Hepatitis B Virus diversity and ribonuclease H inhibitor efficacy

描述（由申请方提供）：B型肝炎病毒（HBV）是一种嗜肝DNA病毒，通过逆转录复制。它慢性感染> 3.5亿人，每年杀死多达120万患者。治疗主要采用核苷（酸）类似物。这些药物能显著抑制HBV，但由于对病毒复制的抑制不完全，很少能治愈患者。此外，由于耐药性突变的演变，HBV的控制常常失败。通过与核苷（酸）类似物联合使用新药进一步抑制HBV，在更多患者中药理学治愈HBV应该是可能的。HBV逆转录需要两种病毒酶活性，它们都位于病毒逆转录酶蛋白上：由核苷（酸）类似物靶向的DNA聚合酶和在病毒RNA复制成DNA后破坏病毒RNA的核糖核酸酶H（RNAseH）。抗HBV RNAseH药物尚未开发，因为不能制备适合于药物筛选的酶。我们最近制造了活性HBV RNAseH并鉴定了13种RNAseH抑制剂。HBV有8种基因型（A-H），抗RNAseH药物必须抑制广泛的HBV毒株才能在临床上有效。我们发现针对基因型D和H分离株鉴定的RNAseH抑制剂可以阻断基因型A分离株的复制，因此跨基因型抑制是可能的。然而，我们测试的基因型D和H RNAseH分离株对RNAseH抑制剂的敏感性不同。我们不知道这些差异是否是由于基因型特异性或分离株特异性差异，因为每个基因型只检测了1个分离株。此外，我们还没有表征来自基因型B和C的重组RNAseH，这是医学上最相关的基因型。在这个小型研究基金（R 03）项目中，我们将评估HBV的高遗传变异影响其对RNAseH抑制剂敏感性的程度。目的1：评估HBV基因型之间和内部的差异如何影响对RNAseH抑制剂的敏感性。我们将在生化和病毒复制试验中检测来自基因型B、C和D的变异HBV RNAseH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。我们将在HBV基因型B、C和D分离株中引入常见核苷类似物耐药突变，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基因型差异是否会使抗RNAseH药物开发复杂化; 2）帮助确定逆转录酶上的2个活性位点之间的通信是否会使核苷（酸）类似物和RNAseH抑制剂的联合治疗复杂化;和3）产生一组变异HBV RNAseH用于筛选交叉基因型功效的候选药物。

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.