Characterization of host restriction factors interfering with replication and persistency of hepatitis B and D viruses

感染乙肝病毒(乙肝病毒)和混合感染丁型肝炎病毒(HDV)仍然是一个主要的全球健康威胁。这两种病毒都可能导致肝脏的急性和慢性(联合)感染。超过2.5亿人慢性感染乙肝病毒，至少1100万人慢性(合并)感染HDV，每年因致命的临床后果造成约100万人伤亡。虽然有一种保护性疫苗可以预防这两种病毒的感染，但目前还没有治愈慢性乙肝感染患者的方法，直接抗HDV疗法还没有完全开发出来。治疗慢性乙肝病毒感染的最大障碍之一是共价闭合环状DNA(CccDNA)，它被用来作为感染肝细胞核中稳定和持久的储存库。HDV作为一种有缺陷的RNA病毒，需要乙肝病毒表面蛋白(HBs Ag)的持续支持，才能包裹和分泌病毒粒子。虽然乙肝病毒已经发展出复杂的机制，但尚未充分研究，以保持“隐形病毒”，HDV激活HDV/乙肝病毒感染细胞中的先天免疫反应，导致抑制乙肝病毒复制。由于慢性HBVHDV感染的细胞感知机制和先天免疫途径仍不清楚，该项目旨在诱导单个下游效应宿主蛋白在HBVHDV持续期间干扰病毒复制周期的作用。为了实现这一目标，我们将使用智能功能获得技术(CRISPRa)直接激活选定的宿主靶基因的转录，以系统地研究激活的细胞基因在体外对乙肝病毒和丁型肝炎病毒复制和持久性的影响。具体来说，我们将(1)研究宿主效应蛋白对HBVHDV感染和复制的影响，(2)分析HDV基因组在88个相关宿主基因感染细胞中的持久性；(3)区分激活宿主基因对HBVHDV复制的直接和间接(调节)作用；(4)比较宿主细胞反应对复制HBVccDNA的影响及其大多数相关HBV型的甲基化特征。确定病毒基因组对抗病毒因子的潜在抵抗力，并提高我们对慢性病毒性肝炎先天免疫反应的理解。

Infections with Hepatitis B virus (HBV), and coinfections with Hepatitis D virus (HDV) are still a major global health threat. Both viruses can lead to acute and chronic (co-)infections of the liver. Over 250 million people are chronically infected with HBV and at least 11 million people are chronically (co-)infected with HDV, causing ca. 1 million casualties annually due to fatal clinical outcomes. While a protective vaccine is available that can prevent infection with both viruses, there is currently no cure for chronically HBV-infected patients, and direct anti-HDV therapies are not yet fully developed. One of the greatest obstacles to a cure for chronic HBV infection is the covalently closed circular DNA (cccDNA) that HBV uses as a stable and persistent reservoir in the nucleus of infected hepatocytes. HDV, as a defective RNA-virus, requires constant support from HBV in the form of surface proteins (HBsAg) for envelopment and secretion of virions. While HBV has developed elaborate, but not yet sufficiently studied mechanisms to remain a “stealth virus”, HDV activates innate immune responses in HDV/HBV infected cells that leads to suppression of HBV replication. Since cellular sensing mechanisms and pathways of innate immunity in chronic HBV/HDV infections still remain elusive, the proposed project aims to elicit the role of individual, downstream effector host proteins that can interfere with viral replicative cycles during HBV/HDV persistence. To pursue this goal, we will directly activate transcription of selected host target genes using a smart gain-of-function technique (CRISPRa) to systematically study the effects of activated cellular genes on replication and persistency of HBV and HDV in vitro. In detail, we will (1) investigate effects of host effector proteins on HBV and HDV infection and replication and (2) analyze persistence of HDV genomes in infected cells with 88 relevant predefined host-genes; (3) distinguish direct vs. indirect (regulatory) effect of activated host genes on HBV/HDV replication; and (4) compare effects of the host-cell response on replicating HBV cccDNA and its methylation profiles of most relevant HBV genotypes.Overall, the proposed project will provide important basic insights into the intracellular interaction of HBV and HDV with their host cells; determine the potential resistance of viral genomes to antiviral factors, and improve our understanding of the innate immune response to chronic viral hepatitis.