Mechanisms by Which the G333D Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Facilitates Dual Resistance to Zidovudine and Lamivudine

Abstract

Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.

Nucleoside reverse transcriptase (RT) inhibitors (NRTI), such as zidovudine (AZT) and lamivudine (3TC), inhibit the replication of human immunodeficiency type 1 (HIV-1). NRTI are deoxynucleoside triphosphate (dNTP) analogs that lack a 3′-hydroxyl group. Once they are incorporated into the nascent viral DNA, in reactions catalyzed by HIV-1 RT, DNA synthesis cannot proceed unless the incorporated NRTI-monophosphate is excised (33). Although combination therapies that contain two or more NRTI have profoundly reduced morbidity and mortality from HIV-1 infection, their long-term efficacy is limited by the selection of drug-resistant variants of HIV-1.

HIV-1 RT is a heterodimer composed of a 66-kDa subunit (p66) and a p66-derived 51-kDa subunit (p51) (22). The catalytically active p66 subunit consists of DNA polymerase (residues 1 to 315), connection (residues 316 to 427), and RNase H (residues 428 to 560) domains. Almost all of the NRTI resistance mutations identified to date are in the DNA polymerase domain of RT, although the connection and RNase H domains of RT have not been routinely analyzed in clinical samples. In fact, none of the genotyping assays available for patient management sequence the entire coding region of RT. Recently, however, strong evidence has emerged that mutations outside the polymerase domain affect NRTI susceptibility (13, 18, 21, 30, 31, 32). Understanding how these mutations reduce NRTI susceptibility at the molecular level is essential to prevent and treat resistant virus effectively and to design new NRTI.

In 1995, dual-NRTI therapy with AZT and 3TC was shown to have significant virological and clinical benefits compared with AZT or 3TC monotherapy (2, 10, 20). The addition of 3TC to AZT delayed AZT resistance in therapy-naive patients and restored viral AZT susceptibility in patients who had previously received AZT alone (23, 24). Virologic and biochemical studies showed that the superior efficacy of AZT-3TC was due to the 3TC-associated M184V mutation antagonizing the phenotypic effects of AZT-associated thymidine analog mutations (TAMs) (3, 15, 28, 29). However, in some AZT-experienced patients, the virological response to AZT and 3TC therapy was not sustained and virus resistant to both drugs could be identified (40). Although substitutions at RT codons 44, 118, 207, and 208 have been associated with increased AZT resistance in viruses that carry both TAMs and M184V (14, 40), in some instances, these substitutions were absent. In this regard, Kemp et al. reported that a G333D/E polymorphism in the connection domain of HIV-1 RT was critical in facilitating dual AZT-3TC resistance in a complex background of mutations that included TAMs and M184V (21). The role of G333D/E in AZT-3TC dual resistance was demonstrated in two ways (21). First, conversion of 333E to G333 in the dually resistant virus derived from patient isolates reversed resistance to AZT. Second, the introduction of G333D in a recombinant virus that contained M41L/M184V/L210W/T215Y resulted in AZT resistance despite the presence of the M184V mutation.

The study by Kemp et al. demonstrated an unequivocal role for G333D/E in dual AZT-3TC resistance (21), but the biochemical mechanism by which this polymorphism restored AZT resistance in RTs containing both M184V and TAMs has not been determined. TAMs and M184V cluster near the polymerase active site of RT (Fig. 1) and have been shown to directly affect 3TC-triphosphate incorporation (M184V) (8, 11) or ATP-mediated excision of AZT-monophosphate from the chain-terminated template/primer (T/P) (TAMs and M184V) (3, 15, 28). By contrast, G333 resides ∼35Å away from the polymerase active site of RT (Fig. 1). Accordingly, the present study was conducted to elucidate how a mutation distant from the polymerase active site of HIV-1 RT can affect NRTI sensitivity.

FIG. 1.

