Hafkemeyer P, Ferrari E, Brecher J, Hübscher U
Department of Pharmacology and Biochemistry, University of Zürich-Irchel, Switzerland.
Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5262-66. doi: 10.1073/pnas.88.12.5262.
The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.
人类免疫缺陷病毒1型的逆转录酶是一种异源二聚体蛋白，由质量分别为66 kDa和51 kDa的两种多肽组成，并且具有作为第二种酶活性的核糖核酸酶H活性。66 kDa的多肽可被病毒编码的蛋白酶切割，产生51 kDa和15 kDa的多肽。后者已被鉴定具有核糖核酸酶H活性[汉森，J.，舒尔茨，T.，梅勒特，W.和默林，K.（1988年）《欧洲分子生物学组织杂志》7，239 - 243]。我们通过一种包括在DEAE - 纤维素、磷酸纤维素和肝素 - 琼脂糖上进行层析的方法，从含有表达载体pJS 3.7的大肠杆菌中同时纯化了含有66/51 kDa多肽的异源二聚体逆转录酶/核糖核酸酶H和15 kDa核糖核酸酶H。在磷酸纤维素上分离出两个核糖核酸酶H和逆转录酶峰，一个与异源二聚体蛋白一致，另一个与15 kDa蛋白一致。基于以下发现，似乎15 kDa的多肽同时具有核糖核酸酶H和逆转录酶活性：（i）它与两种活性一起共纯化；（ii）在SDS/聚丙烯酰胺凝胶电泳后的原位测定中，它作为逆转录酶发挥作用；（iii）针对66 kDa多肽产生的多克隆抗体在免疫印迹中仅与15 kDa多肽反应，而在15 kDa多肽制剂中未见到51 - 66 kDa范围内的免疫反应条带；（iv）只有当使用针对p66羧基末端的特异性抗体时，p15和p66/51逆转录酶才能以酶活性形式被定量沉淀；（v）p15蛋白具有逆转录酶的真正特性，并且能够酶促合成高分子量、耐碱的产物。在测试的各种模板/引物系统上，这两种逆转录酶似乎具有不同的行为。可以想象，人类免疫缺陷病毒1型逆转录酶的不同形式可能在（+）链和（ - ）链复制的各个步骤中被使用。
