参与HBV cccDNA形成的关键宿主因子筛选与鉴定

Persistence of cccDNA is essential for the establishment of chronic infection of HBV, and presents a huge obstacle for the ultimate cure of chronic hepatitis B. Currently, no drug is available which directly targets cccDNA or critical factors required for cccDNA formation. This situation can be attributed, in a large part, to the scarce of clear drug targets, which is in turn caused by the largely unknown mechanism of cccDNA formation. To our knowledge, there is still not a host factor found to be essential for the formation of cccDNA with consistent and solid evidence. In this project, we will try to identify 1 or 2 essential host factors involved in the formation of cccDNA and dissect the mechanism of their actions. Firstly, we will adopt 2 kinds of strategies to screen candidate host factors. One is the sub-genomic scale screening by using a lentiviral shRNA library targeting DNA repair system of host cells. The other is the whole genomic scale screening by using CRISPR/Cas9 technology and 2 unique screening systems, i.e. a cccDNA formation model based on the luciferase reporter gene, and a negative screening system for cccDNA formation. After that, we will characterize these candidates and research deeply their action pathways and identify their interactions with other host factors or viral factors. The findings of this project are expected to provide new information about the events happened during cccDNA formation, and thus help to develop effective strategies interfering with this key step in virus replication.

cccDNA的持续存在是HBV持续感染以及慢乙肝难以彻底治愈的根本原因。目前各种抗HBV治疗方案中，缺少以cccDNA本身或形成环节为靶点的药物。造成这种局面的一个重要原因，是我们对cccDNA形成环节的机制知之甚少，迄今为止，尚无一种宿主因子被确定无疑地认为是HBV cccDNA形成环节所必需。缺乏抗cccDNA的明确靶点，已经严重阻碍了直接针对cccDNA的药物开发。本项目将瞄准cccDNA形成机制问题开展研究，力争鉴定出1-2种参与cccDNA形成的关键宿主因子并解析其作用机制。首先，我们将采用两种分别靶向亚基因组和全基因组的策略来筛选候选因子，然后，对筛选出的宿主因子与cccDNA形成过程的关系进行深入系统研究，解析其作用通路与作用网络，最终为阐明cccDNA的形成机制做出贡献，并为发展干预cccDNA形成的手段奠定基础。

cccDNA的持续存在是HBV持续感染以及慢乙肝难以彻底治愈的根本原因。目前各种抗HBV治疗方案中，缺少以cccDNA本身或形成环节为靶点的药物。因此，研究HBV cccDNA的合成和转录机制，以及开发靶向cccDNA的药物十分必要。为此，我们在本项目研究中，从cccDNA合成和转录相关的靶基因发现、cccDNA表征模型构建以及靶向cccDNA转录的药物筛选三个方面展开研究，取得了如下研究成果：（1）通过亚基因组规模的慢病毒shRNA筛选,从110种参与DNA修复的宿主基因中，筛选到4种可能参与cccDNA合成的候选基因，进一步的验证发现,LIG1/3基因参与了HBV cccDNA的合成过程，而CCNH,BTG2不参与cccDNA的合成；（2）发现了一种构建重组HBV基因组的经验规则，并以此为基础构建了一系列可用于HBV报告病毒产生的HBV重组体，为后续研究提供了极为便利的工具；（3）建立了一种基于生物素连接酶的筛选策略，用于筛选参与HBV核心启动子转录相关因子。通过该策略，发现了一种参与HBV核心启动子转录的新因子STAU1,并对其作用机制进行了解析；（4）建立了一种可以极大改善三分段sfGFP用于蛋白相互作用表征效果的新策略；（5）建立了一种新的靶向HBV核心启动子的抗乙肝药物筛选细胞模型，并以此为基础，筛选到2种可有效抑制HBV核心启动子转录的新型化合物，为后续药物开发奠定了十分良好基础。