Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates

Abstract

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.

FIG. 1

Unblocking of chain-terminated primer-templates by WT or 67/70/215/219 mutant RT through either dinucleoside polyphosphate synthesis or pyrophosphorolysis. (A) Rescue of d4TMP-terminated primer. d4TMP-terminated 5′-³²P-labeled L33 primer-WL50 template (5 nM) was incubated with 200 nM WT (upper panels) or 67/70/215/219 mutant RT (lower panels) and with no substrate (left panels), 3.2 mM ATP (middle panels), or 50 μM PP_i (right panels) for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with exonuclease-free Klenow fragment of E. coli DNA polymerase I and all four dNTPs. Products were fractionated on a 10% denaturing polyacrylamide gel (24). Positions for unextended primer (primer) and for products formed after elongation to the end of the template (ext. primer) are shown at the left of the figure. Lane 1, unextended primer-template. (B) Effect of incubation in the absence of HIV-1 RT. The experiment was performed as described in panel A in the absence of HIV-1 RT (lanes 1 to 4) or with HIV-1 RT (lane 5) with or without added ATP (3.2 mM) or PP_i (50 μM), as indicated in the figure, for 40 min at 37°C. After heat treatment, unblocked primer was extended by addition of exonuclease-free Klenow fragment of E. coli DNA polymerase I and all four dNTPs. (C) Rescue of primer terminated with d4TMP, AZTMP, or ddTMP. Incubations were carried out as in panel A with primer terminated with either d4TMP, AZTMP, or ddTMP in the presence of 3.2 mM ATP (left panels) or 50 μM PP_i (right panels) and WT RT (●) or 67/70/215/219 mutant RT (○). Radioactivity in products of >34 nucleotides (rescued primers) was quantitated by phosphorimaging and expressed as percentage of total radioactivity in the lane. The data were fitted to a hyperbola using Sigmaplot 4.0 (solid lines).

FIG. 2

Ability of the dNTP complementary to the next nucleotide position on the template to inhibit primer rescue and to induce a stable complex with chain-terminated primer-template and 67/70/215/219 mutant RT. (A and B) Inhibition of primer rescue by dGTP. Rescue by 67/70/215/219 mutant RT of primer-templates terminated with d4TMP (▾), ddTMP (○), or AZTMP (●) using either ATP (A) or PP_i (B) as a substrate was performed as described in the legend to Fig. 1, in the presence of the indicated concentrations of dGTP. The results were quantitated by phosphorimaging, and the percent inhibition was plotted versus dGTP concentration and fitted to hyperbolas (solid lines) using Sigmaplot 4.0. (C) Formation of DEC with 67/70/215/219 mutant RT, chain-terminated primer-templates, and dGTP. DEC formation was carried out in the absence of ATP or PP_i and was monitored by gel mobility shift assay as described previously (23, 38). The percent DEC formation was plotted as a function of dGTP concentration. The primer-templates are identified as in panel A. Partial sequences of the WL50 template and L33 primer terminated with a dTMP analogue (T_AN) are shown.

FIG. 3

Primer extension by 67/70/215/219 mutant RT in the presence of d4TTP or AZTTP or in the absence of chain terminator. (A) 5′-³² P-labeled D25 primer-D70 template (5 nM) was incubated for 30 min at 37°C with 200 nM 67/70/215/219 mutant RT, the indicated concentrations of all four dNTPs, and d4TTP (d4TTP/dTTP = 4:1) (upper panel); AZTTP (AZTTP/dTTP = 2:1) (middle panel), or no chain-terminating nucleotide (lower panel), in the absence (−ATP) or presence (+ATP) of 3.2 mM ATP. The products were fractionated on a 10% denaturing polyacrylamide gel. The primer lengths are indicated in nucleotides. Lane 1, untreated primer-template. (B) The results in panel A were quantitated by phosphorimaging, and the 31-mer chain-terminated product formed in the presence of ATP plus d4TTP plus dNTPs (●) or ATP plus AZTTP plus dNTPs (○), expressed as a percentage of the total radioactivity in the lane, was plotted versus the dNTP concentration.

FIG. 4

Effects of dNTPs on ATP-dependent removal of chain terminators in multiple sequence contexts. (A) 5′-³²P-labeled D25 primer-D70 template was incubated for 30 min at 37°C with excess 67/70/215/219 mutant RT, 1 μM concentrations of all four dNTPs, and either d4TTP (2 μM) or AZTTP (1 μM). The resulting mixtures of chain-terminated products were extracted with phenol-chloroform, followed by ethanol precipitation. Excess 67/70/215/219 RT (200 nM) was incubated with either d4TMP-terminated products (5 nM) (upper panel) or AZTMP-terminated products (5 nM) (lower panel) in the absence or presence of 3.2 mM ATP and in the presence or absence of 50 μM of the indicated dNTP for the indicated times at 37°C. After heat inactivation of the HIV-1 RT (5 min at 90°C), unblocked primers were extended by the addition of exonuclease-free Klenow fragment of E. coli DNA polymerase I and 100 μM concentrations of each of the four dNTPs and the incubation at 37°C for 30 min. Product lengths are indicated in nucleotides. A partial sequence of the D70 template is shown. (B) Inhibition of ATP-dependent primer rescue by dNTPs. The results shown in panel A were quantitated by phosphorimaging. The amounts of radioactivity in the 31-mer products (left panels), the 39-mer products (middle panels), and the 41-mer products (right panels) terminated with either d4TMP (upper panels) or AZTMP (lower panels) were normalized to the total radioactivity in each lane. The percent ATP-dependent rescue was calculated by the formula: 100−100(a/b), where a and b are the normalized radioactivity in the band of interest in the presence or absence of ATP, respectively, and plotted versus time. The data (indicated by the symbols shown at the bottom of the figure) were fitted to hyperbolas (solid lines) using Sigmaplot 4.0.