Viral Mutation Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
J Virol. 2010 May;84(10):5238-49. doi: 10.1128/JVI.01545-09. Epub 2010 Mar 10.
Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.
最近，观察到 HIV-1 逆转录酶 (RT) 的连接亚结构域 (CN) 和 RNase H 结构域的突变表现出对核苷和非核苷逆转录酶抑制剂 (NRTIs 和 NNRTIs) 的双重耐药性。为了阐明 CN 和 RH 突变赋予 NNRTIs 耐药性的机制，我们假设这些突变降低了 RNase H 的切割，并为 NNRTI 从 RT 解离提供了更多的时间，从而导致 DNA 合成的恢复和增强了 NNRTI 的耐药性。我们观察到，降低 RNase H 切割对 NNRTI 耐药性的影响取决于每个 NNRTI 与 RT 的亲和力，并且进一步受到 NNRTI 结合口袋 (BP) 突变体的存在的影响。降低 RNase H 切割的 D549N、Q475A 和 Y501A 突变体增强了对奈韦拉平 (NVP) 和地拉韦啶 (DLV) 的耐药性，但对依非韦伦 (EFV) 和依曲韦林 (ETR) 没有耐药性，这与它们对 RT 亲和力的增加一致。将 D549N 突变体与 NNRTI BP 突变体结合使用可使 NNRTI 耐药性进一步增加 30 倍，这支持了 NNRTI-RT 亲和力在我们的 NNRTI 耐药性模型中的作用。我们还证明了先前报道的增强 NRTI 耐药性的治疗经验患者的 CN 也降低了 RNase H 的切割，并增强了患者 RT pol 结构域或野生型 pol 结构域中的 NNRTI 耐药性。这些结果共同证实了我们的 NNRTI 耐药性模型的关键预测，并为 CN 和 RH 突变可表现出双重 NRTI 和 NNRTI 耐药性的统一机制提供了支持。
