Peng C, Chang N T, Chang T W
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
J Virol. 1991 May;65(5):2751-6. doi: 10.1128/JVI.65.5.2751-2756.1991.
Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
构建了三种1型人类免疫缺陷病毒（HIV-1）突变体，其蛋白酶基因发生了突变：AH2-pSVL，有一个同相位缺失；BH27-pSVL，有一个异相位缺失，在缺失位点后立即产生一个终止密码子；以及CA-pSVL，有一个点突变，在假定的蛋白酶活性位点产生天冬氨酸到丙氨酸的替换。野生型HXB2-pSVL和突变的病毒基因组用于转染COS-M6细胞并产生病毒粒子。用针对p24的单克隆抗体（MAb）进行免疫印迹分析表明，所有三种突变体都含有gag前体Pr56gag，其中AH2和CA表达一条约160 kDa的额外条带。用针对HIV-1逆转录酶（RT）的单克隆抗体进行的类似分析还显示，AH2和CA病毒粒子中有一个160 kDa的蛋白，而HXB2病毒粒子中有两个成熟的p66和p51 RT亚基。此外，HXB2、AH2和CA病毒粒子表现出RT活性，而BH27病毒粒子则没有。160 kDa条带中的同一蛋白似乎同时具有p24和RT成分，因为针对p24的单克隆抗体能够免疫吸附RT抗原和酶活性。这些结果表明，在哺乳动物细胞中产生的HIV-1 gag-pol融合蛋白具有显著的RT活性。
