A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.
A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.
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模板切割在减少含有 M184V 突变的人类免疫缺陷病毒 1 型逆转录酶切除链终止核苷酸中的作用。
DOI：
10.1128/jvi.05767-11
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发表时间：
2012
影响因子：
5.4
通讯作者：
Scott,WalterA
中科院分区：
文献类型：
--
作者：
Acosta-Hoyos,AntonioJ;Matsuura,SuzanneE;Meyer,PeterR;Scott,WalterA
对核苷逆转录酶（RT）抑制剂的耐药性是通过胸苷类似物耐药突变（TAM）赋予人类免疫缺陷病毒1型的，这些突变增加了RT在被掺入后切除链终止核苷酸的能力。RT突变M184 V是TAM的有效抑制剂。在含有TAM的RT中，添加M184 V抑制3′-脱氧-3 ′-叠氮胸苷单磷酸（AZTMP）在RNA模板上的切除的程度大于具有相同序列的DNA模板。无催化活性的RNase H突变E478 Q消除了这种差异。切除活性的降低与ATP或焦磷酸作为受体底物相似。AZTMP的切除减少与相对于引物末端的-7位RNA模板的切割增加有关，这导致引物-模板解离增加。无论M184 V是否存在，RT最初都不会结合在-7切割位点。在起始位点的切割之后，在-7切割位点进行RT解离和再结合，当存在M184 V突变时，解离和再结合增强。与M184 V的作用相反，K65 R突变抑制RT对RNA或DNA模板的切除活性至相同程度，并且不改变RNA酶H切割模式。基于这些结果，我们建议，增强RNase H切割附近的引物末端发挥作用，M184 V抑制AZT抗性，而K65 R抑制发生通过不同的机制。
Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3′-deoxy-3′-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position −7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the −7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the −7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.