# Inhibition of the Initiation of HIV-1 Reverse Transcription by 3'-Azido-3'-deoxythymidine

## COMPARISON WITH ELONGATION*

Received for publication, April 17, 2000
Published, JBC Papers in Press, June 22, 2000, DOI 10.1074/jbc.M003262200

Mickaël Rigourd‡, Jean-Marc Lanchy‡§, Stuart F. J. Le Grice¶, Bernard Ehresmann‡, Chantal Ehresmann‡, and Roland Marquet‡||

*From ‡UPR 9002 du CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France and the ¶Resistance Mechanisms Laboratory, NCI-FCRDC, National Institutes of Health, Frederick, Maryland 21702-1206*

Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA<sub>3</sub><sup>Lys</sup>, and reverse transcriptase. Initiation, corresponding to addition of the first six nucleotides to tRNA<sub>3</sub><sup>Lys</sup>, is distinguished from elongation by its high specificity and low efficiency (processivity). Here, we compared the inhibition of initiation and elongation of reverse transcription by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deoxythymidine. We report the first detailed study of nucleotide binding, discrimination, and pyrophosphorolysis by the authentic initiation complex. We showed that the initiation and elongation complexes bound AZTTP and dTTP with the same affinity, while the polymerization rates were reduced by 148–160-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced by the same extent, indicating that the polymerization equilibrium is the same in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis significantly relieved inhibition of DNA synthesis during elongation in the presence of physiological pyrophosphate concentrations. Remarkably, although pyrophosphorolysis of dTMP and AZTMP were equally efficient during elongation, reverse transcriptase was almost totally unable to unblock the AZTMP-terminated primer during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription, despite a reduced selectivity of incorporation.

Reverse transcription is a central event of the retroviral replication cycle during which the single-stranded RNA genome is converted into double-stranded DNA (1). Synthesis of (–) strand DNA is initiated at the primer binding site (PBS)<sup>1</sup> located close to the 5’ end of the RNA, whereas initiation of (+)

strand synthesis takes place in the 3' region of the RNA, at the polypurine tract. Complete synthesis of the DNA provirus requires two strand transfers that lead to duplication of the long terminal repeats (1). In retroviruses, plant pararetroviruses and most long terminal repeat-containing retrotransposons, reverse transcription is primed by a cellular tRNA whose 3' end is complementary to the PBS (2–5). All immunodeficiency viruses, including the type 1 human immunodeficiency virus (HIV-1), utilize tRNA<sub>3</sub><sup>Lys</sup> as primer, while tRNA<sub>3</sub><sup>Trp</sup> and tRNA<sub>3</sub><sup>Pro</sup> are used by most avian and murine retroviruses, respectively.

Several lines of evidence indicated that initiation of HIV-1 reverse transcription strongly differs from the elongation phase that follows it. First, the viral RNA and tRNA<sub>3</sub><sup>Lys</sup> form a complex three-dimensional structure constrained by several intermolecular interactions, in addition to the annealing of the 3' end of the primer tRNA to the PBS (6–8). The integrity of this complex is required for efficient initiation of reverse transcription *in vitro* (9–11), and most likely in cell culture (12–16). Second, initiation of reverse transcription, unlike elongation, is a specific process. Most reverse transcriptases (RTs), including lentiviral RTs, are unable to efficiently initiate reverse transcription from the HIV-1 RNA/tRNA<sub>3</sub><sup>Lys</sup> complex (9, 17). This result implies the existence of specific interactions between HIV-1 RT and its natural primer/template during initiation, in agreement with our structural studies (8). Third, the binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription. As a result, DNA synthesis is processive during elongation, while the initiation of reverse transcription is a distributive process (9, 11). The transition from the distributive to the processive mode of DNA synthesis is a two-step process (18). First, the polymerization rate dramatically increases between addition of the sixth and seventh nucleotide to tRNA<sub>3</sub><sup>Lys</sup>. Then, the dissociation rate of the ternary complex sharply decreases between addition of the 17th and 19th nucleotides.

The specificity and low processivity of the initiation of reverse transcription suggest it might represent a valuable target for anti-retroviral agents. The low efficiency of initiation suggests that this step should be easier to block than elongation. Furthermore, viral escape during treatment with anti-initiation agents should be limited by the specificity constraints, since tRNA<sub>3</sub><sup>Lys</sup> would not be able to co-vary with the viral components of the initiation complex. However, before designing antiviral agents specific for the initiation complex, it is important to evaluate the efficiency of widely used RT inhibitors on the initiation phase. Indeed, the efficiency of nucleoside

<footnote>* This work was supported by a grant from the Agence Nationale de Recherches sur le SIDA and by a Biomed II grant from the European Union. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>§ Present address: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI 53706.</footnote>

<footnote>‖ To whom correspondence should be addressed. Tel.: 33-3-88-41-70-91; Fax: 33-3-88-60-22-18; E-mail: r.marquet@ibmc.u-strasbg.fr.</footnote>

