Contribution of the P51 subunit of HIV-1 reverse transcriptase to enzyme processivity

Functional Characterization of Chimeric Reverse Transcriptases with Polypeptide Subunits of Highly Divergent HIV-1 Group M and O Strains

“…Analysis of RT Activity in Crude Bacterial Lysates-DNA polymerization assays were carried out by mixing 20 l of the obtained lysate and 80 l of 62.5 mM Tris-HCl (pH 8.0) containing 25 mM NaCl, 12.5 mM MgCl 2 , 2 mM DTT, 0.5 g/ml poly(rC)/oligo(dG) [12][13][14][15][16][17][18] , and 10 M [␣-…”

“…RT concentrations were determined using the Bio-Rad protein assay. The specific activity of the purified enzyme was estimated in DNA polymerase assays carried out in 50 l of 50 mM Tris-HCl (pH 8.0) containing 20 mM NaCl, 10 mM MgCl 2 , 8 mM DTT, 5 M dTTP (3-5 Ci/ml [ 3 H]dTTP), and 0.5 M poly(rA)/oligo(dT) [12][13][14][15][16][17][18] (concentration expressed as 3Ј-hydroxyl primer termini) as previously described (22,27). One activity unit is the amount of enzyme that incorporates 1 nmol of [ 3 H]dTMP into acid-insoluble products in 10 min at 37°C.…”

“…RT inhibition assays were carried out with poly(rA)/oligo(dT) [12][13][14][15][16][17][18] or D2-47/PG5-25 under the conditions described for kinetic studies. In assays using poly(rA)/oligo(dT) [12][13][14][15][16][17][18] , incorporation of radioactive dTMP was measured in the presence of increasing amounts of nevirapine. In singlenucleotide extension assays, the RT was preincubated with the inhibitor at 37°C for 10 min before the addition of the D2-47/PG5-25 template-primer.…”

“…1). RNA-dependent DNA polymerase activity was determined using poly(rC)/oligo(dG) [12][13][14][15][16][17][18] and [␣-32 P]dGTP as substrates. All five RTs from this ESP2 sample had nearly identical amino acid sequence in the polymerase domain, and we selected clone 46 as the group O RT prototype in the biochemical studies described in this work.…”

“…In addition to providing structural support to the p66 subunit, p51 appears to be required for loading of the p66 subunit onto the templateprimer (7) and for maintaining the proper p66 conformation during initiation of reverse transcription from primer tRNA 3 Lys (8 -11). It has also been reported that the p66/p51 heterodimer as compared with the p66/p66 homodimer displays higher processivity and strand displacement activity during DNAdependent DNA polymerase activity (12,13). Thus, the process of dimerization and subsequent maturation into the p66/p51 heterodimer is essential for a fully functional RT and constitutes a target for therapeutic intervention.…”