Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R+74V

Abstract

While HIV-1 reverse transcriptase (RT) mutations of M to V at position 184 are commonly observed in the clinic, the double mutation of 65R+74V is rarely seen. It has been demonstrated that rapid R-->K reversion occurs at RT codon 65 during replication of HIV-1 in human peripheral blood mononuclear cells containing 65R+74V mutations and that processivity of the RT is reduced relative to wild type. However, clinical studies show that M184V can be detected after several months of therapy interruption, suggesting more effective processivity. Herein, the in vitro RT processivity of genetically engineered M184V and double mutant 65R+74V was compared. Virion-associated RTs of WT pNL4-3, K65R, L74V, M184V and 65R+74V were used to perform RT processivity assays in the presence of trap, poly(rC)-oligo(dG). Both RTs with 184V and 65R+74V mutations exhibited similar processivity when compared with each other and a significantly decreased processivity as compared to WT RT. Both mutant RTs synthesized shorter cDNA molecules (37-42 nt) relative to WT RT, which made longer (65-70 nt) cDNA molecules. Since these surprising biochemical results cannot explain the clinical phenotype, a hypothesis is presented to explain the discrepancy and suggest new approaches for future testing.

Fig. 1. Reduced processivity of RTs containing mutations 65R, 74V, 65R+74V and 184V

The purified cDNA products were run on a 6% polyacrylamide vertical gel electrophoresis. The cDNA bands were visualized by autoradiography. The image contrast for whole gel was adjusted to visualize the relative cDNA densities among different lanes (A). Lanes: 1, WT (no trap); 2, 3, WT; 4–7, 65R; 8, 65R (no trap); 9, 10, 74V; 11, 12, 65R+74V; 13, 14, 184V. Relative amounts of cDNA synthesized with WT and entire mutant panel were quantified by Intelligent Quantifier (Bio Image Systems, Inc.) software. The groups of 6 cDNA bands from bottom to the top were scanned for each lane and plotted against cDNA length (Figure 1B and 1C).

Fig. 1. Reduced processivity of RTs containing mutations 65R, 74V, 65R+74V and 184V

The purified cDNA products were run on a 6% polyacrylamide vertical gel electrophoresis. The cDNA bands were visualized by autoradiography. The image contrast for whole gel was adjusted to visualize the relative cDNA densities among different lanes (A). Lanes: 1, WT (no trap); 2, 3, WT; 4–7, 65R; 8, 65R (no trap); 9, 10, 74V; 11, 12, 65R+74V; 13, 14, 184V. Relative amounts of cDNA synthesized with WT and entire mutant panel were quantified by Intelligent Quantifier (Bio Image Systems, Inc.) software. The groups of 6 cDNA bands from bottom to the top were scanned for each lane and plotted against cDNA length (Figure 1B and 1C).

Fig. 1. Reduced processivity of RTs containing mutations 65R, 74V, 65R+74V and 184V

The purified cDNA products were run on a 6% polyacrylamide vertical gel electrophoresis. The cDNA bands were visualized by autoradiography. The image contrast for whole gel was adjusted to visualize the relative cDNA densities among different lanes (A). Lanes: 1, WT (no trap); 2, 3, WT; 4–7, 65R; 8, 65R (no trap); 9, 10, 74V; 11, 12, 65R+74V; 13, 14, 184V. Relative amounts of cDNA synthesized with WT and entire mutant panel were quantified by Intelligent Quantifier (Bio Image Systems, Inc.) software. The groups of 6 cDNA bands from bottom to the top were scanned for each lane and plotted against cDNA length (Figure 1B and 1C).

Fig. 2. Structural interactions of activated nucleotide

Positions of residues 65 and 74 as found in a covalently trapped catalytic complex of HIV-1 reverse transcriptase, 1rtd.pdb (12), showing potential hydrogen bonds in magenta. Van der Waal surface around Leu74 shows simultaneous contact with aligning template strand residue and Phe 61 on adjacent strand β3. Strand β3 orientation is defined by position of Phe 61 and Lys 65.

Fig. 3. Flexibility of β3-β4 fingers

Orientation of β3 and β4 varies significantly between a) the catalytic complex, also shown in Figure 2 (12) and b) post incorporation complex derived from 1nq6.pdb by Sarafianos et al. (20). Torsional flexing of finger is centered at the interface of residues 61 and 74. Mutation of L74 to V would be predicted to change mobility vector of β3 and directly impact positioning of residue 65 in the catalytic complex. Reduced rotational freedom of Arg may be responsible for its lessened activity relative to Lys at that position. Residue Met184 is shown positioned beneath the primer nucleotide.