<footnote n="1">¹ The abbreviations used are: PBS, primer binding site; AZT, 3'-azido-3'-deoxythymidine; AZTTP, 3'-azido-3'-deoxythymidine 5'-triphosphate; AZTMP, 3'-azido-3'-deoxythymidine 5'-monophosphate; HIV-1, human immunodeficiency virus type 1; PP<sub>i</sub>, inorganic pyrophos-</footnote>

<footnote>phate; RT, reverse transcriptase; ODN, 18-mer oligodeoxyribonucleotide complementary to the PBS.</footnote>
Viral RNA

**FIG. 1.** *Template and primers used in this study.* Only part of the sequence of the 1–311 HIV-1 RNA used as template is shown. The PBS corresponds to nucleotides 179–196 of this RNA. *Upper* and *lowercase letters* stand for ribo- and deoxyribonucleotides, respectively. Only the 3' sequence of tRNA<sub>3</sub><sup>Lys</sup> and of the derived primers is shown.

<figure><img src="image_1.png" /></figure>

analogs, as well as non-nucleoside inhibitors, has been studied *in vitro* almost exclusively on the elongation of reverse transcription, using both RNA and DNA as template.

Here, we compared the inhibition of initiation and elongation by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), an analog of dTTP that acts as chain terminator (19, 20). AZTTP is the active form of 3'-azido-3'-deoxythymidine (AZT) (20), the first drug approved by the Food and Drug Administration for the treatment of acquired immunodeficiency syndrome (AIDS) (21). We report the first detailed study of nucleotide binding and discrimination using the authentic complex of initiation of reverse transcription. We show that pyrophosphorolysis of the last incorporated nucleotide is much slower during initiation than during elongation. Furthermore, although the pyrophosphorolysis of dTMP- and AZTMP-terminated primers was equally efficient during elongation, the pyrophosphorolysis efficiency of the AZTMP-terminated primer was considerably reduced, as compared with the dTMP-terminated primer, during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription.

MATERIALS AND METHODS

**Template, Primers, and RT**—Natural tRNA<sub>3</sub><sup>Lys</sup> was purified from beef liver as described (22). Its sequence and post-transcriptional modifications are identical to those of human tRNA<sub>3</sub><sup>Lys</sup>. After limited hydrolysis with cobra venom phosphodiesterase, it was labeled by reconstituting its 3'-terminal CCA with [α-³²P]ATP and unlabeled CTP using tRNA nucleotidyltransferase (22). Chimeric primers consisting of tRNA<sub>3</sub><sup>Lys</sup> extended at its 3' end by one (dC) or two (dC-dT or dC-AZT) deoxyribonucleotides were synthesized using the Klenow fragment of the *Escherichia coli* DNA polymerase I and the appropriate DNA template and deoxyribonucleotide triphosphate mixtures (18). Chimeric primers were ethanol-precipitated and purified by denaturing polyacrylamide gel electrophoresis. ODN, an 18-mer oligodeoxyribonucleotide complementary to the PBS, as well as the corresponding primers extended by one or two nucleotides (ODN-dC, ODN-dC-dT, and ODN-dC-AZT) were chemically synthesized and 5' end-labeled with [γ-³²P]ATP and polynucleotide kinase from phage T4 (18).

In all experiments, the template was an RNA encompassing nucleotides 1–311 of HIV-1 genomic RNA (Mal isolate), synthesized by *in vitro* transcription as described (23). The labeled primer was quantitatively heat-annealed with the template RNA at a 1:2 ratio as described previously (22).

Wild-type and RNase H(-) HIV-1 RT bearing the E478Q point mutation were purified essentially as described (24). Unless otherwise stated, the RNase H (-) RT was used in order to prevent cleavage of the RNA template when using the ODN primer. We previously showed that both RTs are equally efficient in initiating reverse transcription (9).

**Purification of AZTTP**—Two AZTTP stock solutions were obtained from Moravek Biochemicals. Possible contamination of the AZTTP by inorganic pyrophosphate (PPᵢ) was tested with a commercially available kit (Sigma) as described (25). Even though AZTTP had been purified by high pressure liquid chromatography by the supplier, we found that both stock solutions were strongly contaminated with PPᵢ. In one case,

the PPᵢ concentration represented more than 20% of the AZTTP concentration. Thus, the AZTTP stock solutions were treated with 0.5 units of pyrophosphatase (Roche Molecular Biochemicals) for 1 h at 37 °C in 100 µl of 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 6 mM MgCl₂, 1 mM dithioerythritol. Pyrophosphatase was removed by filtration through a Centricon 10 (Amicon) device.

**Kinetic Experiments**—In single-turnover experiments, primer/template (2–8 nM) was pre-incubated with excess RT (20 or 50 nM) for 4 min at 37 °C in 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 6 mM MgCl₂, and 1 mM dithioerythritol. Reaction was initiated by addition of dTTP or AZTTP (0.01–75 µM). The reaction was stopped at increasing time intervals by addition of formamide containing 50 mM EDTA to aliquots of the reaction mixture. When tRNA<sub>3</sub><sup>Lys</sup>-dC was used as primer, reaction was slow and required no special equipment. The ODN-dC-primed reaction was fast and required a quenched-flow apparatus. We used a home-built apparatus that allows determination of a complete reaction curve in one stroke (26). This apparatus allows reaction times ranging from 2 to 450 ms. In this case, the reaction was stopped by addition of 25 mM EDTA and 750 mM sodium acetate (final concentrations). Samples were precipitated and dissolved in formamide before analysis on a sequencing gel.

