Wu X, Conway J A, Kim J, Kappes J C
Department of Medicine, University of Alabama at Birmingham 35294.
J Virol. 1994 Oct;68(10):6161-9. doi: 10.1128/JVI.68.10.6161-6169.1994.
Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.
病毒蛋白X（Vpx）是一种2型人类免疫缺陷病毒（HIV-2）和猿猴免疫缺陷病毒的辅助蛋白，它以与Gag蛋白摩尔量相当的量被包装进病毒粒子。为了描绘介导Vpx包装的病毒组装过程，我们使用重组痘苗病毒-T7 RNA聚合酶系统来促进Gag蛋白表达、粒子组装和细胞外释放。将HIV基因置于噬菌体T7启动子的控制之下，并转染到表达T7 RNA聚合酶的HeLa细胞中。蛋白质免疫印迹分析在分别由gag和gag-pol表达产生的纯化粒子中检测到了p55gag及其裂解产物p39和p27。用HIV-2 gag或gag-pol进行vpx的反式表达产生了类似病毒的粒子，其中所含Vpx的量与从高效感染的T细胞产生的HIV-2病毒中检测到的量相似。使用HIV-2 Gag的C末端缺失和截短突变体，我们绘制了Vpx包装决定簇的p15编码序列。该分析揭示了在将Vpx掺入病毒粒子中所需的核衣壳蛋白（NC）下游的一个区域（第439至497位氨基酸）。构建HIV-1/HIV-2 gag嵌合体以进一步表征将Vpx掺入病毒粒子的要求。由HIV-1 p17和p24在C末端与HIV-2 p15框内融合组成的嵌合HIV-1/HIV-2 Gag粒子有效地掺入了Vpx，而由HIV-2 p17和p27在C末端与HIV-1 p15框内融合组成的嵌合HIV-2/HIV-1 Gag粒子则不能。与HIV-1 p17和p24编码区融合的包含第439至497位氨基酸的HIV-2的68个氨基酸序列的表达也产生了能够以与全长HIV-2 Gag相似的量包装Vpx的类似病毒的粒子。蔗糖梯度分析证实了Vpx和Gag蛋白与粒子的结合。这些结果表明，HIV-2 Gag前体（p55）调节Vpx掺入病毒粒子，并表明包装信号位于第439至497位氨基酸残基内。
