宿主蛋白HDAC3调控VSV复制的机制研究

Vesicular stomatitis virus (VSV) has broad cell tropism, one of main reasons is that VSV replicate and propagate by harnessing host metabolism system via host conserved proteins. Based our pilot study, inhibiting/silencing Histone deacetylase 3 (HDAC3) and silencing Nuclear receptor co-repressor 1(NCOR1), which interacts with HDAC3 in the nucleus, could inhibit VSV replication, suggesting HDAC3 functions in chromosome level to regulate VSV replication. However, how HDAC3 regulates the replication of VSV is still largely elusive. For this reason, this project will test the influence of HDAC3 on VSV replication by overexpressing or knocking out Hdac3 gene; make sure whether or how VSV infection affects HDAC3 distribution and expression change by laser confocal fluorescence microscope and Western blot; study HDAC3 enrichment region change across the whole genome through Chromatin Immunoprecipitation sequencing technique, which is followed by identifying the key genes; study the key protein interacted cooperatively with HDAC3 to regulate gene transcription during VSV infection through Co-Immunoprecipitation and Mass Spectrometry technique. The foreseen results will not only uncover the mechanism how HDAC3 regulates VSV replication and propagation, but also provide theoretical guide to search for targets to block VSV infection.

水疱性口炎病毒（VSV）具有广泛的细胞嗜性,主要原因之一是其可借助宿主蛋白参与对机体代谢系统的调控来保障病毒自身的复制周期。本课题组前期研究发现，抑制/沉默宿主细胞组蛋白去乙酰化酶3（HDAC3）及沉默与HDAC3互作的核受体共抑制因子1（NCOR1）均可降低VSV复制，提示HDAC3在染色质水平参与调控作用，但该分子如何影响VSV复制尚不清楚。鉴于此，本项目拟过表达与敲除Hdac3基因，验证其对VSV复制的影响；利用激光共聚焦及Western blot明确VSV感染对HDAC3定位及表达的影响；利用染色质免疫共沉淀-测序，解析VSV感染后HDAC3在全基因组DNA互作位点的变化，鉴定受调控的关键基因；利用免疫共沉淀及质谱技术，明确与HDAC3互作并协同调控转录的蛋白。该项目的预期结果不仅有利于阐明HDAC3对VSV复制增殖的调控机制，而且也为寻找潜在阻止VSV感染的靶点提供理论依据。

水疱性口炎病毒（VSV）具有高度的嗜上皮性，是一种可感染多种动物和人的高度接触性传染病。探索VSV的复制机制有助于防控此种外来疫病。不仅如此，VSV也被视为一种重要的溶瘤病毒，虽然具有很好的开发前景，但潜在的副作用仍是制约其进一步被应用的障碍之一。探索VSV在肿瘤中的复制机制也有助于溶瘤性VSV的开发利用。本项目以宿主细胞中HDAC3为着眼点，着力探究HDAC3对VSV复制增殖影响及机制。明确了HDAC3可促进VSV的复制，而其自身的表达水平和胞内定位并不受影响；HDAC3可在VSV侵染进程中调控干扰素相关基因的表达，其中MX1可抑制VSV；MX1基因启动子区域的H3K27ac修饰在VSV侵染过程中异常性升高，证实在VSV侵染过程中受HDAC3调控表达；HDAC3所在的共抑制复合物中GPS2、NCOR1及TBL1XR1均可调控VSV的复制和干扰素相关基因的表达；HDAC3的功能性抑制可削弱VSV在肿瘤细胞及组织中的复制；HDAC3的功能性抑制也可削弱VSV在肿瘤细胞及组织中诱导的细胞焦亡。预期结果不仅有助于深入揭示VSV的致病机制，为防控水疱性口炎提供理论基础，而且可为VSV作为活病毒载体及抗肿瘤产品的安全应用提供理论指导。