Proc. Nati. Acad. Sci. USAVol. 89, pp. 9720-9724, October 1992Biochemistry

Regulation of the reverse transcriptase of human immunodeficiencyvirus type 1 by dNTPs

(DNA polymerase/noncompetitive inhibition/substrate inhibiton/allostenc regulation/AIDS)

ANTHONY B. WEST*t, THOMAS M. ROBERTS*t, AND RICHARD D. KOLODNER*t*Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; and Departments of tPathology andtBiological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Communicated by Arthur B. Pardee, June 11, 1992 (received for review August 14, 1991)

ABSTRACT Reverse transcriptase (RNA-directed DNApolymerase, EC 2.7.7.49) of human immunodeficiency virustype 1 has been exani ed with respect to the steady-statekinetics of polymerization of dNTPs into product DNA. WithdNTPs as variable substrate, the kinetics of polymerizationdeviated from standard Michaelis-Menten kinetics. Substrateinhibition was observed at high substrate concentrations andnegative cooperativity was seen at lower substrate concentra-tions. Examination of incorporation of substrate dNMPs in thepresence of nucleotides not complementing the template dem-onstrated that dNTPs may act as noncompetitive inhibitors, aswell as substrate. The K; of the enzyme for dNTPs was 104 pAM.A working model is presented that accounts for the substrateinhibition. In this model, the reverse transcriptase is a mul-tisubunit holoenzyme, where noncompetitive inhibition is me-diated by one subunit binding nucleotide and down-regulatingthe enzymatically active 64-kDa subunit. With additionalassumptions, this model can accommodate the negative coop-erativity observed.

The reverse transcriptase of human immunodeficiency virustype 1 (HIV-1) is a holoenzyme containing a 1:1 ratio of51-kDa and 64-kDa subunits (1, 2). These subunits areproducts ofthe modification ofthe gag/pol polyprotein by theviral protease. They share the same N terminus and differ inthat the 51-kDa subunit is missing the C-terminal RNase Hdomain (3, 4). While both subunits possess the catalytic sitefor polymerization, only the 64-kDa subunit appears to beactive (5, 6). The current chemotherapeutic intervention forAIDS primarily utilizes nucleoside analogs (7-10). The poorfidelity of the polymerase results in mutations that allow thevirus to develop resistance to these analogs (11-15). Becausethe details by which nucleoside analogs work is still unclear(16, 17), information regarding the polymerization mecha-nisms of the reverse transcriptase should be of use in thedesign of new therapies.

Previous reports indicate that the kinetics of nucleotideincorporation by the HIV-1 enzyme are straightforward (9,18, 19). The present study indicates that this is not the casefor the baculovirally expressed form of the enzyme. Instead,the kinetics of incorporation of dNMPs into homopolymericRNA primer/templates deviates from Michaelis-Menten ki-netics when dNTPs are the variable substrate. The data showthat dNTPs act both as noncompetitive inhibitors and assubstrates. A working model is proposed in which oneconsequence of the reverse transcriptase existing as a mul-tisubunit holoenzyme is to provide a way to regulate, allo-sterically, the polymerization activity.

MATERIALS AND METHODSNucleotides. The primer/templates were (rA)3soo and

(rC)350o (Boehringer Mannheim) annealed with (dT)15 or

(dG)15 primers (Dana-Farber Cancer Institute Core Molec-ular Biology Facility, Boston), respectively, in a 1:1 molarratio (by nucleotide). The primers were purified by PAGEfollowed by chromatography on a C18 Sep-Pak (Millipore)column. The dNTPs were from Pharmacia and the[a-32P]dNTPs were from New England Nuclear.Enzymes. A baculoviral expression system (20, 21) was

