Mutations of the RNase H C helix of the Moloney murine leukemia virus reverse transcriptase reveal defects in polypurine tract recognition.
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.
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.
David
D
Lim
Marianna
M
Orlova
Stephen P
SP
Goff
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020079:Terminal Repeat Sequences; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H