Persistence of hepatitis delta virus infection in absence of HBV replication

描述（由申请人提供）：丁型肝炎病毒（HDV）是一种重要的人类病原体，全世界有2000万慢性携带者。HDV是人类乙型肝炎病毒（HBV）的一种天然亚病毒因子。HDV利用HBV包膜蛋白形成病毒粒子并感染肝细胞。在自然感染的肝脏中，HDV与HBV共存。在世界范围内，慢性HBV感染是肝细胞癌（HCC）的主要危险因素，与50%的HCC病例相关。伴随的HDV感染能够造成额外的肝脏损害，通常导致肝脏疾病加速和更快/更频繁的肝硬化。慢性HBV/HDV携带者发生HCC的风险是单纯HBV携带者的3倍，发生HCC的时间比单纯HBV携带者早14年。目前，没有直接针对HDV的药物在使用中，许多抗hbv药物不能阻断HDV感染。无论HBV复制标记物是否存在，HBV感染者都支持HDV感染。在慢性HBV感染的肝脏中，高达90%的肝细胞似乎没有HBV复制标记物，但相当数量的肝细胞和大多数hcc含有整合的HBV DNA。即使HBV复制停止，包膜蛋白也可以从整合的HBV DNA中产生。由于HDV仅从HBV中获取包膜蛋白，我们预计如果包膜蛋白可用，HDV可以在没有HBV复制的情况下持续存在。由于在整合的HBV dna和HBV整合物衍生的mrna中经常观察到包膜蛋白（线mrna）的改变（重排、突变、缺失等），因此从线mrna翻译的包膜蛋白是否能够支持HDV组装和传染性仍有待确定。因此，这一应用将验证一个假设，即在没有HBV复制的情况下，持续的HDV感染可以完全通过从整合的HBV DNA中产生功能性包膜蛋白来维持。由于双链线性HBV DNA基因组是整合的主要底物，因此HBV启动子和包膜蛋白的编码序列可能在整合物中保持完整，而HBV多聚(a)信号/位点可能不存在于下游。然而，当邻近的下游宿主序列促进聚腺苷化时，HBV包膜蛋白可以通过整合物表达。我们建议从HBV感染的肝组织和匹配HBV诱导的hcc中克隆表达的ine mrna序列，该序列必须包含HBV序列和poly(A)尾部之间的宿主序列。利用这些克隆的mRNA序列，我们将构建包含整个大包膜蛋白编码序列的载体，从而表达大、中、小三种HBV包膜蛋白（LMS载体）。利用这些LMS载体，我们将首次确定HBV整合剂衍生的包膜蛋白是否支持感染性HDV的有效组装，从而确保HDV在体内完成生命周期。这项研究将促进我们对慢性HDV感染维持机制的理解，并可能证明对慢性HBV/HDV携带者使用HDV靶向药物（除了抗HBV治疗外）是合理的。

DESCRIPTION (provided by applicant): Hepatitis delta virus (HDV) is a significant human pathogen with 20 million chronic carriers worldwide. HDV is a natural subviral agent of human hepatitis B virus (HBV). HDV uses HBV envelope proteins to form virions and infect hepatocytes. In naturally infected liver, HDV co-exists with HBV. Worldwide, chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC) and is associated with >50% of all HCC cases. Concomitant HDV infection is able to inflict additional liver damage, often resulting in accelerated liver disease and more fast/frequent cirrhosis. Chronic HBV/HDV carriers have a three-fold increased risk of HCC incidence, and develop HCC about 14 years earlier as compared to the carriers of HBV only. Currently, no drugs directly targeting HDV are in use, and a number of anti-HBV drugs do not block HDV infection. HBV-infected individuals support HDV infection regardless of the presence of HBV replication markers. In livers chronically infected with HBV, up to 90% of hepatocytes can appear free of HBV replication markers, but a significant number of hepatocytes and most HCCs contain integrated HBV DNA. The envelope proteins can be produced from the integrated HBV DNA even when HBV replication is ceased. Since HDV takes only the envelope proteins from HBV, we anticipate that HDV can persist in absence of HBV replication, if the envelope proteins are available. Since alterations (rearrangements, mutations, deletions, etc.) are frequently observed in integrated HBV DNAs and in HBV integrant-derived mRNAs for the envelope proteins (ine mRNAs), it remains to be determined whether the envelope proteins translated from the ine mRNAs can support HDV assembly and infectivity. Therefore, this application will test the hypothesis that persistent HDV infection can be maintained exclusively via production of functional envelope proteins from integrated HBV DNA in the absence of HBV replication. Since a double-stranded linear HBV DNA genome is a main substrate for integration, HBV promoters and coding sequences for the envelope proteins may remain intact in the integrant, while HBV poly(A) signal/site are likely not present downstream. However, HBV envelope proteins can be expressed from integrants, when polyadenylation is facilitated by adjacent downstream host sequences. We propose to clone from HBV-infected liver tissues and matching HBV-induced HCCs the sequences of the expressed ine mRNAs, which must bear host sequences between the HBV sequence and poly(A) tail. Using these cloned mRNA sequences, we will construct vectors bearing the entire large envelope protein coding sequence, and thus expressing all three HBV envelope proteins, large, middle and small (LMS vectors). Using these LMS vectors, we will determine for the first time whether HBV integrant-derived envelope proteins support the efficient assembly of infectious HDV, and thus ensure in vivo the completion of the HDV life cycle. This study will advance our understanding of the mechanism of the maintenance of chronic HDV infection and it may justify the use of HDV-targeting drugs (in addition to anti-HBV therapies) for chronic HBV/HDV carriers.