Singh S P, Tomkowicz B, Lai D, Cartas M, Mahalingam S, Kalyanaraman V S, Murali R, Srinivasan A
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Virol. 2000 Nov;74(22):10650-7. doi: 10.1128/jvi.74.22.10650-10657.2000.
Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike other mutants and control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G(2), mutant Vpr showed an intermediary effect with the exception of VprDelta35-50 and VprDelta35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.
Vpr由人类免疫缺陷病毒1型基因组编码，含有96个氨基酸，是一种多功能蛋白，其功能包括使细胞周期停滞在G(2)期、核定位、参与前整合复合物的转运、阳离子通道活性、寡聚化以及与细胞蛋白相互作用，此外还能整合到病毒颗粒中。最近，基于核磁共振和圆二色光谱的结构研究表明，Vpr在氨基末端含有一个螺旋（HI）-转角-螺旋（HII）核心，在中间区域含有一个两亲性螺旋（HIII）。尽管已经确定了螺旋结构域HI和HIII对Vpr功能的重要性，但螺旋结构域HII的作用尚不清楚。为了解决这个问题，我们构建了一系列突变体，其中HII结构域通过缺失、插入和/或取代诱变进行了改变。为了能够检测Vpr，将与Flag表位（DYKDDDDK）对应的序列框内添加到Vpr编码序列中。通过体外转录/翻译系统和在细胞中表达的突变体显示出与Vpr缺失相对应的迁移改变。对HII中残基的取代突变分析表明，VprW38S-FL、VprL42G-FL和VprH45W-FL的稳定性降低。采用涉及共转染NLDeltaVpr原病毒DNA和Vpr表达质粒的实验来分析Vpr的病毒体整合特性。含有缺失和特定取代（VprW38S-FL、VprL39G-FL、VprL42G-FL、VprG43P-FL和VprI46G-FL）的突变型Vpr表现出负性病毒体整合表型。此外，含有缺失的突变型Vpr-FL也未能与野生型Vpr结合，表明Vpr的寡聚化特性可能存在缺陷。亚细胞定位研究表明，与其他突变体和对照Vpr-FL不同，突变体VprDelta35-50-H-FL、VprR36W-FL、VprL39G-FL和VprI46G-FL表现出细胞质和细胞核定位。虽然野生型Vpr使细胞周期停滞在G(2)期，但突变型Vpr除VprDelta35-50和VprDelta35-50-H外表现出中间效应。这些结果表明，HII结构域中的残基对Vpr功能至关重要。
