Variation in NTP use by the HCV polymerase and response to therapy

描述（由申请人提供）：丙型肝炎病毒（HCV）感染270万美国人，是慢性肝炎和肝细胞癌的主要原因。用干扰素治疗？加上核苷类似物利巴韦林。利巴韦林的一个有争议的机制是通过HCV RNA聚合酶（RdRp）在细胞酶磷酸化成利巴韦林三磷酸后并入病毒基因组。将利巴韦林掺入HCV RNA会降低病毒适应度并抑制后续的RNA合成。在一项小型试点研究中，我们发现HCV RdRp的自然序列变化导致4名治疗应答者中2名在RNA合成过程中鸟苷相对于尿嘧啶的使用增加（G/U比），但由于GTP使用升高而不是UTP使用减少，4名无应答者中没有一人。这一观察结果具有直接的临床意义，因为利巴韦林是一种鸟苷类似物。假设：HCV RdRp序列的变化导致RNA合成过程中鸟苷和/或利巴韦林的使用变化，并调节利巴韦林治疗的成功。目的1。确定高G/U比值是否与抗病毒治疗的成功相关。高G/U比的RdRps来自干扰素治疗失败的患者。单药治疗，然后用干扰素？联合利巴韦林。这一研究选择的患者对治疗的反应主要是由于利巴韦林的加入，但它排除了高鸟苷使用与治疗初治患者对治疗的反应的关联。因此，我们将测量HCV治疗的Virahep-C试验参与者的RdRps的G/U比率，以确定G/U比率是否与初次治疗患者的治疗成功相关。目标2。确定高G/U比与使用三磷酸利巴韦林的关系。预计高G/U比的RdRps比低G/U比的RdRps将利巴韦林纳入HCV rna的率更高。因此，我们将通过来自Aim 1的重组RdRps来测量三磷酸利巴韦林作为底物的体外使用，并将这种活性与对治疗的反应联系起来。目标3。评估鸟苷和/或利巴韦林使用改变对RdRp结构的影响。分子模型将用于识别可能导致核苷酸使用改变的RdRp变异。预测会增加鸟苷或利巴韦林使用的关键变异将转移到低G/U比的RdRps中，并且将测量鸟苷和利巴韦林的使用以测试结构预测。这些研究将描述HCV RdRp的自然变异如何影响其在RNA合成过程中使用鸟苷和利巴韦林的能力，并将确定鸟苷或利巴韦林的使用升高是否与HCV治疗的成功相关。将抗HCV治疗的成功与RdRp使用利巴韦林联系起来，将为利巴韦林在人类中的作用机制提供强有力的证据，证明利巴韦林是通过并入病毒基因组的，并将解决利巴韦林如何促进HCV清除的争议。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infects 2.7 million Americans and is a leading cause of chronic hepatitis and hepatocellular carcinoma. It is treated with interferon ? plus the nucleoside analog ribavirin. A controversial mechanism for ribavirin is through incorporation into the viral genome by the HCV RNA polymerase (RdRp) following phosphorylation to ribavirin triphosphate by cellular enzymes. Incorporation of ribavirin into HCV RNAs would reduce viral fitness and inhibit subsequent rounds of RNA synthesis. In a small pilot study we found that natural sequence variation in the HCV RdRp led to increased use of guanosine relative to uracil (G/U ratio) during RNA synthesis in 2 of 4 responders to therapy but in none of the 4 non-responders due to elevated GTP use rather than decreased UTP use. This observation has direct clinical implications because ribavirin is a guanosine analog. Hypothesis: Sequence variation in the HCV RdRp leads to variable guanosine and/or ribavirin use during RNA synthesis and modulates success of therapy employing ribavirin. Aim 1. Determine if high G/U ratios are associated with success of antiviral therapy. The RdRps with high G/U ratios were from patients who failed interferon ? monotherapy and were then retreated with interferon ? plus ribavirin. This selected for patients whose response to treatment was primarily due to addition of ribavirin, but it precluded associating high guanosine use with response to therapy in treatment- naive patients. Therefore, we will measure the G/U ratios of RdRps from participants in the Virahep-C trial of therapy for HCV to determine if G/U ratios correlate with success of therapy in treatment-naive patients. Aim 2. Determine the relationship of high G/U ratios and use of ribavirin triphosphate. RdRps with high G/U ratios are predicted to incorporate ribavirin into HCV RNAs at higher rates than RdRps with low G/U ratios. Therefore, we will measure in vitro use of ribavirin triphosphate as a substrate by recombinant RdRps from Aim 1 and correlate this activity with response to therapy. Aim 3. Assess effects of variations associated with altered guanosine and/or ribavirin use on the RdRp structure. Molecular modeling will be used to identify RdRp variations that may cause altered nucleotide use. Key variations predicted to elevate guanosine or ribavirin use will be transferred to RdRps with low G/U ratios and guanosine and ribavirin use will be measured to test the structural predictions. These studies will characterize how natural variation in the HCV RdRp affects its ability to use guanosine and ribavirin during RNA synthesis and will determine if elevated guanosine or ribavirin use correlates with success of HCV therapy. Associating success of anti-HCV therapy with use of ribavirin by the RdRp would provide strong evidence for ribavirin's mechanism in humans as being through incorporation into the viral genome and would resolve the controversy of how ribavirin contributes to HCV clearance.