Elucidation of host-retrovirus interaction by analyzing chromatin dynamics

为了在后基因组时代建立新的病毒学，通过分析宿主和病毒染色质的动力学来阐明表观遗传学的分子基础和了解感染机制是重要的。本项目的目标是通过病毒转录的启动、维持、基因沉默和再激活来阐明逆转录病毒和慢病毒的早期感染和潜伏感染的分子机制，其中涉及染色质重塑因子、SWI/SNF复合体。在本项目的这三年中，我们取得了以下结果：1.我们清楚地证明了在没有TAT的情况下，人SWI/SNF复合体的BRM亚单位对于MLV-LTR或HIV-LTR驱动的稳定和长时间的转录是必不可少的。在BRM表达缺陷的人肿瘤细胞系中，MLV或基于HIV的载体的表达以随机方式迅速沉默。2.利用蛋白质组学，我们已经鉴定了几种与SWI/SNF复合体亚化学计量相互作用的蛋白质。其中，我们仔细研究了p54^&lt；nrb&gt；，因为该蛋白被认为参与转录、剪接和RNA编辑，还因为它可以与HIV RNA的特定区域结合。我们的分析清楚地表明，p54^-lt；nrb&gt；和SWI/SNF复合体移位了几个启动子区域，它们也与启动子或其RNA转录本的下游结合，影响剪接模式。因此，在BRM缺失的细胞系中，一些外显子往往被排除在外。我们现在正在分析p54是否也影响了HIV的剪接，从而产生了几个亚基因组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.