The Role of RNA Structure in the Hepatitis B Virus Lifecycle

总结 在全球范围内，慢性B型肝炎病毒（HBV）感染是肝细胞癌最常见的原因。 坏疽性口炎（肝细胞癌）。据估计，全世界有2.4亿人慢性HBV感染， 很少能治愈HBV的DNA基因组被病毒编码的蛋白质衣壳包围， 蛋白质/脂质包膜。感染肝细胞后，病毒基因组被运送到细胞核并转化为 称为共价闭合环状（ccc）DNA的附加体。cccDNA不是当前抗HBV药物的靶点 它的稳定性与这些治疗停止时的病毒反弹有关。尝试靶向cccDNA 还没有成功，需要新的抗HBV疗法。cccDNA不是直接复制的， HBV RNA转录物模板。其中一个转录本，前基因组（pg）RNA，是双核苷酸化和反向的- 由病毒编码的逆转录酶/聚合酶（RT/Pol）转录以产生HBV DNA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。这些元件中的许多在病毒生命周期中具有关键作用，并且靶向RNA 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， RNA结构在HBV生命周期中的作用在很大程度上还未被探索。多功能HBV pgRNA是模板 用于逆转录，并且还被翻译以产生核心（衣壳）和RT/Pol蛋白。RT/Pol 开放阅读框（ORF）位于pgRNA上核心ORF的3'端，并与核心ORF的3'端重叠; RT/Pol的合成需要通过尚不清楚的机制内部启动翻译。RNA 被称为ε（ε）茎环的结构位于pgRNA的5 ′-和3 ′-末端附近，但只有5 ′ ε（ε）茎环位于pgRNA的5 ′-和3 ′-末端附近。 是pgRNA糖苷化所必需的。由于现有技术的限制，以前的研究集中在当地 HBV RNA转录本的区域，许多问题仍然存在。例如，无论是翻译还是翻译， pgRNA在结构上是不同的，RT/Pol合成的内部起始是如何被调节的，以及为什么只有5' ε 对pgRNA的影响还不完全了解或完全未知。 研究活细胞中RNA结构的新技术，如SHAPE-MaP（选择性2′-羟基 通过引物延伸和突变谱分析的酰化）和DMS-MaP（硫酸二甲酯探测和 突变分析），将RNA化学探针技术与下一代测序结合起来， 在RNA转录物的整个长度上的结构。我们将使用这些技术来定义RNA结构 HBV pgRNA中的元件以及这些元件在HBV生命周期中的作用。我们假设pgRNA 含有在HBV生命周期中起关键作用的结构元件，我们的研究可以识别新的， 高度特异性靶点阻断HBV复制和HCC的发展。

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.