Decreased processivity of human immunodeficiency virus type 1 reverse transcriptase (RT) containing didanosine-selected mutation Leu74Val: a comparative analysis of RT variants Leu74Val and lamivudine-selected Met184Val
Decreased processivity of human immunodeficiency virus type 1 reverse transcriptase (RT) containing didanosine-selected mutation Leu74Val: a comparative analysis of RT variants Leu74Val and lamivudine-selected Met184Val
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DOI：
10.1128/jvi.73.10.8448-8456.1999
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发表时间：
1999-10-01
影响因子：
5.4
通讯作者：
Crumpacker, CS
中科院分区：
文献类型：
--
作者：
Sharma, PL;Crumpacker, CS
我们之前表明，pNL43 背景中的去羟肌苷选择突变赋予人类免疫缺陷病毒 1 型复制劣势，导致复制适应性丧失。这项工作得到了扩展，表明具有 HXBc2 主链和来自含有 Leu74Val 突变的 pNL4-3 的逆转录酶 (RT) 片段的重组病毒在 3 周内产生的 p24 抗原量不断减少。含有来自 pNL4-3 的野生型 RT 的 HXBc2 重组体可有效复制。当含有 Leu74Val 突变的病毒体相关 RT 与同聚物 RNA 模板引物、poly(A) 和oligo(dT) 一起用于 RT 持续进行性测定时，具有改变的 Leu74Val 突变的 RT 持续性较差，与野生型 pNL4-3 RT 相比，产生的 cDNA 产物更少。将具有 Leu74Val 突变的突变体的复制动力学和 RT 持续合成能力与拉米夫定选择的突变体 Met184Val 的复制动力学和 RT 持续性进行比较。在复制动力学测定中，突变体 Leu74Val 的复制速度比突变体 Met184Val 慢。在持续合成能力测定中，两种病毒的突变 RT 显示持续合成能力的相当程度的降低。这些观察结果提供了持续性降低的生化证据，以支持 Leu74Val 或 Met184Val 突变观察到的复制适应性降低。
we previously showed that a didanosine-selected mutation in pNL43 background conferred a replication disadvantage on human immunodeficiency virus type 1, resulting in a loss of replication fitness. This work has been extended by showing that a recombinant virus with the HXBc2 backbone and reverse transcriptase (RT) fragments from pNL4-3 containing the Leu74Val mutation produce decreasing amounts of p24 antigen over a 3-week period. The HXBc2 recombinant containing the wild-type RT from pNL4-3 replicated efficiently. When the virion-associated RT containing the Leu74Val mutation was used in an RT processivity assay with homopolymer RNA template-primer, poly(A), and oligo(dT), the RT with altered Leu74Val mutation was less processive, generating fewer cDNA products in comparison to wild-type pNL4-3 RT. The replication kinetics and RT processivity of the mutant with the Leu74Val mutation were compared to those of a lamivudine-selected mutant Met184Val. In replication kinetics assays, mutant Leu74Val replicated slower than the mutant Met184Val. In a processivity assay, the mutant RTs from both viruses show comparable decreases in processivity. These observations provide biochemical evidence of decreased processivity to support the decrease in replication fitness observed with the Leu74Val or Met184Val mutations.