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

摘要 全世界有超过2.5亿的慢性HBV携带者。慢性HBV感染是导致 肝细胞癌（HCC）。目前还没有治愈HBV的方法。干扰素治疗仅对以下患者提供暂时性益处： 一部分病人尽管FDA批准的核苷（酸）类似物可以极大地抑制HBV复制， 慢性HBV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， 在大多数情况下，表面抗原（HBsAg或HBV包膜蛋白）的丢失。HBV经常反弹 治疗停止后。HBV感染的机制尚未完全了解，这影响了治疗方案。 本研究将分析编码HBsAg的HBV整合子衍生RNA（id-RNA）与HBV DNA之间的关系， HBV的生命周期HBV DNA整合到宿主基因组中被认为对HBV生命周期并不重要， ID-RNA还没有得到很好的研究。分析来自慢性HBV携带者的匹配肝/HCC组织，使用 RT-PCR，我们发现，无论HBV复制：id-RNA积累在所有组织中，并代表45- 在60%的组织（40%的肝脏/80%的HCC）中发现100%的所有HBV RNA; RNA通过隐蔽HBV多聚腺苷酸化 poly（A）信号（可能是整合源性的或由HBV复制产生）在60%的肝脏/40%的肝脏中被发现。 在20%的肝/20%的HCC中，HBV RNA是最丰富的;复制来源的RNA（rd- 使用常规HBV poly（A）信号多聚腺苷酸化的RNA是70%的HBV中丰度最低的种类。 组织（40%肝脏/100% HCC）。此外，通过RNA测序对所选组织的分析还显示， 在大多数分析的样品中id-RNA的丰度。这些数据表明，id-RNAs可能会产生显著的 携带HBV整合体的细胞中HBsAg的分数。如果相同的细胞也支持HBV复制，那么 形成的HBV病毒体的一部分可能携带id-RNA衍生的HBsAg（id-HBsAg）作为其主要成分， 信封因此，这些HBV病毒粒子的感染性及其支持病毒传播的能力将主要取决于 id-HBsAg的性质，而不是HBV复制衍生的HBsAg（rd-HBsAg）的性质。因此，id-RNA 可以调节慢性HBV感染的维持，这可能需要修改目前的模型， HBV感染表明HBV的生命周期是独立的整合。很明显， 抗HBV药物不能实现血清HBsAg的损失，因为大量的HBsAg可以从 id-RNA与HBV复制无关。根据我们的数据，Aim 1使用大量肝脏/HCC组织将发现 如果id-RNA的丰度在慢性HBV感染中是常见的。目标2和3将（i）发现如果id的大部分- RNA不携带HBsAg编码序列的改变，因此可以作为功能性免疫调节剂的相当大的来源。 HBsAg与HBV复制无关，（ii）分析id-RNA的3 '端非翻译区（包括 宿主插入物）影响HBsAg合成，以及（iii）发现id-HBsAg是否可以有效地支持 HBV病毒粒子。该提案将促进我们对id-RNA和id-HBsAg如何影响HBV寿命的理解 周期和HBV发病机制，并可能证明id-RNA作为新的抗病毒干预的重要目标。

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.