Luban J, Alin K B, Bossolt K L, Humaran T, Goff S P
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Virol. 1992 Aug;66(8):5157-60. doi: 10.1128/JVI.66.8.5157-5160.1992.
We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and O. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.
我们建立了一种用于逆转录病毒gag多聚蛋白多聚化的遗传分析方法。该分析方法基于用于研究蛋白质-蛋白质相互作用的GAL4双杂交系统（S. 菲尔兹和O. 宋，《自然》（伦敦）340:245 - 246，1989年）。在我们最初的实验中，我们构建了酿酒酵母质粒，其分别表达与人类免疫缺陷病毒1型（HIV - 1）gag多聚蛋白Pr55gag融合的GAL4 DNA结合结构域和GAL4激活结构域。这两种杂交蛋白在酿酒酵母中的共表达导致GAL4结构域的结合以及整合的GAL4反应性lacZ指示基因的有效激活。用编码GAL4 - 莫洛尼鼠白血病病毒（M - MuLV）gag多聚蛋白杂交蛋白的质粒也获得了类似结果。相反，异源的GAL4 - HIV - 1 gag和GAL4 - M - MuLV gag融合蛋白无法相互作用以诱导lacZ表达。结果表明，该酵母系统为病毒体组装过程中发生的逆转录病毒gag蛋白相互作用提供了一种快速且特异的分析方法。
