Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication.
Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication.
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人类免疫缺陷病毒 1 型 (HIV-1) Vpr 的结构功能分析：亮氨酸残基对 Vpr 介导的反式激活和病毒复制的作用。
DOI：
10.1016/j.virol.2004.07.013
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发表时间：
2004
期刊：
影响因子：
3.7
通讯作者：
Ayyavoo,Velpandi
中科院分区：
文献类型：
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作者：
Thotala,Dineshkumar;Schafer,ElizabethA;Tungaturthi,ParithoshK;Majumder,Biswanath;Janket,MichelleL;Wagner,Marc;Srinivasan,Alagarsamy;Watkins,Simon;Ayyavoo,Velpandi
HIV-1 Vpr已显示通过与包括糖皮质激素受体（GR）在内的几种细胞来源的蛋白质相互作用反式激活LTR定向表达。激活后，类固醇受体与含有特征基序LxxLL的蛋白质结合，易位到细胞核中，与它们的反应元件结合，并激活转录。HIV-1 Vpr中存在这样的基序，促使我们对参与Vpr-GR相互作用的特定亮氨酸残基的作用、亚细胞定位及其对Vpr-GR介导的反式激活的影响进行分析。在Vpr分子中突变存在于H I、II和III中的单个亮氨酸残基，并评估它们与GR相互作用、反式激活GRE和HIV-1 LTR启动子以及它们与GR共定位的能力。和L 68表现出与Vprwt相似且略高的活化水平。有趣的是，与野生型相比，残基L22处的取代导致显着更高的GRE和HIV-1 LTR反式激活（高8至11倍）。共聚焦显微镜表明，Vpr L22 A表现出不同的浓缩核定位模式与Vprwt注意到的核/核周模式。此外，电泳迁移率变动分析（EMSA）显示，与野生型Vpr-GR复合物相比，VprL 22 A-GR复合物具有更高的DNA结合活性。这些结果表明，亮氨酸残基对HIV-1 LTR定向反式激活的作用与其在Vpr中的位置有关。
HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL, translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr–GR interaction, subcellular localization and its effect on Vpr–GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher level of activation compared to Vprwt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift assay (EMSA) revealed that the VprL22A–GR complex had higher DNA-binding activity when compared to the wild type Vpr–GR complex. These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.