The Role of RNA Structure in the Hepatitis B Virus Lifecycle

摘要 在全球范围内，慢性乙肝病毒(乙肝病毒)感染是导致肝细胞癌的最常见原因。 Noma(肝细胞癌)。据估计，全球有2.4亿人慢性感染乙肝病毒，并获得批准的治疗方法 很少能治愈。HBVDNA基因组包裹在病毒编码的蛋白衣壳中，衣壳周围有一个 蛋白质/脂肪包膜。在感染肝细胞后，病毒基因组被送到细胞核并转换 与称为共价闭合环(CCC)DNA的Episome结合。CccDNA不是当前抗乙肝病毒的靶点 当这些疗法停止时，其稳定性与病毒反弹有关。靶向cccDNA的尝试 都没有成功，需要新的抗乙肝疗法。CccDNA不是直接复制的，而是 HBVRNA转录本的模板。其中一个转录本，前基因组(PG)RNA，被封装并反转- 由病毒编码的逆转录酶/聚合酶(RT/POL)转录产生HBVDNA基因组。 RNA结构探测技术的最新进展导致了RNA结构的发现 几种病毒中的元素。其中许多元件在病毒生命周期中具有关键作用，并以RNA为靶标 病毒基因组中的结构可能为药物开发开辟新的途径。尽管有治疗潜力，但 RNA结构在乙肝病毒生命周期中的作用在很大程度上是未知的。以多功能乙肝病毒pgRNA为模板 用于逆转录，也被翻译成核心(衣壳)和RT/POL蛋白。RT/Pol 开放阅读框架(ORF)是pgRNA上核心ORF的3‘端，与核心ORF的3’端重叠； RT/Pol的合成需要通过一种尚不了解的机制在内部启动翻译。核糖核酸 被称为epsilon(ε)茎环的结构位于pgRNA5‘和3’端附近，但只有5‘ε 是pgRNA封装所必需的。由于可用技术的限制，以前的研究主要集中在局部 在HBVRNA转录本中发现了不同的区域，仍然存在许多问题。例如，是否已封装和转换 PgRNA在结构上是不同的，RT/Pol合成的内部启动是如何调节的，以及为什么只有5‘ε 对pgRNA包埋的影响尚不完全了解或完全未知。 检测活细胞中RNA结构的新技术，如SHAPE-MAP(选择性2‘-羟基 用引物延伸和突变图谱分析的酰化反应)和DMS-MAP(硫酸二甲酯探针和 突变分析)，将RNA化学探测技术与下一代测序相结合以识别RNA 整个RNA转录本的结构。我们将使用这些技术来定义RNA结构 乙肝病毒pgRNA中的元素以及这些元素在乙肝病毒生命周期中的作用。我们假设pgRNA 含有在乙肝病毒生命周期中起关键作用的结构元素，我们的研究可以确定新的和 针对阻断乙肝病毒复制和肝细胞癌发展的高度特异性靶点。

SUMMARY Globally, a chronic hepatitis B virus (HBV) infection is the most common cause of hepatocellular carci- noma (HCC). An estimated 240 million people are chronically HBV-infected worldwide, and approved treatments are rarely curative. HBV has a DNA genome enclosed in a virally encoded protein capsid surrounded by a protein/lipid envelope. After infection of a hepatocyte, the viral genome is delivered to the nucleus and converted to an episome referred to as covalently closed circular (ccc)DNA. cccDNA is not targeted by current anti-HBV therapies, and its stability is linked to viral rebound when these therapies are stopped. Attempts to target cccDNA have not succeeded, and there is a need for new anti-HBV therapies. cccDNA is not directly replicated but is the template for HBV RNA transcripts. One of the transcripts, the pregenomic (pg)RNA, is encapsidated and reverse- transcribed by the virally encoded reverse-transcriptase/polymerase (RT/Pol) to generate the HBV DNA genome. Recent advances in RNA structure probing techniques have led to the discovery of RNA structural elements in several viruses. Many of these elements have critical roles in the viral lifecycle, and targeting RNA structures in viral genomes could open new avenues of drug development. Despite the therapeutic potential, the role of RNA structure in the HBV lifecycle is largely unexplored. The multi-functional HBV pgRNA is the template for reverse transcription and is also translated to produce the core (capsid) and RT/Pol proteins. The RT/Pol open-reading frame (ORF) is 3’ of the core ORF on pgRNA and overlaps with the 3’ end of the core ORF; synthesis of RT/Pol requires internal initiation of translation by a mechanism that is not understood. RNA structures referred to as epsilon (ε) stem-loops are located near the 5’- and 3’-ends of pgRNA, yet only the 5’ ε is required for pgRNA encapsidation. Due to limitations of available techniques, prior studies focused on local regions in HBV RNA transcripts, and many questions remain. For example, whether encapsidated and translated pgRNAs are structurally different, how internal initiation of RT/Pol synthesis is regulated, and why only the 5’ ε affects pgRNA encapsidation are incompletely understood or entirely unknown. New techniques to examine RNA structure in living cells, such as SHAPE-MaP (selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling) and DMS-MaP (dimethyl sulfate probing and mutational profiling), couple RNA chemical probing techniques with next-generation sequencing to identify RNA structures over the entire length of an RNA transcript. We will use these techniques to define RNA structural elements in HBV pgRNA and the role of these elements in the HBV lifecycle. We hypothesize that pgRNA harbors structural elements that have critical roles in the HBV lifecycle, and our studies could identify novel and highly specific targets for blocking HBV replication and the development of HCC.