used to overexpress the entire pol open reading frame of thebiologically active HIV-1 strain BH10. In this system, the tworeverse transcriptase subunits and integrase are processedfrom the protease-polymerase-integrase polyprotein. Thereverse transcriptase holoenzyme was subsequently purifiedto >95% purity by immunoaffinity chromatography using amonoclonal antibody specific for the 64-kDa protein andcontained the 51-kDa and 64-kDa subunits in a 1:1 ratio. Bothsubunits had the same N-terminal sequence, which wasidentical to the virion form of the enzyme. Protein concen-trations were determined by the method of Bradford (22).Overproduction and purification of the enzyme is describedelsewhere (23). Avian myeloblastosis virus (AMV) reversetranscriptase was from Pharmacia.Measurement of Initial Velocity. A titration ofeach primer/

template was performed to determine the level that wassaturating when the dNTP concentration was 400 pM. Inboth cases [(rA)35oo (dT)15 and (rC)35oo (dG)1s], 260 nM wassaturating (23).Unless otherwise stated, the final concentration for the

heterodimer form of the reverse transcriptase was 6 nM (50ng per 75-Al reaction mixture). The enzyme was preincubatedat 37°C for 3 min with primer/template in 60 pul of reactionbuffer A (50 mM Tris-HCI, pH 8.1/100 mM NaCl/1 mMdithiothreitol). The reaction was initiated by addition to theenzyme/primer/template mix of 15 pul of a reaction cocktailcontaining MgCl2 (10 mM), one [a-32P]dNTP (specific activ-ity 150 cpm/pmol), and the unlabeled dNTPs in buffer A.

The concentrations of dNTPs are indicated for each exper-iment. At 1-min intervals, 10 ,ul was removed and added to 2.5,ul of 500 mM EDTA on ice. The aliquot for eaqh time pointwas spotted onto DE 81 paper (Whatman), washed threetimes for 5 min each in 2x SSC (0.3 M NaCl/0.03 M sodiumcitrate, pH 7), washed in 95% ethanol, and dried. Theradioactivity remaining was measured in a scintillationcounter and used to determine initial velocity for eachreaction. Previously performed challenged polymerase as-says have demonstrated that velocities measured over thefirst 3 min after initiation of the reaction are equivalent to theprocessive velocity of the enzyme (23). In such an assay,

Abbreviations: HIV, human immunodeficiency virus; AMV, avianmyeloblastosis virus.

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Proc. Natl. Acad. Sci. USA 89 (1992) 9721

FIG. 1. SDS/PAGE analysis ofHIV-1 reverse transcriptase. The bacu-loviral expression system was used tooverexpress the entire pol open readingframe of the HIV strain BH10. The re-verse transcriptase was purified by im-munoaffinity chromatography (23).Shown here is 5 ,g of enzyme containingthe properly proteolytically processedsubunits separated by SDS/PAGE andvisualized by Coomassie blue staining.

reactions are performed with (rA)3500o(dT)15 and dTTP assubstrates as described above; however, upon initiation ofthe reaction, an inhibitory excess of (rC)35oo (dG)15 (30 AM) isadded to eliminate reinitiation by the polymerase. This limitspolymerization to one round of synthesis and activity is seenfor processive synthesis only. In all cases the time course waslinear over the 3-min period. The results obtained withchallenged assays were not significantly different for thatobserved with unchallenged assays over the first 3 min of thereaction. In control experiments, AMV reverse transcriptasewas used at 5 ng per 75-,ul reaction mixture in the samefashion as described for the HIV enzyme.