Kinetics of the pyrophosphorolysis reactions were performed as described above, except that tRNA<sub>3</sub><sup>Lys</sup>-dC-dT, tRNA<sub>3</sub><sup>Lys</sup>-dC-AZT, ODN-dC-dT, and ODN-dC-AZT were used as primers. Reactions were initiated by addition of PPᵢ at increasing concentrations (0.5–8 mM).

For competition experiments between dTTP and AZTTP, 10 nM primer/template complex and 200 nM HIV-1 RT were pre-incubated as described above. Reactions were initiated by addition of dCTP and ddGTP (50 µM each), dTTP (0.1, 5, or 50 µM), NaPPᵢ (0, 75, or 150 µM), and increasing concentrations of AZTTP (from 0.001 µM to 2 mM) for 10 s to 30 min at 37 °C. Reactions were stopped by addition of an equal volume of formamide containing 50 mM EDTA.

For the synthesis of (-) strand strong stop DNA in the presence and absence of AZTTP and PPᵢ, 10 nM primer/template complex were preincubated with 20 nM RT for 4 min at 37 °C. Polymerization was initiated by adding dNTP (50 µM each), AZTTP (0 or 5 µM), and NaPPᵢ (0 or 100 µM). Reaction was stopped as described above.

For all experiments, the reaction products were analyzed on 15% (ODN, ODN+1, and ODN+2 primers) or 8% (tRNA<sub>3</sub><sup>Lys</sup>, tRNA<sub>3</sub><sup>Lys</sup>+1, and tRNA<sub>3</sub><sup>Lys</sup>+2 primers) sequencing gels and quantified with a BioImager BAS 2000 (Fuji). Curve fitting was performed with SigmaPlot software (Jandel Scientific Software).

RESULTS

**Single-turnover Kinetics**—In order to compare the incorporation kinetics of AZTTP and dTTP during initiation and elongation of reverse transcription, we used a 311-nucleotide template corresponding to the 5' end of HIV-1 genomic RNA that contained the PBS. Using this template, dTTP and AZTTP were incorporated at the second position from the PBS (Fig. 1). To compare initiation and elongation of reverse transcription, we used as primer either natural tRNA<sub>3</sub><sup>Lys</sup> extended by dCMP (tRNA<sub>3</sub><sup>Lys</sup>-dC) or ODN-dC (Fig. 1). For each phase of reverse transcription, we determined the polymerization rate ($k_{pol}$) and the binding equilibrium ($K_d$) constant of AZTTP and dTTP by performing single-turnover kinetics at increasing nucleotide
<figure><img src="image_2.png" /></figure>

FIG. 2. Single-turnover kinetics of AZTTP and dTTP incorporation during elongation (A and B) and initiation (C and D) of reverse transcription. A, ODN-dC/viral RNA complex (2.5 nM) was incubated with 100 nM HIV-1 RT and reaction was initiated by addition of 5 (open squares), 10 (filled squares), 20 (open triangles), and 35 (filled triangles) µM AZTTP. Experimental data were fit to equation [ODN+2] = A(1 - e⁻$k_{pol}^{obs}t$), where ODN+2 is the ODN extended by two nucleotides, A is the amplitude of the burst, and $k_{pol}^{obs}$ is the apparent first order rate constant. B, dependence of $k_{pol}^{obs}$ for ODN+2 synthesis on the concentration of dTTP (open squares) and AZTTP (filled squares). The $k_{pol}^{obs}$ values obtained as in A were fit to the hyperbolic equation $k_{pol}^{obs} = (k_{pol}^*[dNTP])/((K_d^* + [dTTP]))$. The curves shown correspond to fits with $k_{pol(dTTP)} = 113 s^{-1}$, $K_{d(dTTP)} = 27.0 \mu M$, and $k_{pol(AZTTP)} = 80 s^{-1}$, $K_{d(AZTTP)} = 28.1 \mu M$. C, tRNA<sub>3</sub><sup>L-ys</sup>-dC/viral RNA complex (2 nM) was incubated with 20 nM HIV-1 RT and reaction was initiated by addition of 0.01 (open squares), 0.05 (filled squares), 0.1 (open triangles), 0.5 (filled triangles), 5 (open diamonds), or 20 (filled diamonds) µM AZTTP. Experimental data were fit to equation [tRNA+2] = A(1 - e⁻$k_{pol}^{slow}t$) + B(1 - e⁻$k_{pol}^{slow}t$), where tRNA+2 is the primer extended by two nucleotides, A and B correspond to the amplitudes of the fast and slow processes, respectively, $k_{pol}^{obs}$ is the apparent extension rate constant, and $k_{slow}$ is the rate constant of the slow process. D, dependence of $k_{pol}^{obs}$ for tRNA+2 synthesis on the concentration of dTTP (open squares) and AZTTP (filled squares). The $k_{pol}^{obs}$ values obtained as in C were fit to a hyperbolic equation as in B. The curves shown correspond to fits with $k_{pol(dTTP)} = 0.76 s^{-1}$, $K_{d(dTTP)} = 26.8 \mu M$, and $k_{pol(AZTTP)} = 0.49 s^{-1}$, $K_{d(AZTTP)} = 29.0 \mu M$.

concentrations (Fig. 2 and Table I).