Structure of HIV-1 RT in complex with AZT-MP chain-terminated DNA/DNA T/P. The DNA polymerase, connection, and RNase H domains of the p66 subunit are yellow, blue, and green, respectively. The p51 subunit is gray. AZT-MP and residues M41, M184, L210, T215, and G333 are shown in spacefill. This figure was generated by using Molecular Operating Environment (Chemical Computing Group, Montreal, Canada) and Brookhaven Protein Databank entry 1N6Q (35).

MATERIALS AND METHODS

Enzymes.

The M41L, K65R, K70E, M184V, L210W, T215Y, and G333D mutations were introduced into wild-type (WT) HIV-1_LAI RT (37) by site-directed mutagenesis with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Full-length sequencing of mutant RTs was performed to confirm the presence of the desired mutations. Both WT and mutant RTs were overexpressed in bacteria and purified to homogeneity as described previously (25, 26). Active-site concentrations of RT were calculated from pre-steady-state burst experiments, also as described previously (19). All of the reactions described below were carried out with corrected active-site concentrations.

Pre-steady-state kinetic analyses of dNTP and NRTI-triphosphate (NRTI-TP) incorporation by WT and mutant HIV-1 RTs on a DNA/DNA T/P.

For all experiments, a [γ-³²P]ATP 5′-end-labeled 20-nucleotide DNA primer (P20; 5′-TCGGGCGCCACTGCTAGAGA-3′) was annealed to a 57-nucleotide DNA template (T57; 5′-CTCAGACCCTTTTAGTCAGAATGGAAANTCTCTAGCAGTGGCG CCCGAACAGGGACA-3′) that contained either an adenine or a guanosine base at position 30 (N). This strategy enabled us to evaluate the kinetics of single-nucleotide incorporation for both AZT-TP (TriLink Biotechnologies, San Diego, CA) and 3TC-TP (Sierra Bioresearch, Tucson, AZ) and the respective dNTP substrates with the same T20 primer. Rapid-quench experiments were carried out with a Kintek RQF-3 instrument (Kintek Corporation, Clarence, PA). In all experiments, 400 nM RT and 40 nM DNA T/P were preincubated in reaction buffer (50 mM Tris-HCl [pH 7.5], 50 mM KCl) prior to mixing with an equivalent volume of nucleotide in the same reaction buffer containing 20 mM MgCl₂. Reactions were terminated at times ranging from 10 ms to 30 min by quenching with 0.3 M EDTA, pH 8.0. The quenched samples were then mixed with an equal volume of gel loading buffer (98% deionized formamide-10 mM EDTA-1 mg/ml each bromophenol blue and xylene cyanol) and denatured at 85°C for 5 min, and the products were separated from the substrates on a 7 M urea-16% polyacrylamide gel. Product formation was analyzed with a Bio-Rad GS525 Molecular Imager (Bio-Rad Laboratories, Inc., Hercules, CA).

Analysis of pre-steady-state incorporation data.

Data obtained from kinetic assays were fitted by nonlinear regression with Sigma Plot software (Jandel Scientific) by using the appropriate equations (17). The apparent burst rate constant (k_obs) for each particular concentration of dNTP was determined by fitting the time courses for the formation of product to the equation [Product] = A[1 − exp(−k_obst)], where A represents the burst amplitude. The turnover number (k_pol) and apparent dissociation constant for dNTP (K_d) were obtained by plotting the apparent catalytic rates, k_obs, against dNTP concentrations and fitting the data with the following hyperbolic equation: k_obs = (k_pol[dNTP])/([dNTP] + K_d). Selectivity and n-fold resistance values were calculated as described previously (38, 39).

Excision of AZT-MP by WT and mutant HIV-1 RTs from a DNA/DNA T/P.

