McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
Retrovirology. 2010 Oct 7;7:80. doi: 10.1186/1742-4690-7-80.
HIV-1 subtype C infections account for over half of global HIV infections, yet the vast focus of HIV-1 research has been on subtype B viruses which represent less than 12% of the global pandemic. Since HIV-1 reverse transcriptase (RT) is a major target of antiviral therapy, and since differential drug resistance pathways have been observed among different HIV subtypes, it is important to study and compare the enzymatic activities of HIV-1 RT derived from each of subtypes B and C as well as to determine the susceptibilities of these enzymes to various RT inhibitors in biochemical assays.
Recombinant subtype B and C HIV-1 RTs in heterodimeric form were purified from Escherichia coli and enzyme activities were compared in cell-free assays. The efficiency of (-) ssDNA synthesis was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3(Lys) as primer. Processivity was assayed under single-cycle conditions using both homopolymeric and heteropolymeric RNA templates. Intrinsic RNase H activity was compared using 5'-end labeled RNA template annealed to 3'-end recessed DNA primer in a time course study in the presence and absence of a heparin trap. A mis-incorporation assay was used to assess the fidelity of the two RT enzymes. Drug susceptibility assays were performed both in cell-free assays using recombinant enzymes and in cell culture phenotyping assays.
The comparative biochemical analyses of recombinant subtype B and subtype C HIV-1 reverse transcriptase indicate that the two enzymes are very similar biochemically in efficiency of tRNA-primed (-) ssDNA synthesis, processivity, fidelity and RNase H activity, and that both enzymes show similar susceptibilities to commonly used NRTIs and NNRTIs. Cell culture phenotyping assays confirmed these results.
Overall enzyme activity and drug susceptibility of HIV-1 subtype C RT are comparable to those of subtype B RT. The use of RT inhibitors (RTIs) against these two HIV-1 enzymes should have comparable effects.
HIV-1 亚型 C 感染占全球 HIV 感染的一半以上，但 HIV-1 研究的绝大部分重点都集中在亚型 B 病毒上，而这种病毒只占全球大流行的不到 12%。由于 HIV-1 逆转录酶（RT）是抗病毒治疗的主要靶点，而且不同 HIV 亚型之间已经观察到了不同的耐药途径，因此研究和比较来自亚型 B 和 C 的 HIV-1 RT 的酶活性以及确定这些酶在生化测定中对各种 RT 抑制剂的敏感性非常重要。
从大肠杆菌中纯化出重组的 HIV-1 亚型 B 和 C RT，并在无细胞测定中比较酶活性。使用 HIV-1 PBS RNA 模板和 tRNA3（Lys）作为引物的凝胶基础测定来测量（-）ssDNA 合成的效率。在单循环条件下，使用均聚和杂聚 RNA 模板测定了连续性。在存在和不存在肝素陷阱的情况下，通过时间过程研究比较了具有 5'-端标记 RNA 模板和 3'-端凹陷 DNA 引物的内在 RNase H 活性。使用错配掺入测定来评估两种 RT 酶的保真度。使用重组酶进行无细胞测定和细胞培养表型测定来进行药物敏感性测定。
对重组 HIV-1 亚型 B 和 C 逆转录酶的比较生化分析表明，两种酶在 tRNA 引发的（-）ssDNA 合成效率、连续性、保真度和 RNase H 活性方面非常相似，并且两种酶对常用的 NRTIs 和 NNRTIs 都表现出相似的敏感性。细胞培养表型测定证实了这些结果。
HIV-1 亚型 C RT 的整体酶活性和药物敏感性与亚型 B RT 相当。使用 RT 抑制剂（RTIs）针对这两种 HIV-1 酶应该具有相当的效果。