Extension of ODN-dC with dTTP (data not shown) and AZTTP (Fig. 2A) was fast and followed mono-exponential kinetics. An apparent polymerization rate was determined for each experiment. A plot of this observed first-order rate constant on the concentration of dTTP and AZTTP then allowed determination of the polymerization rate at saturating nucleotide concentration ($k_{pol}$) and the $K_d$ of AZTTP and dTTP (Fig. 2B). Our results indicate that the incorporation rate of AZTTP was ~30% slower than that of dTTP (Fig. 2B and Table I). However, both nucleotide triphosphates bound the template/primer-RT complex with the same affinity (Fig. 2B and Table I).

As expected from our previous studies, extension of tRNA<sub>3</sub><sup>L-ys</sup>-dC with dTTP (data not shown) and AZTTP (Fig. 2C) was slow and followed biphasic kinetics. Dependence of the observed fast rate constant on the concentration of dTTP and AZTTP indicated that the incorporation rate of AZTTP was reduced by ~35%, as compared with dTTP (Fig. 2D and Table I). The $k_{pol}$ values of dTTP and AZTTP were reduced by 140- and 160-fold, respectively, when comparing initiation with elongation. As for elongation, dTTP and AZTTP bound equally well to the template/tRNA<sub>3</sub><sup>L-ys</sup>-dC·RT complex. Moreover, in contrast with the $k_{pol}$ values, the $K_d$ of these nucleotides were indistinguishable in the initiation and elongation phases (Table I).

The efficiencies of dTTP and AZTTP incorporation during initiation and elongation, defined as $k_{pol}/K_d$, are summarized in

Table I. Due to the differences in $k_{pol}$ in initiation and elongation, incorporation of dTTP and AZTTP was about 150-fold more efficient during elongation than during initiation of reverse transcription. However, the selectivity of HIV-1 RT for AZTTP versus dTTP was almost the same in the two polymerization phases (Table I). Thus, the probability of incorporating AZTTP at a single site in the presence of equal amounts of dTTP would be almost the same during initiation and elongation (38% and 41%, respectively).

**Competitive Incorporation of dTTP and AZTTP**—In order to test these conclusions, we set up an assay allowing direct monitoring of the competitive incorporation of AZTTP and dTTP. Furthermore, as the intracellular $PP_i$ concentration can reach 150 µM in lymphocytes (27), we also used this assay to test the effect of $PP_i$ on AZTTP incorporation. In this assay, we used labeled ODN or tRNA<sub>3</sub><sup>L-ys</sup> primers, and initiated the extension reaction by adding dCTP (50 µM), dTTP (5 µM), ddGTP (50 µM), and increasing concentrations of AZTTP (Fig. 3). The rationale of this assay is that dTTP and AZTTP will compete for incorporation at the second position, and that only those primers having incorporated dTMP can be extended with ddGTP (Fig. 1). After complete reaction, the ratio of the products corresponding to extension of the primers by 2 and 3 nucleotides will reflect the relative incorporation of AZTTP and dTTP. Thus, when this ratio is plotted as a function of the AZTTP to dTTP ratio, a straight line is expected, whose slope
<table><caption>TABLE I
Equilibrium and kinetics constants for binding and incorporation of dTTP and AZTTP during initiation and elongation of HIV-1 reverse transcription</caption><thead><tr><th></th><th>Nucleotide</th><th>k<sub>pol</sub></th><th>K<sub>d</sub></th><th>k<sub>pol</sub>/K<sub>d</sub></th><th>Selectivity<sup>a</sup></th></tr><tr><th></th><th></th><th>s<sup>-1</sup></th><th>μ<sup>M</sup></th><th>μ<sup>M</sup>-<sup>1</sup>s<sup>-1</sup></th><th></th></tr></thead><tbody><tr><td rowspan="2">Elongation</td><td>dTTP</td><td>113 ± 3</td><td>27 ± 6</td><td>4.1 ± 1.0</td><td rowspan="2">0.70 ± 0.3</td></tr><tr><td>AZTTP</td><td>80 ± 2</td><td>28 ± 6</td><td>2.9 ± 0.6</td></tr><tr><td rowspan="2">Initiation</td><td>dTTP</td><td>0.76 ± 0.04</td><td>27 ± 3</td><td>0.028 ± 0.005</td><td rowspan="2">0.60 ± 0.2</td></tr><tr><td>AZTTP</td><td>0.49 ± 0.03</td><td>29 ± 4</td><td>0.017 ± 0.003</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>FIG. 3. Competitive incorporation of dTTP and AZTTP during elongation and initiation of reverse transcription. A, 10 nM ODN/viral RNA complex were incubated with 200 nM RT, 50 μM dCTP, 5 μM dTTP, 50 μM ddGTP, increasing concentrations of AZTTP, and 0 (squares), 75 (triangles), or 150 (diamonds) μM PPᵢ. Reaction was for 10 s (open symbols) or 30 min (filled symbols). A control reaction incubated with inorganic pyrophosphatase for 30 min in the absence of PPᵢ is also included (filled circles). Fit with a linear equation yielded slope values of 0.91 ± 0.03 (open squares), 0.86 ± 0.03 (open triangles), 0.85 ± 0.05 (open diamonds), 0.85 ± 0.03 (filled circles), 0.36 ± 0.01 (filled squares), 0.071 ± 0.003 (filled triangles), and 0.034 ± 0.001 (filled diamonds). B, 10 nM tRNA<sub>3</sub><sup>Lys</sup>/viral RNA complex were incubated as above, except that reaction time was for 3 min (open symbols) or 30 min (filled symbols). The slopes of the straight lines are: 0.39 ± 0.02 (open squares), 0.35 ± 0.02 (open triangles), 0.32 ± 0.02 (open diamonds), 0.33 ± 0.01 (filled squares), 0.35 ± 0.01 (filled triangles), and 0.37 ± 0.01 (filled diamonds).</figcaption></figure>

