Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

• Published in: Biochemistry (Easton) 1996-02, Vol.35 (6), p.1911-1917
• Main Authors: Fan, Naisheng, Rank, Kenneth B, Poppe, Susan M, Tarpley, W. Gary, Sharma, Satish K
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Aspartic Acid Endopeptidases - metabolism; Base Sequence; Cations, Divalent - pharmacology; Dideoxynucleotides; DNA Primers - genetics; DNA, Viral - genetics; Genes, gag; HIV Protease; HIV Reverse Transcriptase; HIV-2 - enzymology; HIV-2 - genetics; human immunodeficiency virus 2; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Poly A - metabolism; Poly T - metabolism; Protein Conformation; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Inhibitors - pharmacology; Ribonuclease H - metabolism; RNA-Directed DNA Polymerase - chemistry; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Substrate Specificity; Thymine Nucleotides - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9516440

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 (K i[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.