核蛋白；通过上调和下调表达研究细胞核蛋白对Npro蛋白核定位、Ⅰ型干扰素应答和猪瘟病毒复制的影响，以及Npro蛋白对细胞核蛋白功能的影响，从而阐明Npro蛋白核定位的分子机制和生物学新功能，为揭示猪瘟病毒复制和致病机制提供科学数据。

The N-termianl autoprotease Npro, which is unique to pestiviruses, is involved in prevention of type I interferon production by promoting proteasomal degradation of interferon regulatory factors 3 or 7. Previously, we demonstrated for the first time the nuclear localization of Npro in living cells using biarsenically labeled classical swine fever virus (CSFV). However, the molecular mechanism and function(s) of nuclear localization of Npro remain unclear. This proposal is aimed to 1) track the nuclear trafficking of Npro in living PK-15 cells infected with tetracysteine-tagged CSFV, 2) determine the nuclear import pathway of Npro using passive diffusion and selective translocation assays, 3) map the nuclear localization signal of Npro using serial deletion mutants of Npro tagged with enhance green fluorescent protein, 4) identify the cellular nucleoprotein(s) interacting with Npro by GST pull-down, co-immunoprecipitation, mammalian cell two-hybrid, co-localization and RNA interference assays, and 5) investigate the effects of cellular nucleoprotein(s) on Npro localization, type I interferon induction and viral replication by overexpression and knock-down. This project is expected to uncover the molecular mechanism of nuclear localization and new function(s) of Npro, and provide useful data for understanding of the replication and pathogenicity mechanisms of CSFV.