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

丙型肝炎病毒(丙型肝炎病毒)感染270万美国人，是慢性肝炎和 肝细胞癌。它是用干扰素a加核苷类似物利巴韦林治疗的。一个有争议的 利巴韦林的作用机制是通过丙型肝炎病毒RNA聚合酶(RdRp)整合到病毒基因组中 在细胞酶将三磷酸利巴韦林磷酸化后。利巴韦林在丙型肝炎病毒中的掺入 RNA会降低病毒的适合度，并抑制随后几轮的RNA合成。在一项小型先导研究中，我们 发现丙型肝炎病毒RdRp中的自然序列变异导致鸟苷相对于尿嘧啶的使用增加 (G/U比)在4个治疗应答者中有2个在RNA合成过程中，但在4个无应答者中没有一个是由于 增加了GTP使用量，而不是减少了UTP使用量。这一观察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：丙型肝炎病毒RdRp序列变异导致变量 鸟苷和/或利巴韦林在RNA合成过程中的使用和调节治疗的成功 利巴韦林。 目的1.确定高G/U比值是否与抗病毒治疗的成功相关。The RdRps 高G/U比率的患者来自单一干扰素治疗失败的患者，然后再接受 干扰素A加利巴韦林。这是为那些对治疗的反应主要是由于 添加利巴韦林，但这排除了在治疗中大量使用鸟苷与治疗反应之间的联系- 天真的病人。因此，我们将测量Virahep-C试验参与者的RdRp的G/U比率 丙型肝炎病毒的治疗，以确定G/U比率是否与未接受治疗的患者的治疗成功相关。 目的2.确定高G/U比值与三磷酸利巴韦林使用的关系。RdRps，带 与低G/U比率的RdRps相比，高G/U比率可使利巴韦林以更高的速率结合到丙型肝炎病毒的RNA中 G/U比值。因此，我们将通过重组技术在体外使用三磷酸利巴韦林作为底物。 来自目标1的RdRps，并将该活动与治疗反应相关联。 目的3.评估与改变鸟苷和/或利巴韦林使用有关的变化对 RdRp结构。分子模拟将被用来识别可能导致改变的RdRp变异 核苷酸的使用。预计将提高鸟苷或利巴韦林使用量的关键变异将转移到RdRps 低G/U比率和鸟苷和利巴韦林的使用将被测量以检验结构预测。 这些研究将表征丙型肝炎病毒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合成中的作用，并将确定鸟苷或利巴韦林的使用增加是否与 丙型肝炎病毒治疗的成功。将抗丙型肝炎病毒治疗的成功与RdRp使用利巴韦林联系起来将 为利巴韦林在人类中的机制提供强有力的证据，因为它是通过整合到病毒中的 并将解决利巴韦林如何促进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.