Lim David, Orlova Marianna, Goff Stephen P
Integrated Program in Cellular, Molecular and Biophysical Studies, Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Virol. 2002 Aug;76(16):8360-73. doi: 10.1128/jvi.76.16.8360-8373.2002.
Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged alpha-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (DeltaC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5' and 3' viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5' end.
莫洛尼鼠白血病病毒（Mo-MLV）逆转录酶（RT）的核糖核酸酶H（RNase H）结构域和大肠杆菌RNase H都拥有一个带正电荷的α螺旋（C螺旋）和一个环，而人类免疫缺陷病毒（HIV）RT或禽肉瘤病毒RT的RNase H结构域中不存在这些结构。虽然缺乏C螺旋的突变型Mo-MLV RT（ΔC RT）在同聚物底物上保留了DNA聚合酶活性和部分RNase H活性，但该病毒RNA基因组在体内的逆转录存在缺陷。为了确定C螺旋的基本特征，构建了一组Mo-MLV RT突变体。对这些突变病毒的分析揭示了H594、I597、R601和G602残基的重要性。检测了这些突变体在急性感染后合成病毒DNA以及形成正确的5'和3'病毒DNA末端的能力。在体外测试了突变RT的外源RT活性、内源性RT反应中的负链强终止DNA合成、非特异性RNase H活性，最后测试了在多聚嘌呤序列-U3连接处的正确切割。R601A突变体在体内和体外都是缺陷最严重的突变体，其RNase H活性极低。H594A、I597A和G602A突变体的RNase H活性及其病毒复制速率都显著降低。许多突变体形成了不正确的病毒DNA末端，并且在体外对多聚嘌呤序列-U3的识别和切割效率较低。数据表明，C螺旋对整体RNase H切割活性起着关键作用。数据还表明，C螺旋可能在多聚嘌呤序列识别和正链DNA 5'端的正确形成中发挥重要作用。
