Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase.
Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.
Fan
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Upjohn Laboratories, Kalamazoo, Michigan 49001, USA.
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D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D054306:Dideoxynucleotides; D015750:Genes, gag; D016333:HIV Protease; D054303:HIV Reverse Transcriptase; D015498:HIV-2 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology
D002413:Cations, Divalent; D017931:DNA Primers; D004279:DNA, Viral; D054306:Dideoxynucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D011061:Poly A; D011071:Poly T; C008950:poly A-T; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C065184:p16 protease, Human immunodeficiency virus 2; C017216:2',3'-dideoxythymidine triphosphate