Schröder H C, Ugarkovic D, Merz H, Kuchino Y, Okamoto T, Müller W E
Institut für Physiologische Chemie, Universität, Mainz, West Germany.
FASEB J. 1990 Oct;4(13):3124-30. doi: 10.1096/fasebj.4.13.1698680.
An expression vector (pU3R-III/2-5AS) of human 2',5'-oligoadenylate (2-5A) synthetase was constructed in which a cDNA encoding an active form of the enzyme was located 3' to a 3'-long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). The LTR-directed expression of this hybrid DNA could be activated in trans by the HIV tat gene product. This vector was used for transfection of HeLa-T4+ cells, which are permissive to HIV infection, as well as of normal HeLa cells. HIV replication after infection of the CD4-receptor-bearing HeLa-T4+ cells with HIV-1 was found to be strongly reduced when drug-selected cells cotransfected with pU3R-III/2-5AS and a hygromycin B resistance gene containing plasmid were used. In nontransfected cultures or after transfection with the selectable marker plasmid only, about 60% p17- and p24-positive cells were found 5 days after infection. However, after stable transfection with pU3R-III/2-5AS the number of positive cells was decreased to about 2%. The reverse transcriptase (RT) activity, as a measure for virus production, was markedly decreased in the culture fluids of pU3R-III/2-5AS transfected cells compared with the mock-transfected controls. In parallel experiments it was established that Tat-mediated trans-activation of HIV-1 LTR-directed 2-5A synthetase expression resulted in a great increase in both 2-5A synthetase mRNA level and activity as well as in cellular 2-5A content. Similar results were found in HeLa-T4+ cells and in HeLa cells (without CD4 receptor) cotransfected with pU3R-III/2-5AS and a tat gene containing plasmid or after introduction of purified Tat protein into the pU3R-III/2-5AS transfected cells by using a modified scrape loading procedure. These results indicate that HIV-trans-activated 2-5A synthetase can selectively inhibit HIV replication in vitro, and might be a promising gene therapeutical approach.
构建了人2′,5′-寡腺苷酸（2-5A）合成酶的表达载体（pU3R-III/2-5AS），其中编码该酶活性形式的cDNA位于人免疫缺陷病毒1型（HIV-1）的3′长末端重复序列（LTR）的3′端。这种杂交DNA的LTR指导的表达可被HIV tat基因产物反式激活。该载体用于转染对HIV感染敏感的HeLa-T4+细胞以及正常HeLa细胞。当使用与pU3R-III/2-5AS和含潮霉素B抗性基因的质粒共转染的药物筛选细胞时，发现用HIV-1感染携带CD4受体的HeLa-T4+细胞后，HIV复制被强烈抑制。在未转染的培养物中或仅用选择标记质粒转染后，感染后5天发现约60%的p17和p24阳性细胞。然而，用pU3R-III/2-5AS稳定转染后阳性细胞数量降至约2%。与mock转染对照相比，pU3R-III/2-5AS转染细胞的培养液中作为病毒产生指标的逆转录酶（RT）活性明显降低。在平行实验中确定，Tat介导的HIV-1 LTR指导的2-5A合成酶表达的反式激活导致2-5A合成酶mRNA水平和活性以及细胞2-5A含量大幅增加。在用pU3R-III/2-5AS和含tat基因的质粒共转染的HeLa-T4+细胞以及HeLa细胞（无CD4受体）中，或通过改良的刮擦加载程序将纯化的Tat蛋白引入pU3R-III/2-5AS转染细胞后，发现了类似结果。这些结果表明，HIV反式激活的2-5A合成酶可在体外选择性抑制HIV复制，可能是一种有前景的基因治疗方法。
