Chen Renxiang, Yokoyama Masaru, Sato Hironori, Reilly Cavan, Mansky Louis M
Institute for Molecular Virology, University of Minnesota, 18-242 Moos Tower, 515 Delaware St. SE, Minneapolis, MN 55455, USA.
J Virol. 2005 Sep;79(18):12045-57. doi: 10.1128/JVI.79.18.12045-12057.2005.
The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. We have previously reported that both drugs and drug-resistant RT mutants can increase virus mutation frequencies. To further assess the contributions of nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and drug-resistant RTs to HIV mutagenesis, a new high-throughput assay system was developed. This assay system was designed to specifically detect frameshift mutations in the luciferase gene in a single virus replication cycle. New drug-resistant RTs were identified that significantly altered virus mutation frequencies. Consistent with our previous observations of NRTIs, abacavir, stavudine, and zalcitabine increased HIV-1 mutation frequencies, supporting the general hypothesis that the NRTIs currently used in antiviral drug therapy increase virus mutation frequencies. Interestingly, similar observations were made with NNRTIs. This is the first report to show that NNRTIs can influence virus mutation frequencies. NNRTI combinations, NRTI-NNRTI combinations, and combinations of drug and drug-resistant RTs led to significant changes in the virus mutation frequencies compared to virus replication of drug-resistant virus in the absence of drug or wild-type virus in the presence of drug. This indicates that combinations of RT drugs or drugs and drug-resistant virus created during the evolution of drug resistance can act together to increase HIV-1 mutation frequencies, which would have important implications for drug therapy regimens. Finally, the influence of drug-resistant RT mutants from CRF01_AE viruses on HIV-1 mutation frequencies was analyzed and it was found that only a highly drug resistant RT led to altered virus mutation frequencies. The results further suggest that high-level drug-resistant RT can significantly influence virus mutation frequencies. A structural model that explains the mutation frequency data is discussed.
抗病毒药物耐药性的产生是治疗人类免疫缺陷病毒1型（HIV-1）感染中的一个重要问题。目前强效抗逆转录病毒疗法用于治疗，通常至少由两种逆转录酶（RT）抑制剂组成。我们之前曾报道，药物和耐药RT突变体均可增加病毒突变频率。为进一步评估核苷类RT抑制剂（NRTIs）、非核苷类RT抑制剂（NNRTIs）及耐药RT对HIV诱变的作用，开发了一种新的高通量检测系统。该检测系统旨在特异性检测单个病毒复制周期中荧光素酶基因的移码突变。鉴定出了可显著改变病毒突变频率的新型耐药RT。与我们之前对NRTIs的观察结果一致，阿巴卡韦、司他夫定和扎西他滨增加了HIV-1突变频率，支持了目前用于抗病毒药物治疗的NRTIs会增加病毒突变频率这一普遍假设。有趣的是，对NNRTIs也有类似观察结果。这是首份表明NNRTIs可影响病毒突变频率的报告。与在无药物情况下耐药病毒的病毒复制或在有药物情况下野生型病毒的病毒复制相比，NNRTI组合、NRTI-NNRTI组合以及药物与耐药RT的组合导致病毒突变频率发生显著变化。这表明RT药物组合或在耐药性演变过程中产生的药物与耐药病毒可共同作用增加HIV-1突变频率，这对药物治疗方案具有重要意义。最后，分析了CRF01_AE病毒的耐药RT突变体对HIV-1突变频率的影响，发现只有高度耐药的RT会导致病毒突变频率改变。结果进一步表明，高水平耐药RT可显著影响病毒突变频率。文中讨论了解释突变频率数据的结构模型。