The P20 primer was 5′ end labeled with [γ-³²P]ATP and annealed to T57 as described above. The primer was chain terminated by incubation with WT RT and 100 μM AZT-TP for 30 min at 37°C. ³²P-labeled, chain-terminated 21-nucleotide primer was further purified by extraction of the appropriate band after 7 M urea-16% acrylamide denaturing gel electrophoresis. The purified chain-terminated primer was reannealed to T57 for use in ATP-mediated phosphorolysis experiments. Excision of AZT-MP was achieved by incubating 200 nM active-site RT with 20 nM chain-terminated T/P in 50 mM Tris-HCl (pH 8.0)-50 mM KCl. The reaction was initiated by the addition of 3.0 mM ATP, 10 mM MgCl₂, 1 μM TTP, and 5 μM ddCTP. After defined incubation periods, aliquots were removed and processed as described above.

Excision of AZT-MP by WT and mutant HIV-1 RTs from an RNA/DNA T/P.

The 5′-end γ-³²P-labeled P20 primer was chain terminated (as described above) and annealed to a 35-nucleotide RNA template (T35; 5′-AGAAUGGAAAAUCUCUAGCAGUGGCGCCCGAACAG-3′). Phosphorolytic removal of AZT-MP was achieved by incubating 200 nM active-site RT with 20 nM chain-terminated T/P in 50 mM Tris-HCl (pH 8.0)-50 mM KCl. The reaction was initiated by the addition of 3.0 mM ATP and 10 mM MgCl₂. After defined incubation periods, aliquots were removed and processed as described above.

RNase H cleavage activity of WT and mutant HIV-1 RTs.

T35 was 5′ end labeled with [γ-³²P]ATP and annealed to P20. P20 was chain terminated as described above, except that an RNase H-deficient RT containing the D443N mutation was used in the chain-terminating reaction. The AZT-MP chain-terminated RNA/DNA T/P was then purified with a QIAGEN nucleotide removal spin column (QIAGEN, Valencia, CA). RNase H activity was assayed by incubating 200 nM active-site RT with 20 nM chain-terminated T/P in 50 mM Tris-HCl (pH 8.0)-50 mM KCl. The reaction was initiated by the addition of 3.0 mM ATP and 10 mM MgCl₂. After defined incubation periods, aliquots were removed and processed as described above.

Gel mobility shift assays.

Gel mobility shift assays were used to evaluate the thermodynamics of RT-T/P interactions. In these assays, the amount of DNA-bound RT present in an equilibrium solution was assayed by native gel electrophoresis. RT (0 to 600 nM total) was equilibrated with 200 nM ³²P-labeled AZT-MP chain-terminated DNA/DNA T/P for 2 h in 50 mM Tris (pH 7.5)-50 mM KCl-10 mM MgCl₂. Samples were then loaded onto a 4% polyacrylamide gel in 40 mM Tris-acetate (pH 8.0)-1 mM EDTA. Gels were run at room temperature for 30 min (100-V constant voltage) and quantified as described above. Discontinuity of sample and gel buffers can cause severe streaking of the bands. To correct for this, the area of the unshifted band was estimated from the lane containing DNA alone and the area between the shifted and unshifted bands was counted as the shifted band. The percent DNA-bound RT was calculated by assuming that the amount of DNA in the shifted band represented a 1:1 complex of RT-T/P. Data from the gel mobility shift assays were analyzed as described previously (16).

RESULTS

NRTI resistance mutations can be broadly categorized into two groups, depending on their phenotypic mechanism of resistance. The mutations K65R, K70E, L74V, Q151M, and M184V increase the selectivity of RT for incorporation of the natural dNTP substrate versus the NRTI-TP (6, 7, 8, 11, 36, 39). In comparison, TAMs, which include M41L, D67N, K70R, L210W, T215F/Y, and K219Q/E, increase the ability of HIV-1 RT to excise a chain-terminating NRTI-monophosphate (NRTI-MP) from a prematurely terminated DNA chain (1, 27). In the experiments described below, we examined both the discrimination and excision phenotypes of different RTs from both kinetic and thermodynamic perspectives to elucidate the mechanism(s) by which G333D facilitates dual AZT-3TC resistance. The enzymes included in this study were WT RT, M184V RT, M41L/L210W/T215Y (AZT^r) RT, AZT^r/M184V RT, and AZT^r/M184V/G333D RT. The enzymes used in control experiments included AZT^r/K65R RT, AZT^r/K70E RT, and AZT^r/K65R/G333D RT.

