Intervention of HBV DNA synthesis and transcription

描述（由申请方提供）：慢性B型肝炎病毒感染影响全球约3亿人，并构成纤维化和肝细胞癌（HCC）的重要风险因素。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，其在细胞核中转化为共价闭合环状DNA（cccDNA），cccDNA用作病毒mRNA合成和蛋白质的模板。cccDNA在当前使用的抗病毒药物存在下持续存在，所述抗病毒药物充当链终止剂。我们（Hostetler和同事）以前已经证明，无环核苷膦酸酯，HPMPC和HPMPA被纳入病毒DNA。含有这些碱基的病毒DNA模板不能容易地复制，并且在DNA结构中具有畸变。在这项研究中，我们建议研究这些药物在从核心颗粒内的RNA前基因组转化和随后合成cccDNA的过程中掺入HBV DNA。在研究资助的R21阶段，我们通过分析核心颗粒内的病毒DNA以及最终cccDNA的形成来研究放射性标记的HPMPA/HPMPC的掺入及其影响。这些药物对病毒的影响 将分析mRNA和蛋白质合成，以检测含有HPMPA/HPMPC的cccDNA的模板活性。在第二个R33阶段，我们建议定义包含这些核苷酸的双链体DNA的变化。我们将合成几种含有HPMPA或HPMPC的模板，并评估其双链DNA与对照DNA双链体的NMR溶液结构。R33阶段的目的4使用广泛用于HBV领域并因其对HBV感染的易感性而被认可的细胞系HepaRG探索这些药物在HBV感染背景下的影响。这些研究将提供一个独特的机会来评估潜在的新方法，以禁用并最终消除HBV cccDNA并阻止其表达。我们的假设是，HMPMA掺入DNA会诱导结构异常，在这种情况下，可能会使cccDNA不稳定，改变其结构并导致其随时间消除。我们的团队结合了最先进的药物化学（Hostetler）和HBV分子生物学（Siddiqui）专业知识，以解决慢性感染中cccDNA的HBV病毒持续存在以及目前流行的抗病毒策略难以治疗的长期问题。

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.