Calculation of the K, for dNTPs. Eq. 1,

Vmax[S](Km + [S]) (1 + [i]/Ki)

derived from Fig. 3 can be transformed to generate theequation for a double reciprocal plot:

1 1 [i]_ = x V+v Vmax VmaxKi

Km

Vmax[S]Km[i]

VmaxKi[S]Here the slope and y intercept of 1/v vs. 1/[S] (see Fig. 3B)

vary by a factor of 1 + [i]/Ki. By plotting the slopes (ml) fromeach of the double reciprocal plots vs. inhibitor concentra-tion, [i], one gets a line (see Fig. 3C) with a slope (M2) whichequals Km/(VmaxKi). Thus, K, = Km/(VmaxM2) = 104 ILM.The Km and Vmax values were derived from the doublereciprocal plot where [i] = 0. To confirm the estimate, the yintercepts from each double reciprocal plot were also plottedvs. [i] to give a line (see Fig. 3C) with the slope (M3) such that

Ki = l/(Vmam3) = 107 ,uM. The two values of K, are within3% of each other.

RESULTS

Fig. 1 shows a Coomassie blue-stained gel ofthe pure enzymeindicating that the enzyme contains an equimolar ratio of the51-kDa and 64-kDa subunits. The identity of the N terminiwas determined by protein sequencing and matched that ofthe virion form. Because this expression system contains theHIV-1 protease and because the pol polyprotein is processedto yield the two reverse transcriptase subunits and theintegrase (23), it is likely the C termini of the two subunits areidentical to those found in the viral form of the enzyme. Thespecific activity is 350 nmol ofdTMP incorporated per mg permin on a poly(rA)-oligo(dT) template/primer.

Kinetics of Nucleotide Incorporation with HomopolymerPrimer/Templates. Fig. 2A shows the activity of the reversetranscriptase on (rA)35oo-(dT)15 and (rC)35oo-(dG)15 primer/templates with dTTP and dGTP as variable substrates,respectively. Two unusual features are observed: (i) sub-strate inhibition at higher nucleotide concentrations and (ii)a deviation from the rectangular hyperbolic curve expectedfor Michaelis-Menten kinetics at low substrate concentra-tions (24). This is visualized on double reciprocal plots (Fig.2B) where the velocity increases less rapidly with increasingsubstrate concentration than in a linear double reciprocalplot. Such a deviation is usually associated with negativecooperativity (25, 26). With single-stranded M13mpl8 DNAas the template and a 17-base primer (3' end at nucleotide5616; 1:1 molar ratio with template) and all four dNTPs as thevariable substrate, the double reciprocal plot also had adownward curvature like that observed with homopolymertemplates, which we interpret as negative cooperativity. Nosubstrate inhibition was observed with the M13 template (23).

In control experiments with AMV reverse transcriptase(Fig. 2C), rectangular hyperbola were obtained indicatingthat deviations seen with the HIV-1 enzyme were not arti-facts of the assay system (23) or contaminating nonspecificinhibitors in the primers or dNTPs.

Substrate Inhibition. To understand the nature of thesubstrate inhibition by dNTPs, the effect of secondary nu-cleotides on the initial velocity of incorporation ofdTMP intoa (rA)3500 (dT)15 primer/template was measured with dTTP assubstrate at 5, 10, and 50 AM. The secondary nucleotideswere a mixture of dATP, dCTP, and dGTP, which are notcomplementary to the template and are likely to serve only aseffector molecules for nucleotide-mediated inhibition. It isnecessary to use homopolymers as template to allow dATP,

200 300 400

dNTP (giM)

Q7B -a- dTTP0.6 * dGTP

0.5

0.4

0.3

0.2

0.1

0.0I I

0.0 0.2O0 0.6 0S i.o 1.2

1/s0.0 0.2 0.4 0.6 0.8 1.0 1.2

1/s

FIG. 2. Effect of variable nucleotide substrate concentration on polymerization. Reverse transcriptase was preincubated with primer/template for 3 min. Reactions were initiated by the addition of Mg2+ and dNTPs. For (rA)3500 (dT)15 and (rC)3500 (dG)15 templates, dTTP anddGTP were the nucleotides used, respectively. Initial velocities were derived as described in Materials and Methods and are shown at varioussubstrate concentrations (A). These data are presented in a double reciprocal plot (B). In control experiments, AMV reverse transcriptase (5ng) was used under the conditions described for the HIV enzyme. The data are presented in a double reciprocal plot (C).