Transient kinetic analyses of 3TC-TP and AZT-TP incorporation by WT and mutant RTs.

To determine whether G333D directly affected the catalytic efficiency of NRTI-TP incorporation, transient kinetic analyses were carried out to assess the interaction of natural dNTP substrates (dCTP and TTP) and NRTI-TP (3TC-TP and AZT-TP) with the polymerase active sites of the WT, M184V, AZT^r, AZT^r/M184V, and AZT^r/M184V/G333D HIV-1 RTs (Table 1). These experiments determined the maximum rates of nucleotide incorporation (k_pol), the nucleotide dissociation constants (K_d), and the catalytic efficiencies of incorporation (k_pol/K_d). The k_pol/K_d values for dCTP and TTP incorporation by the WT and AZT^r RTs were similar. However, as reported by others, the catalytic efficiency (k_pol/K_d) values for RTs containing M184V were decreased about twofold (8, 11). The addition of G333D to the AZT^r/M184V enzyme had no effect on the catalytic efficiencies of dNTP incorporation, suggesting that this mutation does not adversely affect the structure or function of the polymerase active site. The selectivity of RT, which is defined as (k_pol/K_d)_dNTP/(k_pol/K_d)_analog, is an indication of the ability of WT or mutant RT to discriminate between the natural dNTP substrate and the NRTI-TP. Compared with WT RT, the selectivity values for dCTP versus 3TC-TP were 49-fold, 62-fold, and 116-fold higher for M184V RT, AZT^r/M184V RT, and AZT^r/M184V/G333D RT, respectively. These data demonstrate that G333D in combination with M184V can increase by about twofold the selectivity of RT for dCTP versus 3TC-TP. For each of the enzymes containing M184V or M184V/G333D, the greater discrimination against 3TC-TP could primarily be attributed to decreased 3TC-TP binding, although small changes in k_pol were also noted (Table 1). In contrast with 3TC-TP, none of the enzymes effectively discriminated between TTP and AZT-TP.

TABLE 1.

Pre-steady-state kinetic constants for binding and incorporation of the natural dNTP substrates and NRTI-TP by WT and mutant HIV-1 RTs^a

Enzyme	dCTP			3TC-TP			Selectivity	n-Fold resistance	TTP			AZT-TP			Selectivity	n-Fold resistance
	kpol (s−1)	Kd (μM)	kpol/Kd ratio	kpol (s−1)	Kd (μM)	kpol/Kd ratio			kpol (s−1)	Kd (μM)	kpol/Kd ratio	kpol (s−1)	Kd (μM)	kpol/Kd ratio
WT	1.22 ± 0.65	1.14 ± 0.37	1.07	0.023 ± 0.004	0.37 ± 0.02	0.06	17.8		7.75 ± 1.45	2.08 ± 0.55	3.72	8.78 ± 0.24	1.99 ± 0.09	4.41	0.85
M184V	1.33 ± 0.36	2.49 ± 1.55	0.53	0.005 ± 0.001	8.2 ± 3.2	0.00061	868.9	48.8	6.56 ± 0.89	2.85 ± 0.75	2.30	6.99 ± 0.67	2.90 ± 1.05	2.41	0.96	1.13
AZTrb	1.31 ± 0.43	1.23 ± 0.51	1.06	0.018 ± 0.005	0.43 ± 0.16	0.04	26.5	1.5	5.00 ± 0.60	1.20 ± 0.36	4.17	4.90 ± 0.20	1.15 ± 0.14	4.26	0.97	1.14
AZTr/M184V	1.33 ± 0.57	2.99 ± 0.81	0.44	0.004 ± 0.001	9.8 ± 1.8	0.0004	1,100	61.8	5.25 ± 0.85	2.65 ± 1.05	1.98	4.50 ± 0.60	3.50 ± 1.20	1.3	1.50	1.76
AZTr/M184V/G333D	1.45 ± 0.09	2.51 ± 1.10	0.58	0.005 ± 0.001	17.8 ± 4.7	0.00028	2,071	116.3	4.94 ± 0.76	2.90 ± 1.10	1.70	4.22 ± 0.74	3.81 ± 1.86	1.1	1.54	1.81

