Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function.
The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.
Tanese
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Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Telesnitsky
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Goff
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D000818:Animals; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D016296:Mutagenesis; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics; D014771:Virion / Q000201:enzymology / Q000235:genetics; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H