Intervention of HBV DNA synthesis and transcription

描述(申请人提供)：慢性乙肝病毒感染影响全球约3亿人，并构成纤维化和肝细胞癌(肝细胞癌)的重要风险因素。乙肝病毒是一种DNA病毒，它通过前基因组RNA扩增其基因组。这种RNA由乙肝病毒编码的聚合酶蛋白(Poll)转换成DNA，Poll的功能是逆转录酶。该反应的最终产物是部分双链DNA，它在细胞核中转化为共价闭合环状DNA(CccDNA)，作为病毒mRNA合成和蛋白质的模板。CCcDNA在当前使用的作为链终止物的抗病毒药物的存在下持续存在。我们(Hostetler及其同事)此前已经证明，无环核苷膦、HPMPC和HPMPA被整合到病毒DNA中。含有这些碱基的病毒DNA模板不容易复制，DNA结构也有异常。在这项研究中，我们打算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合成过程中掺入HBVDNA的情况。在赠款的R21阶段，我们通过分析核心颗粒中的病毒DNA以及最终形成cccDNA来研究放射性标记HPMPA/HPMPC的掺入及其影响。这些药物对病毒的影响 通过分析mRNA和蛋白质的合成来检测含有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DNA的变化。我们将合成几个含有HPMPA或HPMPC的模板，并评估它们的双链DNA和对照DNA双链的核磁共振溶液结构。R33阶段的目标4使用在乙肝病毒领域广泛使用的细胞系HepaRG探索这些药物在乙肝病毒感染背景下的影响，该细胞系因其对乙肝病毒感染的敏感性而被公认。这些研究将提供一个独特的机会来评估禁用并最终消除HBVcccDNA并阻止其表达的潜在新方法。我们的假设是，将HMPMA掺入DNA会导致结构异常，在这种情况下，可能会破坏ccCDNAs的稳定性，改变其结构，并导致其随着时间的推移而消除。我们的团队结合了最先进的药物化学(Hostetler)和乙肝病毒分子生物学(Siddiqui)的专业知识，以解决这一长期存在的问题，即慢性感染中cccDNA的乙肝病毒持久性，以及对目前流行的抗病毒策略的抵抗力。

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.