^aData are means ± standard deviations from three independent experiments.

^bAZT^r contains the M41L, L210W, and T215Y substitutions.

Excision of AZT-MP by WT and mutant HIV-1 RTs from DNA/DNA T/Ps.

To determine whether G333D directly affected the catalytic efficiency of AZT-MP excision, the ATP-mediated phosphorolytic activities of WT and mutant RTs were investigated with AZT-MP chain-terminated DNA/DNA T/P. Consistent with previously published results, AZT^r RT exhibited an about fourfold greater ability to excise AZT-MP from this T/P compared with either the WT or the M184V enzyme (Fig. 2) (38, 39). The AZT^r/M184V enzyme excised more efficiently than the WT and M184V enzymes but less efficiently than AZT^r RT. The antagonistic effects of M184V on the excision activity of RT containing TAMs, however, were not as pronounced as those resulting from K65R or K70E (Fig. 2B). The rate of AZT-MP excision by AZT^r/M184V/G333D RT was similar to that of the AZT^r/M184V enzyme, suggesting that G333D does not directly affect the enzyme's ATP-mediated AZT-MP excision activity.

FIG. 2.

ATP-mediated excision of AZT-MP from a DNA/DNA T/P by WT and mutant HIV-1 RTs. (A) Isotherm for ATP-mediated excision of AZT-MP by WT (○), M184V (▿), AZT^r (□), AZT^r/M184V (⋄), and AZT^r/M184V/G333D (▵) HIV-1 RTs. Excision products were analyzed at 0, 4, 8, 12, 16, 20, 40, 60, and 90 min, respectively. (B) First-order rate constants for ATP-mediated excision of AZT-MP by WT and mutant HIV-1 RTs. Data are averages of three separate experiments.

Excision of AZT-MP by WT and mutant HIV-1 RTs from RNA/DNA T/P.

Decreased RNase H activity of RT has been proposed to increase AZT resistance by delaying degradation of the RNA/DNA T/P heteroduplex, thereby allowing more time for unblocking of the terminated DNA primer and resumption of polymerization on an intact T/P (32). To determine whether G333D decreased the rate of RNase H cleavage and affected ATP-mediated excision of AZT-MP from a chain-terminated RNA/DNA T/P, we carried out excision and RNase H assays in parallel with the same AZT-MP chain-terminated RNA/DNA T/P (see Materials and Methods for experimental details). The excision results obtained from these experiments (Fig. 3A) were very similar to those obtained for the DNA/DNA T/P (Fig. 2A). Specifically, both the WT and M184V enzymes were inefficient at excising AZT-MP in comparison with AZT^r RT. AZT^r/M184V RT and AZT^r/M184V/G333D RT exhibited similar rates of AZT-MP excision, but these were decreased in comparison with that of AZT^r RT. Analysis of the rate of RNase H cleavage of the 35-nucleotide RNA template showed that the WT and each mutant enzyme showed very similar rates of RNA template degradation (Fig. 3B). Furthermore, no significant differences were evident in the relative rates of the primary and secondary RNase H cleavage events (Fig. 3C).

FIG. 3.