should correspond to *S*, the RT selectivity for AZTTP, as defined in the legend of Table I (28). Furthermore, for incubation times significantly longer than that required to extend the primer by 3 nucleotides, the amount of each product should be constant.

When using ODN as primer and a 10-s reaction time, the ratio of primer extended by 2 and 3 nucleotides was almost independent from the pyrophosphate concentration (Fig. 3A). Furthermore, the selectivity of HIV-1 RT for AZTTP deduced from these experiments, 0.85–0.91 ± 0.03, is in agreement, within the experimental errors, with the selectivity deduced from the single-turnover kinetics (0.7 ± 0.3, see Table I). These data confirm that HIV-1 RT poorly discriminates between AZTTP and dTTP during elongation of reverse transcription. However, when the reaction time was extended to 30 min, the apparent selectivity was dramatically reduced, especially in the presence of PPᵢ (Fig. 3A). At the highest PPᵢ concentration, the apparent selectivity for AZTTP was reduced by ~25-fold. The most likely explanation for that observation is that after incorporation at the second position, AZTMP was significantly removed by pyrophosphorolysis over the 30-min period, thus allowing addition of ddGTP after competitive incorporation of dTTP. In agreement with this explanation, the apparent selectivity did not decrease over time when reverse transcription was performed in the presence of pyrophosphatase (Fig. 3A).

When studying the tRNA<sub>3</sub><sup>Lys</sup>-primed reaction, we used 3- and 30-min incubation times, because primer extension was not complete after 10 s (data not shown). As for the ODN-primed reaction, we observed that, at short incubation time, the selectivity for AZTTP was independent of the PPᵢ concentration (Fig. 3B). However, the apparent selectivity for AZTTP during

initiation of reverse transcription deduced from these experiments, 0.32–0.39 ± 0.02, is significantly lower than that deduced from single-turnover kinetics (0.6 ± 0.2, see Table I). Indeed, these selectivity values are reduced more than 2-fold, as compared with elongation. Moreover, in contrast with elongation, the apparent selectivity of HIV-1 RT for AZTTP during initiation was unaffected when increasing the reaction time, even at high PPᵢ concentration (Fig. 3B). Thus, our results suggested that the rate of pyrophosphorolysis markedly differed during initiation and elongation of reverse transcription.

Comparison of the Pyrophosphorolysis Rate during Initiation and Elongation of Reverse Transcription—In order to determine the rate of pyrophosphorolysis during elongation of reverse transcription, we used 20-mer deoxyribonucleotide primers terminated either by dTMP or by AZTMP, designated ODN-dC-dT and ODN-dC-AZT, respectively (see “Materials and Methods” and Fig. 1). Using these primers, we performed single-turnover kinetics at increasing PPᵢ concentrations. Pyrophosphorolysis of ODN-dC-dT (Fig. 4A) and ODN-dC-AZT (data not shown) followed mono-exponential kinetics. At 100 μM PPᵢ, reaction was almost complete in 5 min. In the case of ODN-dC-dT, a pyrophosphorolysis rate constant, $k_{pyro}$, of 0.7 ± 0.2 s⁻¹ could be determined by plotting the apparent pyrophosphorolysis rate as a function of PPᵢ concentration (Fig. 4B). This plot also allowed to determine the $K_d$, for PPᵢ: 12 ± 6 mM. In the case of primer ODN-dC-AZT, we determined a pyrophosphorolysis rate constant, $k_{pyro}$, of 1.3 ± 0.6 s⁻¹ and a $K_d$, for PPᵢ of 21 ± 12 mM (Fig. 4B). Thus, during elongation, the pyrophosphorolysis efficiency, defined as $k_{pyro}/K_d$, is identical for the ODN-dC-dT and ODN-dC-AZT primers (0.06 s⁻¹ mM⁻¹). Furthermore, the actual pyrophosphorolysis rates measured at

