逆转录酶RNase H活性改变对NRTIs和NNRTIs体外药效学的影响：以RNase H为靶点研发HIV逆转录抑制剂的新思路

Acquired immunodeficiency syndrome (AIDS), which was caused by infection with human immunodeficiency virus (HIV), became a chronic disease. There are more than twenty antiretroviral drugs belong to six classes acting on different stages of viral life cycle. Highly active antiretroviral therapy (HAART) is used for AIDS patients. However, adverse effects and drug resistance would appear after a certain time of therapy. Reverse transcriptase is a crucial enzyme of HIV with RNA dependent DNA polymerase, DNA dependent DNA polymerase and ribonuclease H activities. NRTIs and NNRTIs are important antiretroviral drugs for AIDS treatments by targeting to HIV reverse transcriptase DNA polymerase activity. This study focuses on RNase H activity. We will test NRTIs and/or NNRTIs antiviral effects with RNase H activity increased or decreased. This study will demonstrate the function modes (synergism, additive effect or antagonism) of RNase H inhibitor/activator combined with NRTIs and/or NNRTIs. It will provide a new clue to develop a novel type of reverse transcriptase inhibitors targeting to RNase H.

艾滋病已成为慢性病，虽有六类二十余个药物供选择，但患者因长期服用多种药物，仍会出现耐药及不良反应。HIV逆转录酶为病毒特有，有DNA聚合酶和RNase H酶活性。现有核苷及非核苷类逆转录酶抑制剂（NRTIs和NNRTIs）是重要抗艾滋病药物，是逆转录酶DNA聚合酶活性抑制剂，是高效抗逆转录病毒治疗方案所必需。本项目以逆转录酶RNaseH活性为研究对象，以NRTIs和NNRTIs为基础，研究改变RNase H活性（升高或降低）对NRTIs和NNRTIs单独/组合抗HIV体外药效学的影响。本项目将明确RNase H激动剂/抑制剂与NRTIs和NNRTIs联合作用方式（协同、加和或拮抗），明确以何种方式改变RNase H活性可实现与现有NRTIs和NNRTIs联合使用的抗病毒最佳药效，提供以RNase H为靶点的抗逆转录药物研发的新思路，为以RNase H为靶点的新型逆转录酶抑制剂研发提供依据。

为确认升高HIV- RT RNaseH 活性是否可成为抗HIV-1靶点，在未找到RNaseH 激动剂的情况下，本项目首先通过构建评价临床分离HIV-1毒株，发现HIV-1RT RNaseH 突变6个位点(RTN460D/T468S/K530R/A554T/V559I) RNase H活性升高约20%，而RDDP活性降低约6%。AZT、3TC（NRTIs）和EFV、NVP、ETR（NNRTIs）单个药物抑制HIV-1wt和RNaseH高活性 HIV-1突变株复制的半数抑制浓度无明显差异；AZT/3TC/EFV联合使用抑制HIV-1wt和RNaseH高活性 HIV-1突变株复制，药物间均表现为强协同作用，CI值均小于1分别为0.57和0.61，即AZT/3TC/EFV联合用药抗HIV-1wt体外药效学略优于抗HIV-15329体外药效学。. 本研究通过构建并应用表型混合病毒模型提高HIV-1病毒颗粒中的相对RNase H活性，考察了RNase H活性升高对HIV-1复制和聚合酶抑制剂NRTIs及NNRTIs药效的影响。结果显示，RNase H活性升高抑制HIV-1的复制； RNase H活性升高可提升HIV-1RT-K103N和HIV-1RT-Y181C对NVP的敏感性；RNase H活性升高可提升HIV-1RT-D67N/K70R/T215F对AZT的敏感性。. 本研究还发现当向HIV-1RT-M184V病毒颗粒引入50%的野生型RT时，HIV-1RT-M184V对3TC的敏感性与野生型HIV-1一致，即不再产生耐药，提示表型混合病毒模型可用于研究突变病毒比例与耐药出现的相关性。我们还应用该结果验证了胞浆中的RT进入病毒衣壳结构调节逆转录过程的可能性。结果显示，胞浆中的野生型RT并未提升HIV-1RT-M184V对3TC的敏感性，提示胞浆中的RT无法进入病毒衣壳结构调节逆转录过程。. 本研究结果提示，HIV-1逆转录过程中RT聚合酶活性和RNase H活性的平衡十分重要。 ① RNase H活性升高导致两种酶活性失衡，从而抑制HIV-1复制。② RNase H活性升高有利于聚合酶抑制剂的抗突变HIV-1药效。③ 通过向细胞浆提供外源性RNase H无法达到升高RNase H活性的目的，继续寻找RNase H激活剂是直接有效的方法。