Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H.
We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.
Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032.
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Telesnitsky
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Blain
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000502:physiology*; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics*; D014178:Translocation, Genetic; D014771:Virion / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H