<footnote>a Selectivity is defined as $S = (k_{pol}/K_d)_{AZTTP}/(k_{pol}/K_d)_{dTTP}$.</footnote>
<figure><img src="image_4.png" /></figure>

FIG. 4. Single-turnover kinetics of AZTMP and dTMP pyrophosphorolysis during elongation (A and B) and initiation (C and D) of reverse transcription. A, ODN-dC-dT/viral RNA complex (2 nM) was incubated with 100 nM RT, and reaction was initiated by addition of 0 (open squares), 0.1 (filled squares), 0.5 (open triangles), 2 (filled triangles), and 6 (open diamonds) mM PPᵢ. Experimental data were fit to equation [ODN+2] = A(e⁻kₚyroᵢt + C), where ODN+2 is the ODN extended by two nucleotides, A is the amplitude of the burst, kₚyroᵢᵇъ is the apparent first order rate constant, and C is a constant. B, dependence of kₚyroᵢᵇъ for pyrophosphorolysis of ODN-dC-dT (open squares) and ODN-dC-AZT (filled squares) on the PPᵢ concentration. The kₚyroᵢᵇъ values were fit to equation kₚyroᵢᵇъ = kₚyroᵢ²[PPᵢ]/(Kₐ + [PPᵢ]). The curves correspond to kₚyro(dTMP) = 0.70 s⁻¹ and Kₐ(d,PPᵢ) = 12 mM, and kₚyro(AZTMP) = 1.3 s⁻¹ and Kₐ(d,PPᵢ) = 21 mM. C, tRNA₃<sup>Lys</sup>-dC-AZT/viral RNA (squares) or tRNA₃<sup>Lys</sup>-dC-dT/viral RNA (triangles and diamonds) complexes (2 nM) were incubated with 100 nM RT, and reaction was initiated by addition of 0.5 (open squares and open triangles), 2 (filled triangles), 3 (open diamonds), or 6 mM (filled squares and filled diamonds) PPᵢ. Experimental data were fit to a single exponential as in A. D, dependence of kₚyroᵢᵇъ for pyrophosphorolysis of tRNA₃<sup>Lys</sup>-dC-dT on the PPᵢ concentration. The kₚyroᵢᵇъ values were fit to a hyperbolic equation as in B. The curve corresponds to kₚyro(dTMP) = 0.0048 s⁻¹ and Kₐ(d,PPᵢ) = 4.4 mM.

physiological PPᵢ concentration were similar for the two primers.

Removal of the terminal dTMP from the tRNA₃<sup>Lys</sup>-dC-dT primer by pyrophosphorolysis was much slower than from ODN-dC-dT (Fig. 4C). At 100 µM PPᵢ, which is relevant to the *in vivo* situation, removal of the terminal nucleotide took more than 2 h to be almost complete (data not shown). As for elongation, pyrophosphorolysis in the initiation phase followed monoexponential kinetics (Fig. 4C). Dependence of the observed first-order rate constant on the PPᵢ concentration yielded a kₚyro of 0.0048 ± 0.0004 s⁻¹ and a Kₐ for PPᵢ of 4.4 ± 0.8 mM (Fig. 4D). Thus, the rate of pyrophosphorolysis of the terminal dTMP is ~140-fold slower during initiation than during elongation, while the affinity for PPᵢ does not differ very much in the two phases. More surprising, we found that, whereas removal of dTMP and AZTMP from DNA primers occurred at similar rates, removal of AZTMP from tRNA₃<sup>Lys</sup>-dC-AZT was incomplete, even at the highest PPᵢ concentrations. At 6 mM PPᵢ, a plateau corresponding to removal of ~20% AZTMP was reached after 45 min of incubation (Fig. 4C). The origin of the

incomplete AZTMP removal remains presently unknown.

The direct confirmation of the inefficient AZTMP pyrophosphorolysis during initiation, as compared with elongation of reverse transcription supports our conclusions from the competitive incorporation of AZTTP and dTTP.

Effect of Pyrophosphorolysis on the (–) Strand Strong Stop DNA in the Presence of AZTTP—In order to test whether the inefficient pyrophosphorolysis during initiation might affect the inhibition of the synthesis of the (–) strand strong stop DNA by AZT, we performed reverse transcription in the absence and in the presence of PPᵢ, using either ODN or tRNA₃<sup>Lys</sup> as primers.

In the absence of AZTTP, addition of 100 µM PPᵢ did not significantly affect synthesis of the (–) strand strong stop DNA from the ODN primer (Fig. 5A, *left part*); neither the amount of (–) strand strong stop DNA, nor the profile of pausing sites were modified by addition of PPᵢ. The same observation holds true when tRNA₃<sup>Lys</sup> was used as primer (Fig. 5B, *left part*), even though the pausing profile was not the same as with the ODN primer.
FIG. 5. Minus strand strong stop DNA synthesis in the absence and in the presence of AZTTP and PPᵢ. Ten nM ODN/viral RNA (A) or tRNA₃Lys/viral RNA (B) complexes were incubated with 20 nM RT, and reaction was initiated by addition of 50 µM amounts of each dNTP, and in some experiments AZTTP and/or PPᵢ. The PPᵢ and AZTTP concentrations are indicated above each gel. *Lanes 1–12* correspond to increasing reaction times: 0, 1, 2.5, 15, 30, 40, 50, 60, 90, 120, 150, and 180 min, respectively. Positions +2, +5, and +11, corresponding to the first three sites of AZTTP incorporation, are indicated by *asterisks*. Their different location in *panels A* and *B* is due to the differences in primer length (18 or 76 nucleotides) and gel reticulation (see “Materials and Methods”).

