Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization.
Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization.
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HIV-1 Vpr 的功能分析：鉴定亚细胞定位所必需的决定因素。
DOI：
10.1006/viro.1995.1490
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Srinivasan,A
中科院分区：
文献类型：
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作者：
Mahalingam,S;Collman,RG;Patel,M;Monken,CE;Srinivasan,A
Vpr是一种保守的HIV-1辅助蛋白，定位于细胞的核区域。Vpr也存在于病毒体中，并且当与Gag共表达时，其被引导到组装病毒中。这两个本地化活动可能是重要的Vpr功能，我们最近确定了Vpr的区域，是病毒体掺入的关键。在这项研究中，我们分析了参与亚细胞定位的Vpr结构域。转染细胞的免疫荧光染色显示，野生型Vpr只定位于核区域。设计用于破坏预测的α-螺旋结构的N-末端结构域中的突变导致异常定位，而保守取代显示野生型模式。蛋白质的中心部分的区域也具有螺旋结构的潜力，并且该结构域中的两个保守氨基酸（A59，H71）的诱变损害了定位，而第三个（Q65）的取代没有。与此相反，无论是保守的甘氨酸和半胱氨酸的位置75-76，也不是C-末端的碱性残基（R87，K95）是必要的核定位。此外，两个残基插入内和之间的两个假定的螺旋破坏本地化，但插入在C-末端区域没有。因此，Vpr的亚细胞定位功能取决于两个推定的螺旋结构域，但独立于保守的Gly-Cys基序和特定的C-末端碱性残基。
Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted α-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.