Kohl N E, Emini E A, Schleif W A, Davis L J, Heimbach J C, Dixon R A, Scolnick E M, Sigal I S
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686-90. doi: 10.1073/pnas.85.13.4686.
Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.
逆转录病毒蛋白作为多蛋白前体被合成，这些前体经过蛋白水解切割以产生成熟的病毒蛋白。通过在蛋白酶基因中使用定点突变来研究人类免疫缺陷病毒（HIV）蛋白酶在病毒复制周期中的作用。HIV蛋白酶基因产物在大肠杆菌中表达，并观察到在体外将HIV gag p55切割为gag p24和gag p17。将该蛋白的天冬氨酸残基25（Asp-25）替换为天冬酰胺残基不影响该蛋白的表达，但消除了针对HIV gag p55的可检测体外蛋白水解活性。构建了一种突变型HIV前病毒，其蛋白酶基因内含有Asn-25突变。用Asn-25突变型前病毒DNA转染的SW480人结肠癌细胞产生的病毒粒子含有gag p55但不含有gag p24，而用野生型DNA转染的细胞产生的病毒粒子同时含有gag p55和gag p24。突变型病毒粒子不能感染MT-4淋巴细胞。相反，这些细胞对野生型病毒粒子的感染高度敏感。这些结果表明，HIV蛋白酶是一种必需的病毒酶，因此是抗HIV药物的一个有吸引力的靶点。