kDa

112-96-

68-_

45-

24-

*1

I

Biochemistry: West et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

20 30 40 50 60 -0.1 0.0 0.1

dTTP (M) 1/S

qAua

10 20 30 40

[dATP+dCTP+dGTPJ (9M)

FIG. 3. Inhibition by secondary dNTPs. The reactions were performed as in Fig. 2 with dTTP at 0, 5, 10, and 50 AM in the presence of anequimolar mixture of dATP, dCTP, and dGTP at a cumulative concentration of 0, 15, 30, or 45 AM. The experiment was performed at 370C(A) and was plotted on a double reciprocal plot (B). The cumulative concentration of dATP, dCTP, and dGTP in MM is indicated for each line.The slopes (o) and y intercepts (e) from the data were plotted at each concentration of the inhibitor nucleotides (C).

dCTP, and dGTP to act as unincorporatable effector mole-cules.

Fig. 3A demonstrates that unincorporatable effector nu-cleotides cause an inhibition of initial velocity of dTMPincorporation. On a double reciprocal plot (Fig. 3B) thetrajectories of each dTTP titration curve have the same xintercept but different y intercepts. Fig. 3C shows that boththe y intercept and the slope of the double reciprocal plotincrease as the concentration of the inhibitory nucleotidesincreases. The pattern of inhibition where Km is unchanged,is indicative of noncompetitive inhibition (27). This suggeststhat dNTPs may act as inhibitory effector molecules byinteracting with the enzyme at a different site than thevariable substrate, binding of the inhibitory nucleotide beingindependent of substrate binding. One possible explanation isthat dNTPs compete with the primer/template binding site.Inhibition by dNTPs is not overcome by increased concen-trations of the homopolymer primer/template combinationstested above, excluding this possibility (data not shown andref. 23).

K, of the Reverse Transcriptase for dNTPs. The noncom-petitive inhibition demonstrated by Fig. 3 indicates that therate equation (see Eq. 1) for complete noncompetitive inhi-bition may be applied for polymerization (28). For Fig. 3,dTTP acts as the substrate and dATP, dCTP, and dGTP areinhibitors. By solving for Km and Vm,, at zero inhibitorconcentration and assuming that [i] = [dATP + dCTP +dGTP], we estimate that the Ki of the reverse transcriptasefor dNTPs is 104 ,uM (see Materials and Methods).The Rate Equation Predicts Substrate Inhibition. Applica-

tion of the rate equation and the constants derived from theexperiment shown in Fig. 3 to the case where dTTP is theonly dNTP present generates the theoretical curves shown inFig. 4. If noncompetitive inhibition is assumed to be absent,a standard rectangular hyperbola is generated. However, ifdTTP is both substrate (S) and inhibitor (i), substrate inhi-bition is predicted. The data are also shown on a doublereciprocal plot (Fig. 4B). Clearly, by assuming that dTTP actsas both noncompetitive inhibitor and substrate, one canre-create reasonably well the general substrate inhibition atconcentrations seen in Fig. 2.

Inhibition Is Temperature-Sensitive. It is possible that thedeviations from Michaelis-Menten kinetics in Fig. 2 are dueto allosterically mediated noncompetitive inhibition bydNTPs. If this is due to an allosteric interaction betweendifferent subunits or distinct domains of the holoenzyme,then the negative cooperativity and inhibition should besensitive to treatments such as increased temperature, whichdisrupt noncovalent interactions between the subunits ordomains (29-31). This appears to be the case for the HIV-1

reverse transcriptase. Fig. 5 shows there is no inhibition bythe secondary nucleotides (dATP, dCTP, and dGTP) at 44TCwith (rA)350w(dT)15 as the template, as compared to thesituation at 37TC. On the double reciprocal plot (Fig. 5B),there is effectively no change in either the y intercept or theslope as the secondary nucleotides are added to the titrationcurves.

