Intervention of HBV DNA synthesis and transcription

描述（由申请人提供）：慢性乙型肝炎病毒感染影响全球约3亿人，是纤维化和肝细胞癌（HCC）的重要危险因素。HBV是一种DNA病毒，通过前基因组RNA扩增其基因组。这种RNA被hbv编码的聚合酶蛋白（pol）转化为DNA， pol起逆转录酶的作用。该反应的最终产物是部分双链DNA，在细胞核中转化为共价闭合环状DNA (cccDNA)， cccDNA作为病毒mRNA合成和蛋白质的模板。cccDNA在目前使用的抗病毒药物存在下持续存在，这些药物作为链终止剂。我们（Hostetler和同事）先前已经证明无环核苷膦酸盐、HPMPC和HPMPA被纳入病毒DNA。含有这些碱基的病毒DNA模板不容易复制，并且在DNA结构上有畸变。在这项研究中，我们建议研究这些药物在核心颗粒内的RNA前基因组转化和随后的cccDNA合成过程中与HBV DNA的结合。在R21阶段，我们通过分析核心颗粒内的病毒DNA和最终形成cccDNA来研究放射性标记HPMPA/HPMPC的掺入及其影响。这些药物对病毒的作用

DESCRIPTION (provided by applicant): Chronic hepatitis B virus infections affect about 300 million people worldwide and constitute a significant risk factor for fibrosis and hepatocellular carcinoma (HCC). HBV, a DNA virus, amplifies its genome via a pre-genomic RNA. This RNA is converted by HBV-encoded polymerase protein (pol), which functions as a reverse transcriptase, into DNA. The final product of this reaction is a partially double stranded DNA, which is converted to covalently closed circular DNA (cccDNA) in the nucleus cccDNA serves as a template for viral mRNA synthesis and proteins. cccDNA persists in the presence of the antivirals in current use, which act as chain terminators. We (Hostetler and colleagues) have previously shown that the acyclic nucleoside phosphonates, HPMPC and HPMPA are incorporated into viral DNA. Viral DNA templates containing these bases cannot be copied readily and have aberrations in DNA structure. In this study, we propose to investigate the incorporation of these agents into HBV DNA during the conversion from the RNA pregenome within the core particles and subsequent synthesis of cccDNA. In the R21 phase of the grant, we investigate the incorporation of radiolabeled HPMPA/HPMPC and its impact by analysis of viral DNA within core particles and finally the formation of cccDNA. Effect of these drugs on viral mRNA and protein synthesis will be analyzed to test the template activity of cccDNA containing HPMPA/HPMPC. In the second R33 phase, we propose to define the changes in duplex DNA containing these nucleotides. We will synthesize several templates containing HPMPA or HPMPC and assess the NMR solution structures of their duplex DNA versus control DNA duplexes. Aim 4 of R33 phase explores the impact of these drugs in the context of HBV infection using a cell line HepaRG that is widely used in the HBV field and recognized for its susceptibility to HBV infection. These studies will provide a unique opportunity to assess the potential new way to disable and ultimately eliminate the HBV cccDNA and prevent its expression. Our hypothesis is that incorporation of HMPMA into the DNA induces structural abnormalities, and in this case possibly destabilizing cccDNA, altering its structure and leading to its elimination over time. Our team combines the state of the art medicinal chemistry (Hostetler) and HBV molecular biology (Siddiqui) expertise to address this long standing problem of HBV viral persistence of cccDNA in chronic infection and being refractory to antiviral strategies currently in vogue.