ATP-mediated excision of AZT-MP from an RNA/DNA T/P by WT and mutant HIV-1 RTs. (A) Isotherm for ATP-mediated excision of AZT-MP by WT (○), M184V (▿), AZT^r (□), AZT^r/M184V (⋄), and AZT^r/M184V/G333D (▵) HIV-1 RTs. Excision products were analyzed at 0, 2, 5, 8, 12.5, 20, 30, and 45 min, respectively. Data are averages of three separate experiments. (B) Rate of degradation of RNA template by RNase H activity of WT and mutant HIV-1 RTs. Products were analyzed at 0, 2, 5, 8, 12.5, 20, 30, and 45 min, respectively. Rates were determined by scanning the uncleaved RNA substrate (panel C) as a function of time. (C) Representative autoradiograms of RNase H activities of WT and mutant HIV-1 RTs. Assays were carried out as described in Materials and Methods. The time points in this experiment were 0, 5, 8, 12.5, 20, 30, and 45 min, respectively. nt, nucleotides.

Effects of RT-T/P interactions on ATP-mediated excision reactions.

In the excision reactions described above, the concentration of RT was in excess of the concentration of chain-terminated T/P (200 nM versus 20 nM). These conditions ensure that all T/P is bound by RT and that the kinetic rates determined can be directly related to the single-turnover rate of ATP-mediated excision. To evaluate the role of RT-T/P interactions in the ATP-mediated excision reaction, we first carried out excision reactions under steady-state conditions (i.e., [T/P] [tmt] [RT]) in which the rate of T/P dissociation is rate limiting (19). The results demonstrate that the magnitude of the difference observed in excision activity between the WT and AZT^r enzymes was significantly larger in the steady-state assays (Fig. 4A and C) compared with the single-turnover assays (Fig. 2 and 3). Interestingly, AZT^r/M184V RT also exhibited a markedly decreased capacity to excise AZT-MP compared with AZT^r RT in the steady-state assay (Fig. 4A), which was not observed in the single-turnover assay (Fig. 2 and 3). In addition, the excision activity of AZT^r/M184V/G333D RT was enhanced compared to that of AZT^r/M184V RT, suggesting that under the conditions of the steady-state assays, G333D reverses the antagonistic effects of M184V on TAMs (Fig. 4A and C). Additional studies showed that G333D did not reverse the antagonistic effects of K65R on TAMs (Fig. 4B), indicating that the effect of G333D appeared to be specific for RT with M184V and TAMs. Taken together, these data suggest that M184V might antagonize TAMs by promoting RT-T/P dissociation and that G333D compensates for this binding defect.

FIG. 4.

Steady-state ATP-mediated excision of AZT-MP from a DNA/DNA T/P by WT and mutant HIV-1 RTs. (A) Isotherm for ATP-mediated excision of AZT-MP by WT (○), M184V (▿), AZT^r (□), AZT^r/M184V (⋄), and AZT^r/M184V/G333D (▵) HIV-1 RTs. (B) Isotherm for ATP-mediated excision of AZT-MP by WT (○), AZT^r/M184V (⋄), AZT^r/K65R (▾), AZT^r/M184V/G333D (▵), and AZT^r/K65R/G333D (•) HIV-1 RTs. For both isotherms, excision products were analyzed at 0, 15, 30, 45, 60, 90, 120, 150, and 180 min, respectively. Data are averages of three separate experiments. (C) Representative autoradiographs indicating the relative abilities of WT and mutant HIV-1 RTs to excise AZT-MP and rescue DNA synthesis under steady-state conditions. The time points in this experiment were 15, 30, 45, 60, 120, 150, and 180 min, respectively.

Gel mobility shift assays to assess RT-T/P interactions.

