Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3).
We have examined the specificity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase-associated RNase H in removing the tRNA(Lys3) (-)-strand primer in vitro using a model substrate. This substrate represents an intermediate in the reverse transcription process where the tRNA(Lys3) primer has not yet been removed after (+)-strand strong stop DNA synthesis. The substrate consists of an RNA oligonucleotide corresponding to the 3'-terminal 17 nucleotides of the tRNA(Lys3) linked to U5 DNA and annealed to single-stranded DNA containing the U5 and the primer-binding site. Upon incubation with HIV-1 reverse transcriptase p66/p51 heterodimer, the minus-strand DNA product resulting from RNase H cleavage retained the 3'-rA from the model tRNA primer. Changing the 3'-terminal AMP of the model tRNA primer from rA to dA did not alter the RNase H cleavage site. Further, the retention of AMP was not dependent on recognition of adjacent U5 sequences or the CCA terminus of the model tRNA(Lys3). The synthetic RNA primer was released as an intact species by a single endonucleolytic cleavage 5' of the rA. The cleavage patterns of Moloney murine leukemia virus and avian myoblastosis virus RNase H activities on the HIV-1 model substrate were more heterogeneous compared to HIV-1 RNase H. This specificity of HIV-1 RNase H would result in linear DNA molecules with a single rA at the U5 terminus and would provide two bases adjacent to the conserved CA dinucleotide to be cleaved away during the integration process.
Smith
J S
JS
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Roth
M J
MJ
D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004279:DNA, Viral / Q000096:biosynthesis; D016325:HIV Long Terminal Repeat; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D004279:DNA, Viral; D009841:Oligonucleotides; D012357:RNA, Transfer, Lys; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H