HBV RNaseH inhibitors: Effects on HBV biology and resistance development

乙肝病毒是一种嗜肝的DNA病毒，通过逆转录进行复制。它会慢性感染 每年约有2.9亿人死亡，约90万人死亡。治疗主要使用核素(T)类似物来靶向 病毒DNA聚合酶(P)，但只有少数患者清除病毒，所以治疗是终身的。反向 转录是通过合成DNA和DNA的病毒DNA聚合酶(RT)的协调作用来催化的 核糖核酸酶H(RNAseH)，在RNA被复制到DNA中后破坏它。RT和RNAseH 包括P的相邻结构域，并且这两种酶活性是变构的和/或在 复制。新合成的乙肝病毒基因组有三种命运：成为核共价闭合环状DNA (CccDNA)分子，在病毒粒子内分泌，或整合到细胞基因组中。P也是一个监管机构 聚集在内质网并帮助抑制干扰素反应的蛋白质。 RT在乙肝病毒复制中的作用已被很好地了解，但对RNAseH如何 对乙肝病毒生物学的贡献。我们最近表达了适合于机制分析的重组HBVRNAseH 以及第一次发现药物。然后我们开发了第一个筛选HBVRNAseH抑制剂的流水线 并确定了通过抑制RNAseH来阻止乙肝病毒复制的&gt；130化合物。重组RNAseH 而抑制剂是探索RNAseH对病毒生物学贡献的独特新工具。 前提：我们的重组HBVRNAseH、RNAseH抑制剂和RNAseH检测使研究能够揭示 RNAseH如何对病毒生物学做出贡献，以及酶如何进化出对RNAseH抑制剂的抵抗力。 目的1.抑制RNAseH对乙肝病毒逆转录有何影响？我们将评估如何 RNAseH抑制剂影响RNA的包埋，当RNAseH在逆转录过程中RNA的命运 是被抑制的，RNAseH抑制剂对病毒基因组损伤的可逆性，如何阻断RNAseH 影响cccDNA的合成，以及没有RNAseH活性的病毒粒子的特异性感染性。 目的2.对RNAseH抑制剂产生耐药性的可能性是什么？我们将选择抗药性突变来 细胞培养中的RNAseH抑制剂，然后确定它们对病毒适合性和对抑制剂的选择性的影响 来自3种化学型。 目的3.抑制RNAseH如何影响衣壳和乙肝病毒与细胞的相互作用？我们将定义 通过抑制RNAseH产生的RNA：DNA异源双链如何影响乙肝病毒衣壳，RNA：DNA的影响 RNAseH抑制剂在干扰素反应中诱导的异源双链，以及在没有RNAseH抑制剂的情况下异源双链是如何产生的 RNAseH活性影响HBVDNA与宿主基因组的整合。 这些数据将定义HBVRNAseH如何对病毒生物学做出贡献。这一信息将加深我们的 了解乙肝病毒与细胞的相互作用，并为我们正在进行的 RNAseH抑制剂作为新药旨在改善慢性感染患者的治疗。

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects about 290 million people and kills ~900,000 annually. Therapy primarily employs nucleos(t)ide analogs that target the viral DNA polymerase (P), but only a few percent of patients clear the virus so therapy is life-long. Reverse transcription is catalyzed by coordinate action of the viral DNA polymerase (RT) that synthesized the DNA and the ribonuclease H (RNaseH) that destroys the RNA after it has been copied into DNA. The RT and RNaseH comprise adjacent domains of P, and the 2 enzymatic activities are allosteric and/or kinetically linked during replication. Newly synthesized HBV genomes have 3 fates: to become nuclear covalently closed circular DNA (cccDNA) molecules, be secreted within virions, or be integrated into the cellular genome. P is also a regulatory protein that accumulates at the endoplasmic reticulum and helps suppress interferon responses. The role of the RT in HBV replication is fairly well understood, but very little is known about how the RNaseH contributes to HBV biology. We recently expressed recombinant HBV RNaseH suitable for mechanistic analyses and drug discovery for the first time. We then developed the first screening pipeline for HBV RNaseH inhibitors and identified >130 compounds that block HBV replication by inhibiting the RNaseH. The recombinant RNaseH and inhibitors are unique new tools to probe contributions of the RNaseH to viral biology. Premise: Our recombinant HBV RNaseH, RNaseH inhibitors, and RNaseH assays enable studies to reveal how the RNaseH contributes to viral biology and how the enzyme can evolve resistance to RNaseH inhibitors. Aim 1. What are the effects of inhibiting the RNaseH on HBV reverse transcription? We will evaluate how RNaseH inhibitors affect RNA encapsidation, the fate of the RNA during reverse transcription when the RNaseH is inhibited, reversibility of damage to the viral genome induced by RNaseH inhibitors, how blocking the RNaseH affects cccDNA synthesis, and the specific infectivity of virions made without RNaseH activity. Aim 2. What is the potential for resistance to RNaseH inhibitors? We will select resistance mutations to RNaseH inhibitors in cell culture and then define their effects on viral fitness and selectivity against inhibitors from 3 chemotypes. Aim 3. How does inhibiting the RNaseH affect capsids and HBV’s interaction with cells? We will define how RNA:DNA heteroduplexes generated by inhibiting the RNaseH affect HBV capsids, the effects of RNA:DNA heteroduplexes induced by RNaseH inhibitors on interferon responses, and how heteroduplexes made without RNaseH activity affect integration of HBV DNA into the host genome. These data will define how the HBV RNaseH contributes to viral biology. This information will deepen our understanding of HBV’s interaction with cells and provide essential context for our ongoing development of RNaseH inhibitors as novel drugs intended to improve therapy for chronically infected patients.