To directly determine whether the M184V, TAMs, and G333D mutations affect RT-T/P binding, gel mobility shift assays were carried out. In these assays, various concentrations of RT (0 to 500 nM total) were equilibrated with 200 nM ³²P-labeled AZT-MP chain-terminated T/P in 50 mM Tris (pH 7.5)-50 mM KCl-10 mM MgCl₂ and the amount of DNA-bound RT present in the equilibrium solution was assayed by native gel electrophoresis. The results (Fig. 5; Table 2) show that AZT^r/M184V RT exhibits a decreased ability to bind the AZT-MP-terminated T/P compared to WT RT or AZT^r RT. Furthermore, the addition of G333D to the AZT^r/M184V enzymes reversed this decrease in binding affinity. By contrast, K65R did not impact the affinity of AZT^r RT for T/P; the WT, AZT^r, AZT^r/K65R, and AZT^r/K65R/G333D RTs all exhibited similar constants of dissociation from AZT-MP chain-terminated T/P (Table 2).

FIG. 5.

Binding of AZT-MP chain-terminated DNA/DNA T/P to WT (open circles), AZT^r (open squares), AZT^r/M184V (inverted open triangles), AZT^r/M184V/G333D (open diamonds), AZT^r/K65R (filled triangles), and AZT^r/K65R/G333D (filled hexagons) HIV-1 RTs. The inset is a representative autoradiograph of a gel shift assay. Data are averages of three separate experiments.

TABLE 2.

Equilibrium dissociation constants for WT and mutant HIV-1 RTs from AZT-MP chain-terminated DNA/DNA T/P^a

Enzyme	Kd (nM)	Mutant/WT ratio
WT	19.8 ± 3.3
M184V	76.9 ± 6.8	3.8
AZTrb	18.8 ± 2.5	1.0
AZTr/M184V	98.4 ± 5.9	5.0
AZTr/M184V/G333D	46.2 ± 4.2	2.3
AZTr/K65R	20.5 ± 1.8	1.0
AZTr/K65R/G333D	22.3 ± 2.0	1.1

^aData are means ± standard deviations from three independent experiments.

^bAZT^r contains the M41L, L210W, and T215Y substitutions.

DISCUSSION

Although the catalytically active p66 subunit of HIV-1 RT encodes polymerase, connection, and RNase H domains, most commercial genotyping assays do not sequence the latter two domains to identify drug resistance-associated mutations. Recently, however, a growing body of evidence has emerged that implicates mutations outside of the polymerase domain in NRTI resistance. Insight into how these mutations affect NRTI sensitivity is essential to understand and manage resistance effectively. We therefore investigated the biochemical mechanism(s) by which G333D in the connection domain of HIV-1 RT facilitates dual 3TC-AZT resistance in an enzyme that contains both TAMs (M41L, L210W, T215Y) and M184V. Although G333D is a polymorphism (it is present in 6% of treatment-naive individuals) that is selected in a minority (12%) of individuals receiving NRTI therapy (12), we chose to study this mutation because, in comparison with other NRTI resistance mutations that have tentatively been identified in the C terminus of RT, its role in AZT-3TC resistance has been clearly defined both in clinical isolates and in recombinant infectious viruses derived from molecular constructs (21).

