Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase.
To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild-type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently.
Blain
S W
SW
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Goff
S P
SP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D004279:DNA, Viral / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000502:physiology*; D016914:Ribonuclease H / Q000502:physiology*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000032:analysis; D014771:Virion / Q000737:chemistry; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H