If interaction by dNTPs at another site is responsible forthe negative cooperativity in addition to noncompetitive

8

I

U,100 200 300 4 DO

dNTP (

1.0

0.5 ----- i___+1

0. -

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

1/S

FIG. 4. The rate equation and derived Ki account for substrateinhibition. Shown are theoretical curves that result from the assump-tion that dNTPs act both as substrate and as noncompetitive inhib-itor. These curves are the product of Eq. 1 and result when inhibitor(i) concentration equals 0 (-) or when inhibitor concentrationequals substrate concentration (-) (A). The data from A wereplotted on a double reciprocal plot (B) to give a straight line (---) ora curve for noncompetitive substrate inhibition (-).

.2

II

I

,&

m

IA

6 --

5

4 '

35

2

1 ________

+1

9722 Biochemistry: West et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 9723

IU

8

To

.2

a

ii

8.2

2

dTTP (pM) dTTP (pM

0.1

1/s03

FIG. 5. Inhibition by secondary dNTPs is disrupted at hightemperature. This experiment was the same as that of Fig. 3 exceptthat incubation was at 440C (A). Data were also plotted on a doublereciprocal plot (B). The cumulative concentration of dATP, dCTP,and dGTP (,uM) is indicated for each line.

substrate inhibition, then at the higher temperature, thekinetics should conform to a rectangular hyperbola as pre-dicted for a polymerizing enzyme obeying Michaelis-Mentenkinetics. Fig. 6A shows the effect that performing the poly-merization reaction at 44TC with dTTP as the only nucleotidepresent has on the initial velocity ofdTMP incorporation intoa poly(rA)-oligo(dT) primer/template. On a correspondingdouble reciprocal plot, the substrate inhibition appearsgreatly reduced and the trajectory shifts to a more linear formrelative to the same experiment performed at 370C, as dem-onstrated by the Hill constant (32) increasing from 0.6 at 37TCto 0.87 at 440C (23).

DISCUSSIONThe HIV-1 reverse transcriptase as assayed on homopolymertemplates displays deviation from standard Michaelis-Menten kinetics: at high concentrations of dNTPs there issubstrate inhibition, and downward curvature is seen in thedouble reciprocal plot (25, 26). We have observed a similardownward curvature on a double reciprocal plot with asingle-stranded M13 DNA template, indicating that the re-sults observed here are not an artifact of using homopolymertemplates (23).The following model is presented to explain these obser-

vations. It is based on the observation that inhibition bydNTPs can occur in a noncompetitive fashion with respect tothe nucleotide acting as substrate. Noncompetitive inhibitionoccurs when the inhibitor combines with a different site onthe enzyme than the variable substrate and that binding isindependent of the binding of the substrate. This model

1/S

FIG. 6. Effect of high temperature on kinetics of dTMP incor-poration into (rA)3soo5(dT)15 primer/template. The reaction wasperformed as in Fig. 2 with incubation at 440C. Initial velocities ofincorporation are shown at various substrate concentrations (A).These data are presented in a double reciprocal plot (B). The titrationcurve at 370C is presented for comparison. The data point at 2 AuMis not plotted on the curve at 440C, to not alter the scale ofcomparison between the two different curves. However, the valueobtained for this point is consistent with the displayed results.

provides the rate equation and allows for an evaluation of aKi for dNTPs.There is no indication that the enzymatically active 64-kDa

subunit has more than one polymerization site (33, 34). Thus,the location of the inhibitor interaction on the holoenzymemay be at the nucleotide binding site ofanother subunit of theholoenzyme. Consistent with the notion that the inhibitorybinding site is on a separate subunit is the observation thatallosteric effects are disrupted at higher temperatures. Sucha mechanism for noncompetitive inhibition is outlined in Fig.7. In this model the subunit binding the inhibitory nucleotideis shown as distinct from the catalytic subunit. In this case,the inhibiting nucleotide would change the velocity in a waythat cannot be overcome by saturation with the variablesubstrate, a prerequisite of noncompetitive inhibition.The assumption for the rate equation is that a = 1 and i =

