Bachand F, Yao X J, Hrimech M, Rougeau N, Cohen E A
Laboratoire de rétrovirologie humaine, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
J Biol Chem. 1999 Mar 26;274(13):9083-91. doi: 10.1074/jbc.274.13.9083.
The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.
96个氨基酸的Vpr蛋白是1型人类免疫缺陷病毒（HIV-1）主要的病毒体相关辅助蛋白。由于Vpr不是p55 Gag多聚蛋白前体（Pr55(gag)）的一部分，其整合需要一个锚定物才能与正在组装的病毒颗粒结合。虽然目前分子机制尚不清楚，但Pr55(gag)中对应于p6结构域的C末端区域似乎构成了Vpr蛋白整合所必需的这样一个锚定物。为了阐明Vpr辅助蛋白转整合到子代病毒体颗粒中的机制，我们使用酵母双杂交系统和麦芽糖结合蛋白下拉试验检测了HIV-1 Vpr是否与Pr55(gag)相互作用。本研究提供了遗传和生化证据，表明Pr55(gag)可与Vpr蛋白发生物理相互作用。此外，影响p6(gag)保守的L-X-S-L-F-G基序完整性的点突变完全消除了Vpr与Pr55(gag)之间的相互作用，结果阻止了Vpr整合到病毒体中。与其他研究不同，影响NCp7锌指结构完整性的突变既不损害Vpr整合到病毒体中，也不影响Vpr与Pr55(gag)之间的结合。相反，Vpr中的氨基酸取代表明完整的N末端α-螺旋结构对于Vpr-Pr55(gag)相互作用至关重要。Vpr和Pr55(gag)在体外表现出强烈的相互作用，高达900 mM的盐浓度也不能破坏这种相互作用。最后，使用抗Vpr血清可有效竞争这种相互作用。总之，这些结果强烈表明，Vpr转整合到HIV-1颗粒中需要其N末端区域与p6(gag)的C末端区域之间的直接相互作用。Pr55(gag)-Vpr相互作用检测方法的开发可能有助于筛选能够阻止Vpr辅助蛋白整合到HIV-1病毒体中从而抑制其早期功能的分子。
