Luban J, Goff S P
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Virol. 1991 Jun;65(6):3203-12. doi: 10.1128/JVI.65.6.3203-3212.1991.
We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.
我们在T7噬菌体基因10启动子的控制下，在细菌中表达了人类免疫缺陷病毒1型（HIV-1）的gag多聚蛋白（Pr55gag）。当编码病毒蛋白酶的基因以顺式包含在-1读框中时，会产生预期的蛋白水解切割产物MA和CA。蛋白酶编码序列的破坏会阻止蛋白水解加工，并产生全长多聚蛋白。通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）从细菌蛋白中分离出Pr55gag，并固定在硝酸纤维素膜上，它能结合含有HIV-1基因组5'端序列的RNA。这种结合在很宽的pH和温度范围内都能耐受，但对盐有明显的偏好。我们确定了一些条件，这些条件可以防止RNA与细菌蛋白的非特异性结合，但仍允许其与Pr55gag结合。在这些条件下，缺乏HIV-1序列的无关RNA探针与Pr55gag的结合效率较低。通过对带有缺失突变的HIV-1 RNA探针与Pr55gag结合的定量分析表明，HIV-1 gag基因内有两个区域可独立促进RNA与Pr55gag的结合。