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

In the presence of 5 µM AZTTP and in the absence of PPᵢ, synthesis of the (-) strand strong stop DNA from the ODN primer was almost totally inhibited (Fig. 5A). The remaining faint bands correspond to ~2% of the (-) strand strong stop DNA obtained in the absence of AZTTP. Addition of AZTTP also totally changed the pausing pattern, since the intermediate products obtained under these conditions correspond to AZTMP-terminated DNA chains. As expected, the inhibition efficiency increased with the chain length, since the probability of incorporating one AZTTP is proportional to the number of adenines in the copied template.

Interestingly, including PPᵢ in the reaction partially restored synthesis of longer DNA chains, including the (-) strand strong stop DNA. Indeed, the amount of (-) strand strong stop DNA obtained in the presence of PPᵢ and AZTTP corresponds to ~15% of that observed in the absence of chain terminator. Pyrophosphorolysis of the incorporated AZTMP is partially, if not totally, responsible for the rescue. Indeed, whereas the intensity of the bands corresponding to incorporation of AZTTP at positions +2, +5, and +11 in the template reached a plateau when PPᵢ was omitted, they reached a maximum and then strongly decreased in the presence of physiological concentrations of PPᵢ (Fig. 5A). This strongly suggests that AZTTP was incorporated into the nascent DNA chain, but was eventually

removed by pyrophosphorolysis, thus allowing DNA elongation to resume.

When using tRNA₃Lys as primer and in the absence of PPᵢ, synthesis of (-) strand strong stop DNA was totally inhibited in the presence of 5 µM AZTTP (Fig. 5B, right panel). Remarkably, synthesis of (-) strand strong stop DNA was not restored in the presence of physiological concentration of PPᵢ (Fig. 5B). Indeed, the synthesis of long DNA chains from tRNA₃Lys was only marginally improved in the presence of PPᵢ. The difference in the synthesis of (-) strand strong stop DNA observed with ODN and tRNA₃Lys in the presence of AZTTP and PPᵢ can, at least in part, be explained by the inefficient pyrophosphorolysis of AZTMP during initiation when DNA synthesis was primed by tRNA₃Lys. Indeed, the intensity of the bands corresponding to incorporation of AZTTP in front of the first two As in the template did not decrease over time, even in the presence of PPᵢ (Fig. 5B, right panel). As already mentioned, the first two As are located at position +2 and +5 from the PBS (Fig. 1), i.e. in the initiation phase observed in the tRNA-primed reverse transcription.

Thus, the inefficient pyrophosphorolysis of AZTMP-terminated chain that we had observed in single-turnover kinetics was also observed during (-) strand strong stop DNA synthesis. Furthermore, this inefficient pyrophosphorolysis during
initiation had macroscopic effects on the level of (-) strand strong stop DNA synthesis.

DISCUSSION

AZT was the first drug approved by the Food and Drug Administration for treatment of AIDS (21) and is still widely used in combination with other antiviral drugs (29). Despite numerous detailed mechanistic studies (27, 30–39), little is known about the ability of AZTTP to inhibit initiation of reverse transcription. Indeed, in the only study dealing with the inhibition of the RNA-primed RNA-dependent DNA synthesis by AZTTP, an 18-mer RNA complementary to the PBS was used instead of the natural tRNA<sub>3</sub><sup>Lys</sup> primer (39). Thus, our work represents the first detailed study of AZTTP binding, incorporation, and removal by the authentic HIV-1 complex of initiation of reverse transcription. In addition to demonstrating unexpected differences in the inhibition of initiation and elongation of reverse transcription, the present work improves our fundamental understanding of the initiation step.

Our present data show that incorporation of dTTP and AZTTP during initiation occurred at a rate of 0.76 and 0.49 s<sup>-1</sup>, respectively, at saturating nucleotide concentration. These polymerization rates, which represent an ~100-fold decrease compared with elongation, are in good agreement with previous data obtained using a 50 µM dNTP concentration (11, 18). Since the complex interactions between tRNA<sub>3</sub><sup>Lys</sup> and the viral RNA (6–8) are essential for efficient initiation of reverse transcription *in vitro* (6, 10, 11) and *in vivo* (12–16), it can appear surprising that similar rate constants were also measured when an 18-mer RNA complementary to the PBS was used as primer (40, 41). However, previous studies indicated that the affinity of HIV-1 RT for the RNA/RNA primer/template complex was increased by 2 orders of magnitude when using the natural primer (11, 40). In addition, our present study reveals that the *K*<sub>d</sub> for the second nucleotide (dTTP or AZTTP) added to tRNA<sub>3</sub><sup>Lys</sup> is in the range of 25–30 µM. These values are 6–10-fold lower than those obtained using an 18-mer RNA primer complementary to the PBS (39, 40). Thus, the intricate structure of the tRNA<sub>3</sub><sup>Lys</sup>/HIV-1 RNA complex increases the efficiency of reverse transcription by increasing its affinity for both RT and the incoming nucleotide, without affecting the polymerization rate measured under saturating nucleotide and primer/template concentrations.

