Kappes J C, Parkin J S, Conway J A, Kim J, Brouillette C G, Shaw G M, Hahn B H
Department of Medicine, University of Alabama, Birmingham 35294.
Virology. 1993 Mar;193(1):222-33. doi: 10.1006/viro.1993.1118.
Viral protein X (vpx) is a virion-associated HIV-2/SIV accessory protein that enhances viral infectivity and replication in natural target cells. To investigate whether other viral components affect its biosynthesis, subcellular localization, and virion incorporation, we expressed HIV-2 vpx in a mammalian cell system and examined its transport and packaging requirements using an in trans complementation assay. The complete vpx coding region of HIV-2ST was placed under the control of a high-efficiency promoter system (SR alpha) which contained both an SV40 promoter/enhancer region and R/U5 elements of the HTLV-1 LTR. Following transfection of Cos-1 cells, this construct (pSR alpha-vpx) facilitated high level expression of vpx, as demonstrated by Western blot analysis of transfected cell lysates. Moreover, indirect immunofluorescence analysis revealed an intense vpx staining pattern distributed evenly throughout the cytoplasm of transfected cells. This distribution differed markedly from cells expressing wild-type HIV-2 in which vpx localized to the inner surface of the plasma membrane. To determine whether other HIV components were required for this surface localization, we expressed vpx in the context of replication competent HIV-1 and HIV-2 proviruses. Following cotransfection with a vpx-deficient HIV-2 provirus (pXM7), eukaryotically expressed vpx targeted to the plasma membrane and colocalized with HIV-2 p27 gag in a pattern indistinguishable from wild-type HIV-2. Moreover, progeny virions from cotransfected Cos-1 cells contained wild-type amounts of vpx protein, demonstrating that vpx could be efficiently packaged in trans. Under the same experimental conditions, cotransfection of vpx with wild-type HIV-1 (pHXB2) and with vpr-deficient HIV-1 (pR2) failed to result in detectable cell surface targeting or virion incorporation of vpx despite its high level cellular expression. These results demonstrate that efficient intracellular transport and packaging of vpx require interaction with other type-specific virus components.
病毒蛋白X（vpx）是一种与病毒粒子相关的HIV-2/SIV辅助蛋白，可增强病毒在天然靶细胞中的感染性和复制能力。为了研究其他病毒成分是否会影响其生物合成、亚细胞定位和病毒粒子整合，我们在哺乳动物细胞系统中表达了HIV-2 vpx，并使用反式互补试验检测其运输和包装需求。HIV-2ST的完整vpx编码区置于高效启动子系统（SRα）的控制之下，该系统包含SV40启动子/增强子区域以及HTLV-1 LTR的R/U5元件。转染Cos-1细胞后，通过对转染细胞裂解物进行蛋白质免疫印迹分析表明，该构建体（pSRα-vpx）促进了vpx的高水平表达。此外，间接免疫荧光分析显示，vpx染色强烈，均匀分布在转染细胞的整个细胞质中。这种分布与表达野生型HIV-2的细胞明显不同，在野生型HIV-2细胞中，vpx定位于质膜内表面。为了确定这种表面定位是否需要其他HIV成分，我们在具有复制能力的HIV-1和HIV-2前病毒的背景下表达vpx。在用缺乏vpx的HIV-2前病毒（pXM7）共转染后，真核表达的vpx靶向质膜，并与HIV-2 p27 gag共定位，其模式与野生型HIV-2无法区分。此外，来自共转染Cos-1细胞的子代病毒粒子含有野生型数量的vpx蛋白，表明vpx可以有效地反式包装。在相同的实验条件下，尽管vpx在细胞中高水平表达，但将vpx与野生型HIV-1（pHXB2）以及缺乏vpr的HIV-1（pR2）共转染，均未能导致可检测到的vpx细胞表面靶向或病毒粒子整合。这些结果表明，vpx的有效细胞内运输和包装需要与其他类型特异性病毒成分相互作用。
