Wang L, Mukherjee S, Narayan O, Zhao L J
Marion Merrell Dow Foundation, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160-7424, USA.
Gene. 1996 Oct 31;178(1-2):7-13. doi: 10.1016/0378-1119(96)00312-5.
Human immunodeficiency virus type 1 (HIV-1) replicates productively in vitro in CD4(+)-T cells and/or macrophages. In the host, however, HIV-1 replication may be restricted by the quiescence of susceptible cells. Vpr is a 15-kDa late viral gene product, which is assembled in the virion and suspected to enhance HIV-1 replication in the infected host. We demonstrated previously that Vpr interacted specifically with the cellular transcription factor Sp1, and activated transcription from the HIV-1 long-terminal-repeat. Both Vpr-Sp1 interaction and trans-activation by Vpr required a central Leu/Ile-rich domain (LR domain, aa 60-81) in Vpr. This domain of Vpr was also found critical for Vpr interaction with another cellular protein of 180 kDa. We now provide biochemical evidence that the Vpr LR-domain has a leucine-zipper-like structure. The leucine-zipper structure has been found in a variety of cellular transcription factors, which use the leucine-zipper domain to form a specific dimer before they can bind to DNA through an upstream basic domain. The LR domain of HIV-1 Vpr, when fused to the basic domain of the cellular transcription factor CREB, was capable of supporting specific DNA binding by the CREB basic domain. Point mutational analysis of the Leu/Ile residues in the LR domain suggested that multiple Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues critical for Vpr interaction with the cellular 180-kDa protein. These results suggested that the leucine-zipper-like domain may be an important functional determinant for HIV-1 Vpr.
1型人类免疫缺陷病毒（HIV-1）可在体外CD4(+)T细胞和/或巨噬细胞中高效复制。然而，在宿主体内，HIV-1的复制可能会受到易感细胞静止状态的限制。Vpr是一种15千道尔顿的晚期病毒基因产物，它组装在病毒颗粒中，并被怀疑可增强HIV-1在受感染宿主体内的复制。我们之前证明，Vpr与细胞转录因子Sp1特异性相互作用，并激活HIV-1长末端重复序列的转录。Vpr-Sp1相互作用以及Vpr的反式激活都需要Vpr中一个富含亮氨酸/异亮氨酸的中央结构域（LR结构域，氨基酸60-81）。Vpr的这个结构域对于Vpr与另一种180千道尔顿的细胞蛋白相互作用也至关重要。我们现在提供生化证据表明，Vpr的LR结构域具有类似亮氨酸拉链的结构。亮氨酸拉链结构已在多种细胞转录因子中发现，这些转录因子利用亮氨酸拉链结构域形成特定的二聚体，然后才能通过上游碱性结构域与DNA结合。当HIV-1 Vpr的LR结构域与细胞转录因子CREB的碱性结构域融合时，能够支持CREB碱性结构域特异性结合DNA。对LR结构域中亮氨酸/异亮氨酸残基的点突变分析表明，多个亮氨酸/异亮氨酸残基可能参与维持类似亮氨酸拉链的结构。在全长Vpr背景下进行的诱变也有助于确定参与维持类似亮氨酸拉链结构的亮氨酸/异亮氨酸残基。在全长Vpr背景下进行的诱变还有助于确定对Vpr与细胞180千道尔顿蛋白相互作用至关重要的亮氨酸/异亮氨酸残基。这些结果表明，类似亮氨酸拉链的结构域可能是HIV-1 Vpr的一个重要功能决定因素。
