Coutsinos Dimitrios, Invernizzi Cédric F, Xu Hongtao, Brenner Bluma G, Wainberg Mark A
McGill University AIDS Center, Jewish General Hospital, Montréal, QC, Canada.
Antivir Chem Chemother. 2010 Jan 5;20(3):117-31. doi: 10.3851/IMP1443.
We have shown that the K65R resistance mutation in HIV type-1 (HIV-1) reverse transcriptase (RT) is selected more rapidly in subtype C than subtype B HIV-1 in biochemical, cell culture and clinical studies. Template-usage experiments demonstrated that subtype C nucleotide coding sequences caused RT to preferentially pause, leading to K65R acquisition. This new study now further establishes the basis for differential occurrence of both K65R and thymidine analogue mutations (TAMs) between subtypes.
Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65.
When positive double-stranded DNA synthesis was evaluated from a negative single-stranded DNA template, pausing at the 67 region, which is linked to occurrence of TAMs, was alleviated with both subtype B and C templates at high dCTP concentrations, but this alleviation was more pronounced with the subtype C template. By contrast, pausing at the 65 region on the subtype C but not subtype B template always occurred and was not alleviated at high levels of nucleotide triphosphates or by other means. Furthermore, templates containing repeats of the homopolymeric sequence spanning codons 64-66 of pol showed corresponding pausing repeats at the 65 region with the subtype C template only. Inverted RNA and DNA templates both displayed pausing at position K65 for the subtype C template and a ladder of pausing events culminating at codon 67 for the subtype B templates.
These results further establish a mechanistic basis for the exclusion of both K65R and TAMs on single templates as well as the preferential acquisition of K65R in subtype C viruses.
我们已经表明，在生化、细胞培养和临床研究中，1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）中的K65R耐药突变在C亚型中比B亚型HIV-1中选择得更快。模板使用实验表明，C亚型核苷酸编码序列导致RT优先停顿，从而导致K65R的获得。这项新研究现在进一步确立了亚型之间K65R和胸苷类似物突变（TAM）差异发生的基础。
基于凝胶的核苷酸延伸测定法用于研究K65周围的同聚物序列。
当从负链单链DNA模板评估正链双链DNA合成时，与TAM发生相关的67区域的停顿在高dCTP浓度下，B亚型和C亚型模板都有所缓解，但C亚型模板的这种缓解更明显。相比之下，C亚型模板而非B亚型模板上65区域的停顿总是发生，并且在高浓度三磷酸核苷酸或通过其他方式都没有得到缓解。此外，包含跨越pol基因64 - 66密码子的同聚物序列重复的模板仅在C亚型模板的65区域显示出相应的停顿重复。反向RNA和DNA模板在C亚型模板的K65位置均显示停顿，而B亚型模板在密码子67处出现一系列停顿事件。
这些结果进一步确立了在单个模板上排除K65R和TAM以及C亚型病毒中优先获得K65R的机制基础。