0 for the model in Fig. 7. This is the case in completenoncompetitive inhibition and predicts the observed sub-strate inhibition (27). Here, the binding of substrate at onesite on the holoenzyme negatively affects the activity of theenzyme by inhibiting the rate of polymerization. The kineticsobserved at the higher temperature support the notion thatthe allosteric regulation occurs between the subunits of theholoenzyme. If the holoenzyme is a heterodimer of the 64-and 51-kDa subunits, then the 51-kDa subunit is a goodcandidate for having the noncompetitive inhibitory bindingsite. However, ifthe holoenzyme exists as a higher oligomer,then the allosteric interaction could be mediated between two

Biochemistry: West et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

Q

B k (Tn+1A) B

BN - , (Tn+lA) BN

aFIG. 7. Model for noncompetitive inhibition of polymerizing

activity by dNTP. Here the enzyme is defined as two subunits, A andB; thus the holoenzyme is AB. The (TnA) B represents the enzymeplus the primer/template complex, (Tn+1A) B the enzyme/primer/template complex with an additional dNMP incorporated, S thenucleotide substrate, Q the pyrophosphate produced by hydrolysis ofthe dNTP, and N the nucleotide acting as inhibitor. Here processivesynthesis in the preformed TnAS complex is inhibited by thesubsequent interaction of another nucleotide (N) with the B subunit.For the sake of clarity, the reaction is written as a single substratereaction. This is a legitimate assumption because in this case only theinitial velocity of the reverse transcriptase on a preformed enzyme/primer/template complex (with the primer/template in excess) ismonitored.

64-kDa subunits. The negative cooperativity also appears tobe reduced at the higher temperature. Whether the secondsubstrate binding site on the holoenzyme which is responsiblefor substrate inhibition is also responsible for apparent neg-ative cooperativity remains to be determined.One function of homotropic allosteric mechanisms is to

reduce the dependence of an enzyme on substrate concen-tration so that its activity is not a hyperbolic function ofsubstrate concentration (35, 36). The velocity is kept in a verynarrow range regardless of the nucleotide concentration orthe species of substrate. This indicates that the incorporationrate of HIV-1 reverse transcriptase has a higher tolerance fordifferences in nucleotide concentration than if it obeyedstandard Michaelis-Menten kinetics. Typically, substrateinhibition is considered an artifact of in vitro conditionsbecause it usually occurs at substrate levels too high to be ofphysiological relevance. However, the Ki of 104 ,uM fordNTPs is close to the cytoplasmic concentration of dNTPs,90 AuM (36). Thus, the substrate ranges where the allostericeffects are observed are within physiologic levels.

Oncoviruses typically can only infect dividing cells (37),whereas the lentivirus HIV can infect both dividing andnondividing cells (38) in a variety of tissue types (39). Onedifference between dividing and nondividing cells is in theoverall nucleotide concentrations and ratios (36, 40). Thus,one obstacle for HIV polymerase is dealing with the diversenucleotide pools it encounters in the cytoplasms of differentcell types. One function of allosteric modulation may be tobuffer the HIV polymerase against the effects of differentsubstrate concentrations and ratios encountered.

We thank Drs. Henry Paulus, Michael Corbley, David Norris, andArlen Johnson for help in manuscript preparation. This work wassupported by National Institutes of Health Grant A127336 to R.D.K.and T.M.R. Use of the Dana-Farber Cancer Institute Core Facilitywas supported by National Institutes ofHealth Core Grants CA06516and A128691.

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S

s7 (TnAS)

aKi

A aKs (TnAS)

(TnA) B

TNnA Ki

(TnA) BN

9724 Biochemistry: West et al.