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

Abstract

Human immunodeficiency virus (HIV)-Lys -primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

“…The increased sensitivity of the mutant enzymes to the denaturing (inactivating) effect of urea is due to an easier conversion of the mutant (heterodimeric) enzymes to their monomeric state, rather than to easier unfolding of their secondary structure by urea. Indeed, SluisCremer et al [24] and Menéndez-Arias et al [12] have shown that urea at concentrations less than 2 M (as is the case in our experiments) results in a decreased catalytic activity of the RT enzyme due to dissociation of the heterodimer to monomers, and not to destruction (unfolding) of the secondary structure of the enzyme (which occurs at higher urea concentrations). Our observations are also in agreement with earlier published results that focus on the importance of an intact b7-b8 loop in the p51 subunit to retain efficient catalytic activity of the enzyme [10,11].…”

“…For determination of the 50% inhibitory concentration (IC 50 ) of the test compounds against HIV-1 RT, the RNA-dependent DNA polymerase assay was performed as follows: the reaction mixture (50 ll) contained 50 mM Tris-HCl (pH 7.8), 0.06% Triton X-100, 5 mM DTT, 0.3 mM glutathione, 150 mM KCl, 5 mM MgCl 2 , 1.25 mg/ml BSA, 0.5 mM EDTA, 0.1 mM template/primer poly(rC)AEoligo(dG) [12][13][14][15][16][17][18] (Amersham Biosciences), a fixed concentration of the labeled substrate [8-3 H]dGTP (1.6 lM, 1 lCi; specific activity, 12.6 Ci/mmol; Amersham Biosciences), 5 ll of inhibitor solution [containing various concentrations (10-fold dilutions) of the compounds], and 5 ll of the RT preparations. In the case, where d4TTP (or PFA) were evaluated for their inhibitory activity, 0.15 mM poly(rA)AEoligo(dT) [12][13][14][15][16][17][18] was used as the template/primer, and 1.6 lM [ 3 H]dTTP as the radiolabeled substrate.…”

“…In the case, where d4TTP (or PFA) were evaluated for their inhibitory activity, 0.15 mM poly(rA)AEoligo(dT) [12][13][14][15][16][17][18] was used as the template/primer, and 1.6 lM [ 3 H]dTTP as the radiolabeled substrate. The reaction mixtures were incubated at 37°C for 30 min, at which time 200 ll of yeast RNA (2 mg/ml) and 1 ml of trichloroacetic acid (TCA) (5% in 20 mM Na 4 P 2 O 7 ) were added.…”

“…The tip of this loop also contributes to the formation of the ÔfloorÕ of the NNRTI binding pocket. The amino acids of this loop in p51 that make contact with the p66 subunit (including P95) are I135, N136, N137, E138 and P140 [12]. The importance of this loop for structural support and polymerase function of the heterodimeric RT was studied by either deletion, or by alanine substitution of amino acids N136, N137, E138 and T139 [10].…”

“…In addition, we replaced two prolines by an alanine (P95 in p66 and P140 in p51) in two separate RT enzymes. These amino acids (i) are highly conserved among all lentiviruses, (ii) make up an important part of the dimerization interface [10][11][12], (iii) contribute to the formation of the bottom of the NNRTI pocket [11,12], and (iv) have not yet been reported as susceptible to mutation under the selective pressure of NNRTIs (or any other drugs) in cell culture. Given these important characteristics, we investigated whether these amino acids may act as potential targets for the design of novel NNRTI or p66/p51 dimerization inhibitors.…”