The biochemical mechanism of antagonism between M184V and TAMs has been investigated previously (3, 28, 29). In two of these studies, M184V was shown to decrease the ability of HIV-1 RT carrying TAMs to effectively excise AZT-MP (3, 28). In the third study, however, M184V was found not to compromise the enzyme's AZT-MP excision activity (29). The results from our study show that the addition of M184V into RT containing TAMs decreases the enzyme's capacity to unblock AZT-MP chain-terminated primers annealed to either DNA or RNA templates (Fig. 2 and 3). However, the decrease in AZT-MP excision activity from M184V is small in comparison with the decreases observed for K65R or K70E in the same TAM background (Fig. 2B). It is therefore unlikely that the negative effect of M184V on the NRTI-MP excision activity is the sole explanation for the resensitization of viruses carrying both TAMs and M184V to AZT in cell culture (29). In this regard, our data clearly show that M184V attenuates the ability of RT to excise AZT-MP excision in assays carried out under steady-state conditions (in which the rate-limiting or slowest step of the reaction is T/P dissociation) and decreases the affinity of RT for AZT-MP chain-terminated T/P (Fig. 4 and 5; Table 2). For AZT-MP excision to occur, RT must bind the chain-terminated T/P long enough to allow the chain-terminating NRTI-MP to be excised (Fig. 6) (5). The kinetic rates of RT-T/P dissociation (i.e., k_off) have been calculated to be in the range of 0.0025 min⁻¹ to 0.1 min⁻¹ (9, 16, 34). By comparison, the kinetic rates of ATP-mediated AZT-MP excision range from 0.01 min⁻¹ to 0.20 min⁻¹, depending on the T/P (RNA or DNA), the sequence of the T/P, and the enzyme used in the assay (i.e., WT versus mutant) (27, 38, 39). As the rate of T/P dissociation approaches the rate of ATP-mediated excision, the latter reaction will become less efficient. Therefore, any mutation—such as M184V—that decreases RT's affinity for T/P will also indirectly impact the enzyme's ability to excise a chain-terminating NRTI-MP.

FIG. 6.

Kinetic pathway for ATP-mediated AZT-MP excision illustrating the steps at which K65R, K70E, M184V, and G333D impact on the efficiency of the reaction.

Our data demonstrated that G333D does not directly reverse the decreased rate of AZT-MP excision caused by M184V (Fig. 2 and 3). Instead, G333D increased the affinity of RT with TAMs and M184V for AZT-terminated T/P (Fig. 5; Table 2) and increased the excision of AZT-MP under steady-state assay conditions (Fig. 4). This suggests that the primary mechanism by which G333D facilitates AZT resistance in RTs containing TAMs and M184V is by compensating for the decreased ability of AZT^r/M184V RT to bind AZT-MP chain-terminated T/P. By contrast, G333D did not reverse the negative effect of K65R on AZT-MP by RT containing TAMs, but unlike M184V, K65R does not impact RT-T/P interactions (Table 2). In addition to the effect of G333D on RT-T/P interactions, our transient kinetic data also show that G333D in combination with M184V increases the selectivity of HIV-1 RT for dCTP versus 3TC-TP (Table 1). Although this increase in 3TC resistance conferred by G333D is unlikely to be clinically significant, it clearly demonstrates that mutations outside of the DNA polymerase domain of RT can directly affect the enzyme's ability to recognize and incorporate nucleotide analogs at the enzyme's DNA polymerase active site. In this regard, we speculate that this effect must result from conformational changes in the connection domain, which are communicated to the polymerase active site of the enzyme via long-range interactions. Because nucleotide selectivity is a function of both active-site and nucleotide structures, it is anticipated that these conformational changes will negatively impact on 3TC-TP incorporation but not on other NRTI-TPs, such as AZT-TP. In this regard, G333 in the RT p66 subunit is positioned close to the base of the thumb region (Fig. 1), which is involved in T/P interactions. Thus, it is possible that changes at residue 333 alter the positioning of the thumb region and subsequently that of the T/P in the active site of the polymerase domain. However, crystal structures of mutant RT enzymes with G333D together with TAMs and M184V are needed to more clearly define the structural mechanism of 3TC-TP resistance.

In conclusion, this study is the first to show at the enzyme level that a mutation (G333D) in the connection domain of HIV-1 RT can impact both the excision and discrimination phenotypes of HIV-1 for NRTI resistance. Of note, recent studies have demonstrated the important role of other residues (e.g., G335C, N348I, and A360I) in the connection domain of HIV-1 RTs in AZT resistance (31). In this regard, preliminary studies have demonstrated that many of these mutations affect the AZT excision phenotype by decreasing the enzyme's RNase H activity (4, 41). Taken together, these data suggest that there may be multiple mechanisms by which mutations in the connection domain of HIV-1 RT can augment NRTI resistance. However, additional in-depth biochemical studies are needed to confirm this.