Influence of integrant-derived HBV RNAs encoding the envelope proteins on HBV life cycle

摘要 全世界有超过2.5亿慢性乙肝病毒携带者。慢性乙肝病毒感染是导致 肝细胞癌(HCC)。目前还没有治愈乙肝病毒的方法。干扰素治疗只对以下患者有暂时的好处 病人的一个子集。不管FDA批准的Negos(T)ide类似物可以极大地抑制HBV复制， 慢性乙肝病毒感染没有得到很好的控制。没有一种药物对所有患者都有效，也不能达到 大多数情况下表面抗原(乙肝表面抗原或乙肝病毒包膜蛋白)的丢失。乙肝病毒频繁反弹 在治疗停止后。乙肝病毒感染的机制还不完全清楚，这影响了治疗选择。 这项建议将分析编码乙肝表面抗原的乙肝病毒组成成分衍生RNA(id-RNAs)之间的关系 和乙肝病毒的生命周期。HBVDNA整合到宿主基因组中对乙肝病毒生命周期并不重要，而且 ID-RNAs的研究还不够深入。慢性乙肝病毒携带者配对的肝/肝细胞癌组织分析 RT-PCR，我们发现，与乙肝病毒复制无关：ID-RNAs在所有组织中积累，代表45- 在60%的组织(40%的肝脏/80%的肝细胞)中发现100%的所有HBVRNA；通过隐匿的HBVRNA多腺化 在60%/40%的肝脏中发现Poly(A)信号(可能是整合体来源的或通过乙肝病毒复制产生的) Hcs，是20%肝脏/20%Hcs中最丰富的HBVRNA； 使用传统的乙肝病毒多聚(A)信号进行多腺化的RNA)是70%最不丰富的物种 组织(40%肝脏/100%肝细胞癌)。此外，对选定的组织进行RNA测序分析也表明 ID-RNAs在大多数分析样品中的丰度。数据表明，id-rna可能会产生显著的 携带乙肝病毒整合子的细胞中的乙肝表面抗原的比例。如果同样的细胞也支持乙肝病毒复制，那么一个很棒的 部分形成的乙肝病毒粒子可能会携带id-RNA衍生的乙肝表面抗原(id-HBs Ag)作为其主要成分 信封。因此，这些乙肝病毒粒子的传染性和它们支持病毒传播的能力将主要取决于 Id-HBs Ag的性质，而不是乙肝病毒复制衍生的HBs Ag(RD-HBs Ag)的性质。因此，id-RNA 可以调节慢性乙肝感染的维持，这可能需要修改目前的模式 乙肝病毒感染表明，乙肝病毒的生命周期是独立的整合。也很明显， 抗乙肝药物不能实现血清乙肝表面抗原的损失，因为大部分的乙肝表面抗原可以从 ID-RNAs，与乙肝病毒复制无关。根据我们的数据，目标1使用大量的肝脏/肝细胞癌组织将发现 如果id-RNAs的丰富在慢性乙肝病毒感染中是常见的。目标2和目标3将(I)发现如果大部分id- RNA在乙肝表面抗原编码序列中不发生改变，因此可以作为相当大的功能来源。 与乙肝病毒复制无关的乙肝表面抗原，(Ii)分析id-RNAs的3‘端非翻译区(包括 宿主插入)影响乙肝表面抗原的合成，以及(Iii)发现id-乙肝表面抗原是否能有效地支持病毒的组装和感染性 乙肝病毒粒子。这项建议将增进我们对id-RNAs和id-HBs Ag如何影响乙肝病毒生命的理解。 周期和乙肝的发病机制，并可能证明id-RNAs作为新的抗病毒干预的重要靶点是合理的。

Abstract There are more than 250 million of chronic HBV carriers worldwide. Chronic HBV infection is the main cause of hepatocellular carcinoma (HCC). There is no cure for HBV. Interferon therapy gives temporary benefits only to a subset of patients. Regardless that FDA-approved nucleos(t)ide analogs can greatly inhibit HBV replication, chronic HBV infection is not well under control. None of the drugs works in all patients, and is able to achieve the loss of the surface antigen (HBsAg, or HBV envelope proteins) in most cases. HBV frequently rebounds after treatment is stopped. Mechanism of HBV infection is not fully understood, which affects treatment options. This proposal will analyze the relationship between HBV integrant-derived RNAs (id-RNAs) coding for HBsAg and HBV life cycle. HBV DNA integration into host genome is not considered important for HBV life cycle, and id-RNAs are not well studied. Analyzing matching liver/HCC tissues from chronic HBV carrier humans using RT-PCR, we found that regardless of HBV replication: id-RNAs accumulated in all tissues and represented 45- 100% of all HBV RNAs found in 60% of tissues (40% livers/80% HCCs); RNAs polyadenylated via cryptic HBV poly(A) signal (could be integrant-derived or produced by HBV replication) were found in 60% livers/40% HCCs, and were the most abundant HBV RNAs in 20% livers/20% HCCs; and replication-derived RNAs (rd- RNAs) polyadenylated using conventional HBV poly(A) signal were the least abundant species in 70% of tissues (40% livers/100% HCCs). Furthermore, analysis of selected tissues by RNA sequencing also showed id-RNAs' abundance in most of the analyzed samples. The data suggest that id-RNAs may produce significant fraction of HBsAg in cells bearing HBV integrants. If the same cells also support HBV replication, then a great fraction of formed HBV virions likely will bear id-RNA-derived HBsAg (id-HBsAg) as a major component of their envelopes. Thus, infectivity of these HBV virions, and their ability to support virus spread will mostly depend on the properties of id-HBsAg, and not on those of HBV replication-derived HBsAg (rd-HBsAg). Thus, id-RNAs could regulate the maintenance of chronic HBV infection, which may call for revision of the current model of HBV infection that suggests that HBV life cycle is independent of the integration. It also became apparent that anti-HBV drugs fail to achieve loss of serum HBsAg, because major amounts of HBsAg can be produced from id-RNAs regardless of HBV replication. In light of our data, Aim 1 using a large set of liver/HCC tissues will find if id-RNAs' abundance is common during chronic HBV infection. Aims 2 and 3 will (i) find if large fraction of id- RNAs do not bear alterations in HBsAg-coding sequences, and thus can serve as sizeable source of functional HBsAg regardless of HBV replication, (ii) analyze how 3'-end untranslated regions of id-RNAs (including the host inserts) affect HBsAg synthesis, and (iii) find if id-HBsAg can efficiently support assembly and infectivity of HBV virions. The proposal will advance our understanding on how id-RNAs and id-HBsAg can impact HBV life cycle and HBV pathogenesis, and may justify id-RNAs as important targets for novel antiviral interventions.