University of Rochester, School of Medicine and Dentistry, Department of Medicine, Rochester, NY, USA.
University of Rochester, School of Medicine and Dentistry, Department of Microbiology and Immunology, Rochester, NY, USA.
J Gen Virol. 2013 Oct;94(Pt 10):2297-2308. doi: 10.1099/vir.0.054999-0. Epub 2013 Jun 26.
Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.
先前我们小组的工作表明，含有非核苷类逆转录酶抑制剂（NNRTI）耐药突变的人类免疫缺陷病毒 1 型（HIV-1）逆转录酶（RT）在 RNase H 活性以及病毒粒子中的 RT 蛋白含量方面存在缺陷。这些缺陷与缺乏 NNRTI 时的复制适应性相关。与野生型相比，含有突变组合 K101E+G190S 的病毒在存在 NNRTI 时的复制能力更好。NNRTI 对病毒生长的刺激发生在病毒生命周期的早期阶段，并且受耐药突变出现的 RT 骨架序列调节。我们想确定 RT 骨架序列在缺乏 NNRTI 时对 RT 含量、聚合和 RNase H 活性的影响。我们比较了含有 K101E+G190S 的 NL4-3 RT 与含有 K101E+G190S 的患者分离株 RT 序列 D10。我们在这里表明，与 NL4-3 骨架不同，与野生型相比，D10 骨架序列降低了纯化重组 RT 的 RNA 依赖性 DNA 聚合活性。相比之下，与 NL4-3 骨架中的野生型和 K101E+G190S 相比，含有 D10 骨架的 RT 的 RNase H 活性增加。与 NL4-3 骨架中的 K101E+G190S 相比，D10 病毒粒子中的 RT 含量也增加。我们得出结论，RT 的骨架序列可以改变 NNRTI 耐药突变体 K101E+G190S 的活性，确定负责的氨基酸将有助于理解 NNRTI 耐药突变体如何改变适应性以及 NNRTI 如何刺激 HIV-1 病毒复制的机制。
