Hepatitis B Virus diversity and ribonuclease H inhibitor efficacy

乙肝病毒是一种嗜肝的DNA病毒，通过逆转录进行复制。它长期存在于 该病毒每年感染3.5亿人，导致多达120万患者死亡。治疗主要使用核素(T) 类比。这些药物深刻地抑制了乙肝病毒，但由于对病毒的不完全抑制，它们很少能治愈患者 病毒复制。此外，由于耐药突变的进化，对乙肝病毒的控制经常失败。 通过使用新药进一步抑制乙肝病毒，在更多的患者中进行药物治疗应该是可能的 与核(T)化物类似物结合使用。 乙肝病毒逆转录需要两种位于病毒逆转录上的病毒酶活性 转录酶蛋白：核糖核酸酶类似物和核糖核酸酶H靶向的DNA聚合酶。 (RNAseH)在病毒RNA被复制到DNA中后将其销毁的一种物质。抗乙肝病毒的RNAseH药物还没有 因为不能制造出适合药物筛选的酶而被开发出来。我们最近把活动 HBVRNAseH，并鉴定出13种RNAseH抑制剂。 乙肝病毒有8种基因型(A-H)，抗RNAseH药物必须抑制广泛的乙肝病毒株才能 临床疗效显著。我们发现针对D型和H型分离株的RNAseH抑制剂可以阻断 A型分离株的复制，因此跨基因抑制是可能的。然而，D和H基因 我们测试的RNAseH分离株对RNAseH抑制剂具有不同的敏感性。我们不知道这些是否 差异是由于特定于基因型或特定于隔离的差异，因为只检查了1 为每个基因型别进行隔离。此外，我们还没有鉴定来自B型的重组RNAseH 和C，这是医学上最相关的基因类型。在这个小型研究资助(R03)项目中，我们将 评估乙肝病毒的高基因变异对其对RNAseH抑制剂的敏感性的影响程度。 目的1：评估不同类型的乙肝病毒对RNAseH敏感性的影响 抑制剂。我们将测试来自B、C和D基因的变异的HBVRNAseH序列对一组 生物化学和病毒复制分析中的RNAseH拮抗剂。 目的2：确定核苷类似物耐药性突变如何影响对RNAseH的敏感性 抑制力。我们将把常见的nulceos(T)ide类似耐药突变引入乙肝病毒的B、C和 D分离并评估它们如何影响病毒复制对RNAseH抑制剂的敏感性。 该项目将：1)揭示乙肝病毒基因差异是否会使抗RNAseH药物复杂化 开发；2)帮助确定两个活动站点之间的通信是否相反 转录酶可能使核素类似物和RNAseH抑制剂的联合治疗复杂化；以及3) 产生一组变异的乙肝病毒RNAseH，用于筛选具有跨基因疗效的候选药物。

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people and kills up to 1.2 million patients annually. Therapy primarily employs nucleos(t)ide analogs. These drugs profoundly suppress HBV but they rarely cure patients due to incomplete inhibition of viral replication. Furthermore, control of HBV often fails due to evolution of drug resistance mutations. Pharmacologically curing HBV in more patients should be possible by suppressing HBV further with new drugs to be used in combination with the nucleos(t)ide analogs. HBV reverse transcription requires 2 viral enzymatic activities that are both located on the viral reverse transcriptase protein: the DNA polymerase that is targeted by the nucleos(t)ide analogs and the ribonuclease H (RNAseH) that destroys the viral RNA after it has been copied into DNA. Anti-HBV RNAseH drugs have not been developed because enzyme suitable for drug screening could not be made. We recently made active HBV RNAseH and identified 13 inhibitors of the RNAseH. HBV has 8 genotypes (A-H), and anti-RNAseH drugs must inhibit a wide range of HBV strains to be clinically effective. We found that RNAseH inhibitors identified against genotype D and H isolates can block replication of a genotype A isolate, so cross-genotypic inhibition is possible. However, the genotype D and H RNAseH isolates we tested are differentially sensitive to RNAseH inhibitors. We do not know whether these dissimilarities are due to genotype-specific or isolate-specific differences because have examined only 1 isolate for each genotype. Furthermore, we have not characterized recombinant RNAseH from genotypes B and C, which are the most medically-relevant genotypes. In this Small Research Grant (R03) project we will evaluate the degree to which HBV's high genetic variation affects its sensitivity to RNAseH inhibitors. Aim 1: Evaluate how differences between and within HBV's genotypes affect sensitivity to RNAseH inhibitors. We will test sensitivity of variant HBV RNAseH sequences from genotypes B, C, and D to a set of RNAseH antagonists in biochemical and viral replication assays. Aim 2: Determine how nucleoside analog resistance mutations affect sensitivity to RNAseH inhibition. We will introduce common nulceos(t)ide analog resistance mutations into HBV genotype B, C, and D isolates and evaluate how they affect sensitivity of viral replication to an RNAseH inhibitor. This project will: 1) Reveal whether HBV genotypic differences will complicate anti-RNAseH drug development; 2) Help determine whether communication between the 2 active sites on the reverse transcriptase may complicate combination therapy with nucleos(t)ide analogs and RNAseH inhibitors; and 3) Generate a set of variant HBV RNAseHs for screening drug candidates for cross-genotypic efficacy.