Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations.
Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations.
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人类免疫缺陷病毒 1 型逆转录酶进行引物解封闭反应的细胞内底物：静态和活化淋巴细胞亚群提取物的检测和定量。
DOI：
10.1128/aac.49.5.1761-1769.2005
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发表时间：
2005
影响因子：
4.9
通讯作者：
Scott,WalterA
中科院分区：
文献类型：
--
作者：
Smith,AnthonyJ;Meyer,PeterR;Asthana,Deshratn;Ashman,MargaritaR;Scott,WalterA
用3′-叠氮基-3 ′-脱氧胸苷（AZT）治疗人类免疫缺陷病毒1型（HIV-1）感染的患者，可选择病毒逆转录酶（RT）的突变形式，其从封闭的DNA链中去除链终止核苷酸的能力增强。我们测试了各种细胞提取物中是否存在该反应的内源性受体底物。细胞提取物与HIV-1 RT和[32 P] ddAMP-终止的DNA引物/模板孵育，产生32 P-标记的腺苷2′，3 ′-双脱氧腺苷5′，5 ′-P1，P4-四磷酸（Ap 4ddA）、ddATP、Gp 4ddA和Ap 3ddA，分别对应于[32 P]ddAMP向ATP、PPi、GTP和ADP的转移。与[32 P]AZT单磷酸盐（AZTMP）终止的引物/模板孵育产生类似的32 P标记的AZT衍生物。基于特异性切除产物的形成速率，确定ATP和PPi水平：H9细胞、巨噬细胞和未刺激的CD 4+或CD 8 +T细胞中ATP的浓度为1.3至2.2 mM，而PPi的浓度为7至15 μM。在这些条件下，ATP依赖性反应占主导地位，AZT抗性突变体RT的切除比野生型RT更有效。活化的CD 4+或CD 8 +T细胞含有1.4至2.7 mM ATP和55至79 μM PPi。这些细胞PPconcentrations低于以前报道的，尽管如此，PPi依赖性反应占主导地位的提取物从活化的T细胞，切除突变体和野生型RT发生类似的效率。虽然PPi依赖性切除可能有助于体内AZT抗性，但AZT抗性突变体的选择可能主要发生在ATP依赖性反应占主导地位的环境中。
Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3′-azido-3′-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [32P]ddAMP-terminated DNA primer/template gave rise to32P-labeled adenosine 2′,3′-dideoxyadenosine 5′,5′′′−P1,P4-tetraphosphate (Ap4ddA), ddATP, Gp4ddA, and Ap3ddA, corresponding to the transfer of [32P]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [32P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPilevels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4+or CD8+T cells, while PPiwas present at 7 to 15 μM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4+or CD8+T cells contained 1.4 to 2.7 mM ATP and 55 to 79 μM PPi. These cellular PPiconcentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.