肠道病毒71型介导的宿主组蛋白H3切割及其在感染中的作用研究

Histone PTMs play a vital role in virus infection. The success of viral infection requires manipulation of host histone proteins in order to promote viral replication and to suppress antiviral responses. Our previous studies revealed that the N-terminal tail of histone H3 was clipped during EV71 virus infection of RD cells, and the clipping site was identified as H3K23 by middle-down quantitative proteomics. We proved that the viral 3Cpro entered host nucleus when the clipping took place. Histone H3 was also cleaved in vitro by 3Cpro. Thus, we hypothesized that 3Cpro cleaved histone H3 in host nucleus during EV71 infection. Here, we will employ point mutation approach to verify the clipping site identified by middle-down proteomics; we will combine over-expression, nucleus-cytoplasm separation and immunofluorescence to prove direct histone H3 clipping by 3C; we will investigate histone H3 clipping-induced changes in host cell transcription and try to elucidate the possible epigenetic regulatory mechanism; we will also try to investigate whether it is a common mechanism for other enteroviruses and small RNA viruses interaction with host cells.This proposal will provide valuable information in elucidating the epigenetic regulatory mechanism in virus-host interactions.

组蛋白修饰在病毒感染中起着重要作用，病毒通过调控宿主组蛋白，激活有利于病毒复制的基因并抑制抗病毒基因。前期工作中我们发现EV71病毒感染引发宿主组蛋白H3切割，通过蛋白质组学分析鉴定切割位点是K23；通过核质分离和免疫荧光实验发现感染中EV71病毒3C蛋白酶入核，与H3切割发生时间吻合，体外实验也证明3C能切割H3，因此我们推测EV71感染中3C蛋白酶入核并切割组蛋白H3。为深入研究病毒与宿主互作的表观遗传调控机制，本项目拟通过点突变切割位点，验证EV71感染引发H3切割的确切位点；结合过表达、核质分离和免疫荧光等手段，证明3C在感染中入核切割H3；通过蛋白质组学、RNA-Seg和CHIP-seq研究H3切割对病毒感染和宿主免疫的影响及可能表观调控机制；通过对其他肠道病毒和小RNA病毒感染的研究，探讨类似病毒与宿主作用的规律性。本项目将为阐明病毒与宿主相互作用的表观遗传调控机制提供新思路。

组蛋白修饰在病毒感染中起着重要作用，病毒通过调控宿主组蛋白，激活有利于病毒复制的基因并抑制抗病毒基因，引起宿主免疫的改变。相比DNA病毒和一些逆转录病毒，我们对RNA病毒感染引发宿主表观遗传变化知之甚少。在本课题中我们发现宿主组蛋白H3在RNA病毒EV71感染时发生了N末端蛋白酶切割现象，因此我们结合多种研究手段对病毒感染引发组蛋白切割进行了系统性研究。我们开发并优化middle-down蛋白质组学研究方法，分析鉴定切割位点为H3K23，并通过点突变实验验证明确了该切割位点。我们也建立和优化了middle-down和bottom-up蛋白组学研究方法综合分析了组蛋白的修饰水平，发现切割位点附近甲基化（如H3K27me）显著升高，乙酰化（如H3K18ac）显著降低。我们通过过表达和构建多种突变质粒、核质分离、免疫荧光、co-IP和体外实验等，证明感染后EV71病毒的3C蛋白进入宿主细胞核内并切割组蛋白H3的N末端。我们通过蛋白质组学、RNA-Seq和CHIP-Seq综合分析，筛选与H3切割密切相关的宿主基因和信号通路，探讨关键基因在病毒感染和宿主免疫中的作用。本项目发现了RNA病毒与宿主相互作用的新方式，为研究RNA病毒通过表观遗传调控宿主基因表达的新规律提供依据。