Elucidation of host-retrovirus interaction by analyzing chromatin dynamics

通过对宿主和病毒染色质动态的分析，阐明表观遗传学的分子基础，理解感染机制，对于后基因组时代新病毒学的建立具有重要意义。本项目的目标是通过逆转录病毒和慢病毒的转录起始、维持、基因沉默和再激活等途径，阐明逆转录病毒和慢病毒早期感染、潜伏感染的分子机制，其中涉及染色质重塑因子SWI/SNF复合物。本课题在三年的研究中取得了以下结果：1.我们明确证明了人SWI/SNF复合物的Brm亚基在达特缺失的情况下，对MLV-LTR或HIV-LTR驱动的稳定和延长的转录是必不可少的。在Brm表达缺陷的人肿瘤细胞系中，基于MLV或HIV的载体的表达以随机方式迅速沉默。2.利用蛋白质组学，我们鉴定了几种与SWI/SNF复合物亚化学计量相互作用的蛋白质。其中，我们对p54蛋白进行了研究<nrb>，因为该蛋白被认为参与转录、剪接和RNA编辑，也因为它可以结合到HIV RNA的特定区域。我们的分析清楚地表明，p54 ~+<nrb>和SWI/SNF复合物转位了几个启动子区域，它们也结合到启动子或其RNA转录本的下游，影响剪接模式，因此，在Brm缺陷细胞系中，一些外显子往往被排除。我们现在正在分析p54是否<nrb>也影响HIV的剪接，产生几个亚基因组RNA。

For the establishment of new virology after the post-genome era, it is important to elucidate molecular basis of epigenetics and to understand infection mechanisms by analyzing dynamics of host and viral chromatin. The goal of this project is to elucidate molecular mechanisms of early infection, latent infection of retrovirus and lentiviruses through viral transcriptional initiation, maintenance, gene silencing and reactivation, which involves chromatin remodeling factor, SWI/SNF complex. During these three years of this project, we obtained following results.1.We clearly demonstrated that Brm subunit of human SWI/SNF complex is essential for stable and prolonged transcription drived by MLV-LTR or HIV-LTR in the absence of tat. In human tumor cell lines that are deficient in Brm expression, the expression of MLV- or HIV- based vectors are rapidly silenced in a stochastic manner.2.Using proteomics, we have identified several proteins that substoichiometorically interact with SWI/SNF complex. Among them, we scrutinized p54^<nrb>, because this protein is suggested to be involve in transcription, splicing and RNA editing and also because it can bind to a specific region if HIV RNA. Our analysis clearly indicated that p54^<nrb> and SWI/SNF complex translocates several promoter regions and they also bind to the downstream of the promoter or its RNA transcripts, affecting splicing patterns.In Brm deficient cell lines, therefore, some exons were tend to be excluded. We are now analyzing whether p54^<nrb> is also affect the splicing of HIV that produces several subgenomic RNA.