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

丙型肝炎病毒（HCV）感染270万美国人，是慢性肝炎的主要原因， 肝细胞癌它是用干扰素加核苷类似物利巴韦林治疗。一个有争议 利巴韦林的作用机制是通过HCV RNA聚合酶（RdRp）掺入病毒基因组 随后被细胞酶磷酸化为三磷酸利巴韦林。利巴韦林掺入HCV RNA会降低病毒的适应性，并抑制随后几轮的RNA合成。在一项小型试点研究中， 发现HCV RdRp中的天然序列变异导致鸟苷相对于尿嘧啶的使用增加 4例治疗应答者中有2例的RNA合成期间（G/U比），但4例非应答者中无1例， GTP使用增加而不是UTP使用减少。这一观察结果具有直接的临床意义，因为 利巴韦林是鸟苷类似物。假设：HCV RdRp中的序列变异导致可变的 在RNA合成期间使用鸟苷和/或利巴韦林并调节采用 利巴韦林。 目标1.确定高G/U比值是否与抗病毒治疗的成功相关。TheRdRps G/U比值高的患者来自干扰素a单药治疗失败的患者， 干扰素A加利巴韦林。这选择了对治疗的反应主要是由于以下原因的患者 添加利巴韦林，但它排除了高鸟苷使用与治疗反应的相关性- 天真的病人因此，我们将测量Virahep-C试验参与者的RdRps的G/U比， HCV的治疗，以确定G/U比值是否与治疗初治患者的治疗成功相关。 目标2.确定高G/U比值与使用三磷酸利巴韦林的关系。RdRps， 较高的G/U比率预测以比RdRps更高的速率将利巴韦林掺入HCV RNA中， G/U比率。因此，我们将在体外使用三磷酸利巴韦林作为底物， RdRps，并将该活性与对治疗的反应相关联。 目标3.评估与鸟苷和/或利巴韦林使用改变相关的变异对 RdRp结构。将使用分子建模来鉴定可能引起改变的RdRp变异。 核苷酸的使用。预计增加鸟苷或利巴韦林使用的关键变化将转移到RdRps 将测量低G/U比和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使用鸟苷的能力 和利巴韦林在RNA合成过程中，并将确定是否升高鸟苷或利巴韦林的使用与 HCV治疗的成功RdRp将抗HCV治疗的成功与利巴韦林的使用联系起来， 为利巴韦林在人体内的作用机制提供了强有力的证据， 基因组，并将解决如何利巴韦林有助于丙型肝炎病毒清除的争议。

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 a 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. TheRdRps with high G/U ratios were from patients who failed interferon a monotherapy and were then retreated with interferon a 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/Uratios 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.