Hansen J, Schulze T, Mellert W, Moelling K
Max-Planck-Institut für Molekulare Genetik, Abt. Schuster, Berlin, FRG.
EMBO J. 1988 Jan;7(1):239-43. doi: 10.1002/j.1460-2075.1988.tb02805.x.
Human immune deficiency virus (HIV) replicates by conversion of the RNA genome into the double-stranded DNA provirus. The reverse transcriptase is not the only enzymatic function crucial in DNA-provirus synthesis. A viral-coded RNase H activity which specifically degrades RNA in RNA-DNA hybrids has been shown to be essential as well. Here we demonstrate that the HIV-reverse transcriptase which consists of a two-polypeptide complex, p66 and p51, copurifies with an RNase H activity which exhibits properties of a processive exonuclease. Only the p66 molecule, not p51, is active as polymerase as evidenced by activated gel analysis. p66 exhibits RNase H activity when precipitated as immune complex by a monoclonal antibody raised against a bacterially expressed carboxy-terminal portion of p66. The monoclonal antibody which does not interfere with enzyme activity also precipitates a second population of molecules with RNase H activity which is of low mol. wt, p15. This RNase H appears therefore to be derived from the carboxy terminus of p66 during processing to the p51 polypeptide. It exhibits low template-binding ability and is of a non-processing mode of action which may be due to the absence of the reverse transcriptase domain. These results lend experimental support to the hypothesis that the RNase H gene maps at the carboxy terminus of the reverse transcriptase. Since both RNase H populations are virus-coded they may be essential for retrovirus replication in general and useful targets for chemotherapeutic agents.
人类免疫缺陷病毒（HIV）通过将RNA基因组转化为双链DNA原病毒进行复制。逆转录酶并非DNA原病毒合成中唯一至关重要的酶促功能。一种病毒编码的核糖核酸酶H（RNase H）活性，其可特异性降解RNA-DNA杂交体中的RNA，也已被证明是必不可少的。在此我们证明，由p66和p51两种多肽复合物组成的HIV逆转录酶与一种具有持续性核酸外切酶特性的RNase H活性共同纯化。如活性凝胶分析所示，只有p66分子具有聚合酶活性，而p51没有。当用针对细菌表达的p66羧基末端部分产生的单克隆抗体沉淀为免疫复合物时，p66表现出RNase H活性。该单克隆抗体不干扰酶活性，同时也沉淀出第二种具有RNase H活性的低分子量分子群体，即p15。因此，这种RNase H似乎是在加工成p51多肽的过程中从p66的羧基末端衍生而来。它表现出低模板结合能力，且作用模式为非持续性，这可能是由于缺乏逆转录酶结构域所致。这些结果为RNase H基因定位于逆转录酶羧基末端这一假说提供了实验支持。由于两种RNase H群体均由病毒编码，它们可能对一般逆转录病毒复制至关重要，并且是化疗药物的有用靶点。
