Persistence of hepatitis delta virus infection in absence of HBV replication

描述(申请人提供)：丁型肝炎病毒(HDV)是一种重要的人类病原体，全球有2000万慢性携带者。HDV是人类乙肝病毒的一种天然亚病毒制剂。HDV使用HBV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与乙肝病毒共存。在世界范围内，慢性乙肝病毒感染是肝细胞癌的主要危险因素，并且与50%的肝细胞癌病例有关。伴随的HDV感染能够造成额外的肝脏损害，通常导致加速的肝病和更快/更频繁的肝硬变。慢性HBVHDV携带者发生肝细胞癌的风险是单纯携带HBV者的三倍，并且与仅携带HBV者相比，发生肝癌的时间要早约14年。目前，没有直接针对HDV的药物在使用，一些抗HBV药并不能阻断HDV的感染。无论是否存在乙肝病毒复制标志物，乙肝病毒感染者都支持HDV感染。在慢性感染乙肝病毒的肝脏中，高达90%的肝细胞不存在乙肝病毒复制标志物，但相当数量的肝细胞和大多数肝细胞含有整合的HBVDNA。即使当乙肝病毒复制停止时，整合的HBVDNA也可以产生包膜蛋白。由于HDV只从乙肝病毒中提取包膜蛋白，我们预计，如果包膜蛋白可用，HDV可以在没有乙肝病毒复制的情况下持续存在。由于改变(重排、突变、删除等)虽然在整合的HBVDNA和乙肝病毒整合体衍生的包膜蛋白(Ine MRNAs)中经常观察到HDV，但从ine mRNAs翻译的包膜蛋白是否支持HDV组装和感染性仍有待确定。因此，这一应用将检验这样一种假设，即在没有乙肝病毒复制的情况下，通过从整合的HBVDNA中产生功能性包膜蛋白，可以完全维持持续的HDV感染。由于双链的线性HBVDNA基因组是整合的主要底物，因此整合的HBV启动子和包膜蛋白的编码序列可能保持完整，而HBVPolyA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多腺苷基化时，乙肝病毒包膜蛋白可以从整合体中表达出来。我们建议从乙肝病毒感染的肝组织和与乙肝病毒诱导的肝细胞相匹配的肝细胞中克隆表达的ine mRNAs的序列，该序列必须带有位于HBVDNA序列和PolyA尾部之间的宿主序列。利用这些克隆的mRNA序列，我们将构建携带完整大包膜蛋白编码序列的载体，从而表达所有三种乙肝病毒包膜蛋白，大、中、小三种蛋白(LMS载体)。利用这些LMS载体，我们将首次确定乙肝病毒整合子来源的包膜蛋白是否支持传染性HDV的有效组装，从而确保在体内完成HDV生命周期。这项研究将促进我们对维持慢性HDV感染的机制的理解，并可能证明对慢性HBVHDV携带者使用HDV靶向药物(除抗HBV药物外)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.