HDV spread during chronic infection as a novel target for antiviral intervention

描述（由申请人提供）：慢性乙型肝炎病毒（HBV）感染是全球肝细胞癌（HCC）的主要原因，占所有HCC病例的50-80%。在目前的4亿慢性HBV携带者中，约有6500万人将死于晚期肝脏疾病或HCC。丁型肝炎病毒（HDV）的合并感染通常会增强肝脏的发病机制。HDV是HBV的一种亚病毒因子，在本质上总是与其辅助HBV共存，并利用HBV包膜蛋白形成病毒粒子，通过HBV受体感染肝细胞。全世界慢性HDV携带者的数量约为2000万。HDV是一种重要的人类病原体。慢性HBV/HDV携带者发生HCC的风险增加3倍，并且比单纯HBV携带者早约14年发生HCC。持续的HDV复制是肝脏相关死亡率的预测因素。目前，没有针对HDV的治疗方法，许多抗hbv药物不能阻断HDV感染。HBV和HDV携带者不能通过HBV疫苗得到帮助，迫切需要新的医疗干预目标和新的治疗方法。我们建议利用慢性HDV感染机制的独特性来寻找抗病毒干预的新靶点。简而言之，尽管研究表明慢性感染期间辅助肝炎病毒（HBV）的细胞间传播是不太可能发生的，但慢性感染期间HBV或HDV传播的发生既没有得到证实，也没有得到反驳。然而，我们对转染细胞和短暂感染动物肝脏中HDV mRNA的分析表明，在没有传播的情况下，HDV基因组在丁型抗原的开放阅读框（ORF）中积累突变。Ag)（唯一的HDV蛋白，对HDV复制至关重要），阻止HDV自我持续复制。因此，这一应用将验证HDV必须依靠持续的细胞间传播来维持慢性感染的假设。我们的目标是确定HDV传播是否是干预慢性HDV感染的有效新靶点。我们将在存在或不存在传播的情况下进行体外和体内HDV感染。为了分析HDV传播的三个关键方面，我们建议：(i)测定？抗原ORF突变产生？Ag无法支持HDV复制（目标1），（ii）测量HDV基因组的复制能力（目标1），以及（iii）比较慢性感染早期和晚期HDV病毒的感染性（目标2）。我们将首次确定，在没有传播的感染肝细胞中，HDV基因组是否由于？Ag ORF，以及是否正在进行的传播选择了编码非功能性的基因组？Ags。总的来说，这项研究将极大地促进我们对慢性HBV/HDV感染机制的理解，并可能(i)确定HDV传播是发病机制和HCC风险的一个因素，以及（ii）促进使用组装和进入抑制剂作为HDV携带者的新型抗病毒药物。此外，如果证明HBV在慢性感染期间传播，病毒进入抑制剂将对HBV携带者有效，无论HBV突变体对当前抗HBV药物是否具有耐药性。

DESCRIPTION (provided by applicant): Chronic hepatitis B virus (HBV) infection is the main cause of hepatocellular carcinoma (HCC) worldwide and is responsible for 50-80% of all HCC cases. Of the 400 million current chronic HBV carriers, about 65 million will die from an advanced liver disease or HCC. Concomitant infection with hepatitis delta virus (HDV) usually enhances liver pathogenesis. HDV, a subviral agent of HBV, always co-exists with its helper HBV in nature and uses HBV envelope proteins to form the virions and infect hepatocytes via HBV receptor(s). The number of chronic HDV carriers worldwide is about 20 million. HDV is a significant human pathogen. Chronic carriers of HBV/HDV have 3-fold increased risk of HCC incidence, and develop HCC approximately 14 years earlier than carriers of HBV only. Persistent HDV replication is a predictive factor for liver-associated mortality. Currently, there s no therapy against HDV, and a number of anti-HBV drugs do not block HDV infection. Carriers of HBV and HDV cannot be helped by HBV vaccine and desperately need novel targets for medical interventions and new treatments. We propose to exploit the uniqueness of the mechanism of chronic HDV infection in a search for a novel target for antiviral intervention. Briefly, despite studies suggesting that cell-to-cell spread of a helper hepadnavirus (HBV) during chronic infection is an unlikely event, the occurrence of the spread of either HBV or HDV during chronic infections was neither demonstrated nor disproved. However, our analysis of HDV mRNA in transfected cells and in the liver of transiently infected animal suggests that in absence of the spread, HDV genomes accumulate mutations in the open reading frame (ORF) for delta antigen (?Ag) (the only HDV protein and is essential for HDV replication) that preclude self-sustained HDV replication. Therefore, this application will test the hypothesis that HDV must rely on continuous cell-to-cell spread to maintain chronic infection. Our goal is to determine if HDV spread is a valid novel target for intervention against chronic HDV infection. We will conduct in vitro and in vivo HDV infections either in the presence or in the absence of the spread. To analyze three critical aspects of HDV spread, we propose to: (i) assay the ?Ag ORF for mutations that make ?Ag unable to support HDV replication (Aim 1), (ii) measure the replication capacity of HDV genomes (Aim 1), and (iii) compare infectivities of HDV virions early and late in chronic infection (Aim 2). We will determine for the first time whether, in infected hepatocytes in the absence of spread, HDV genomes lose the ability for self-sustained replication due to mutations in ?Ag ORF, and whether ongoing spread selects against the genomes encoding non-functional ?Ags. Overall, this study will greatly advance our understanding of the mechanism of chronic HBV/HDV infection, and will likely (i) identify HDV spread as a factor of pathogenesis and HCC risk, and (ii) facilitate the use of inhibitors of the assembly and entry as novel antivirals for HDV carriers. Furthermore, if HBV spread during chronic infection is demonstrated, virus entry inhibitors will work for HBV carriers regardless of HBV mutants resistant to current anti-HBV drugs. PUBLIC HEALTH RELEVANCE: Our study will likely identify HDV spread as an important determinant for maintenance of chronic infection, and thus, (i) as a risk factor of HCC, and (ii) a a new target for medical intervention. It will likely facilitate the use of inhibitors of the assemly and entry (targeting HDV cell-to-cell spread) as novel antiviral and anticancer drugs.