Julias John G, McWilliams Mary Jane, Sarafianos Stefan G, Arnold Edward, Hughes Stephen H
HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, P.O. Box B, Frederick, MD 21702-1201, USA.
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9515-20. doi: 10.1073/pnas.142123199. Epub 2002 Jul 1.
Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.
逆转录病毒逆转录酶含有一种DNA聚合酶活性，可复制RNA或DNA模板，以及一种核糖核酸酶H（RNase H）活性，在逆转录过程中可降解病毒RNA基因组。RNase H进行特异性和非特异性切割；特异性切割用于生成和去除用于正链DNA合成的多聚嘌呤序列引物，并去除用于负链DNA合成的tRNA引物。我们在基于HIV-1的载体中产生突变，以改变RNase H结构域中与RNA和DNA链接触的氨基酸。其中一些突变影响了DNA合成的起始，这表明在病毒DNA合成过程中，HIV-1逆转录的聚合酶和RNase H活性相互依赖。HIV-1基因组线性DNA形式的末端由去除正链和负链引物的特异性RNase H切割所定义；这些末端可以连接形成两个长末端重复序列环。对两个长末端重复序列环连接处的分析表明，RNase H结构域中的突变会影响RNase H切割的特异性。