Our study also provides the first determination of the rate of pyrophosphorolysis of a natural nucleotide (dTMP) during initiation. The measured value (0.0048 s⁻¹) corresponds to a 158-fold reduction of the pyrophosphorolysis rate, as compared with elongation (0.70 s⁻¹). Remarkably, we found the same reduction (161-fold) of the polymerization rate of dTTP during initiation, compared with elongation (0.76 and 113 s⁻¹, respectively). Thus, the polymerization equilibrium constant, which corresponds to *k*<sub>po</sub>/k<sub>pyro</sub>, is identical during initiation and elongation. According to the Arrhenius equation, the reduction of the polymerization and pyrophosphorolysis rates during initiation indicates that the transition state is destabilized by 13 kJ/mol, as compared with elongation.

Our single-turnover kinetics indicates that RT poorly discriminates between AZTTP and dTTP during elongation. The selectivity determined from these data (S = 0.70 ± 0.3) is in agreement with the value obtained from our direct competition experiments (S = 0.87 ± 0.04), and with a previous pre-steady-state kinetics study using a DNA/RNA primer/template (33) (S = 0.69). Concerning initiation, our single-turnover kinetics and direct competitions yielded significantly different selectivity values (S = 0.6 ± 0.2 and 0.35 ± 0.04, respectively). The complex (bi-exponential) kinetics of initiation likely biased the corresponding S value, since this value only takes the fast

process into account. In keeping with this interpretation, kinetic data on an 18-mer RNA primer/viral RNA template following single-exponential kinetics yielded S = 0.37, in agreement with our direct competition experiments (39). Thus, our results strongly suggest that HIV-1 RT discriminates slightly better between AZTTP and dTTP during initiation than during the RNA-dependent elongation that follows.

Our single-turnover kinetics and competition experiments showed that wild-type HIV-1 RT can efficiently remove AZTMP incorporated during elongation by pyrophosphorolysis. The observation that wild-type RT can unblock AZTMP-terminated primers is not new (28, 30, 35), but it has been usually assumed that pyrophosphorolysis is too slow to play a significant role *in vivo* (at least with wild-type RT) (28, 30). However, we showed that, when using a DNA primer, pyrophosphorolysis was able to significantly relieve the inhibition of (-) strand strong stop DNA synthesis by AZTTP. Since we used an AZTTP to dTTP ratio 50–250-fold higher than that observed in patients (42), pyrophosphorolysis might play a significant role *in vivo*, even when considering the synthesis of the complete provirus (which is ~100-fold larger than the (-) strand strong stop DNA). Thus, our results indicate that it should be advantageous to combine AZT with a pyrophosphate analog, from the start of anti-HIV-1 treatments. This “natural resistance” of wild type HIV-1 RT toward AZTTP via pyrophosphorolysis likely explains the synergistic effects of AZT and foscarnet (a PP<sub>i</sub> analog) observed in early AIDS treatments (43, 44).

One of the most unexpected results of this work is the finding that HIV-1 RT was almost totally unable to unblock the AZTMP-terminated primer in the initiation phase. Indeed, whereas removal of AZTMP and dTTP was equally efficient during elongation, less than 20% of AZTMP was phosphorylated during initiation, even after a 2-h incubation in the presence of 8 mM PP<sub>i</sub>. Remarkably, the limited AZTMP pyrophosphorolysis during initiation had a noticeable effect on the synthesis of the (-) strand strong stop DNA when reverse transcription was conducted in the presence of AZTTP and PP<sub>i</sub>. Thus, even though the initial selectivity for AZTTP was decreased during initiation, inhibition of reverse transcription was eventually more efficient due to the inefficient unblocking of the primer. This finding reinforces the notion that the initiation phase of reverse transcription might be a valuable target for antiviral agents.

Finally, increasing evidence indicates that unblocking of AZTMP-terminated primer, either by PP<sub>i</sub> (35, 36) or by NTP (37, 38), is central to the mechanism of drug-induced resistance to AZT by RT bearing mutations at positions 67, 70, 215, and 219 (and sometimes 41). These mechanistic studies are in keeping with the observation that AZT resistance is suppressed by mutations conferring resistance to foscarnet (45). These data, combined with the finding that wild type RT is unable to unblock the AZTMP-terminated primer during initiation, suggest that the AZT-resistant RT might remain sensitive toward AZT during initiation of reverse transcription. We are presently testing this hypothesis.

*Acknowledgments*—We thank G. Keith and G. Bec for their help with the purification of natural tRNA<sub>3</sub><sup>Lys</sup> and C. Isel for critical reading of the manuscript.

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