Hepatitis B Virus diversity and ribonuclease H inhibitor efficacy

描述(申请人提供)：乙肝病毒(乙肝)是一种嗜肝的DNA病毒，通过逆转录进行复制。它慢性感染了3.5亿人，每年导致多达120万患者死亡。治疗主要使用核素(T)类似物。这些药物深刻地抑制了乙肝病毒，但由于对病毒复制的不完全抑制，它们很少能治愈患者。此外，由于耐药突变的进化，对乙肝病毒的控制经常失败。在更多的患者中，药物治疗乙肝病毒应该是可能的，方法是进一步抑制乙肝病毒，并与核素类似物联合使用。乙肝病毒的逆转录需要两种病毒酶活性，这两种酶都位于病毒逆转录酶蛋白上：核糖核酸酶H(RNAseH)和核糖核酸酶H(RNAseH)，这两种酶都位于病毒逆转录酶蛋白上：DNA聚合酶是核糖核酸类似物的靶标，核糖核酸酶H在病毒RNA复制到DNA后将其摧毁。由于不能制备适合于药物筛选的酶，目前还没有开发出抗HBVRNAseH药物。我们最近制作了活性的HBVRNAseH，并鉴定了13种RNAseH的抑制剂。乙肝病毒有8种基因型(A-H)，抗RNAseH药物必须抑制多种乙肝病毒株才能发挥临床疗效。我们发现针对D型和H型分离株的RNAseH抑制剂可以阻止A型分离株的复制，因此跨基因型别的抑制是可能的。然而，我们测试的D型和H型RNAseH分离株对RNAseH抑制剂具有不同的敏感性。我们不知道这些不同是由于特定的基因型还是特定的分离物造成的，因为我们只检查了每个基因型的一个分离物。此外，我们还没有鉴定来自B和C基因的重组RNAseH，这两种基因是医学上最相关的基因。在这个小型研究资助项目(R03)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：评价不同类型的乙肝病毒对RNAseH抑制剂敏感性的影响。我们将在生化和病毒复制检测中测试B、C和D型的变异的HBVRNAseH序列对一组RNAseH拮抗剂的敏感性。目的2：确定核苷类似物耐药突变如何影响对RNAseH抑制的敏感性。我们将把常见的nulceos(T)ide类似耐药突变引入到B、C和D型乙肝病毒分离株中，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HBV型差异是否会使抗RNAseH药物的开发复杂化；2)帮助确定逆转录酶上两个活性位点之间的联系是否会使与核素类似物和RNAseH抑制剂的联合治疗复杂化；以及3)产生一组变异的HBVRNAseH，用于筛选跨基因型疗效的候选药物。

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people and kills up t 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.