J. Biol. Chem.-1993-Wu-9980-5

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 14, Issue of May 15, pp. 9980-9985,1993 Printed in U.S.A.

Monoclonal Antibody-mediated Inhibition of HIV- 1 Reverse Transcriptase Polymerase Activity INTERACTION WITH A POSSIBLE DEOXYNUCLEOSIDE TRIPHOSPHATE BINDING DOMAIN*

(Received for publication, December 1, 1992, and in revised form, January 27, 1993)

Jiong Wu, Emily Amandoron, Xuguang Li, Mark A. Wainberg, and Michael A. ParniakS From the Lady Davis Institute for Medical Research, Sir Mortimer B. Dauis-Jewish General Hospital, and McGill AIDS Centre, McGill Uniuersity, Montreal, Quebec H3T lE2, Canada

A series of monoclonal antibodies against p51/p66 human immunodeficiency virus- 1 (HIV- 1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated lE8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT- primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3’-azido,3’- deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.

The human immunodeficiency virus-1 reverse transcriptase

* This work was supported in part by grants (to M. A. W. and M. A. P.) from Health and Welfare Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence and requests for reprints should be addressed Lady Davis Inst. for Medical Research, Jewish General Hospital, 3755 Cote Ste-Catherine Rd., Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 5294); Fax: 514-340-7502.

(HIV-1 RT)’ is a virus-specific protein responsible for con- verting viral RNA into DNA. Because this enzyme plays an obligatory role in retroviral replication, it provides a logical target for the development of potentially selective antiviral agents. R T is a multifunctional protein, with a number of related yet distinct enzymatic activities including RNA-dependent and DNA-dependent polymerase activities as well as an intrinsic RNase H function. Development of increasingly specific inhibitors of R T requires detailed knowledge of the tertiary structure of the enzyme. Modeling studies show that HIV-1 R T may comprise three major functional domains (Barber et al., 1990; Jacobo-Molina and Arnold, 1991; Lind- borg, 1992; Narasimhan and Maggiora, 1992): (i) the N- terminal polymerization domain (amino acid residues 1-266), (ii) the tether domain (residues 267-440), and (iii) the RNase H domain (residues 440-560). Recent crystallographic studies have provided a much more refined picture of HIV-1 RT structure (Kohlstaedt et al., 1992). However, the organization of enzymatically active motifs on the enzyme molecule is still not completely understood. As an alternative approach to the study of HIV-1 RT structure, we have prepared a series of monoclonal antibodies for use in mapping the tertiary conformation of the enzyme. We report here the characterization of one of these antibodies, designated 1E8, which is able to significantly inhibit the polymerase activity of HIV-1 RT. This antibody recognizes a linear surface epitope within residues 65-73 of the reverse transcriptase. Two mutations within this region have been identified with resistance to the antiviral agent, 3’-azido,3‘-deoxythymidine. This region of HIV-1 R T may be at or near the polymerase active site of the enzyme.

MATERIALS AND METHODS

HIV-1 Reverse Transcriptase: Preparation and Assay Recombinant p66 HIV-1 RT was purified from lysates of Esche-

richia coli JM109 transformed with plasmid pKRT2 (D’Aquila and Summers, 1989), obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, courtesy of Drs. R. T. D’Aquila and W. C. Summers. The purification involved a modification of the method of Clark et al. (1990). Briefly, bacterial lysate proteins (prepared by treatment of 2 h isopropyl-l-thio-P-D-thiogalactopyranoside-inducedpKRTP-transformed E. coli with lysozyme, 0.5% Nonidet P-40, and DNase) were fractionated by a 25-65% (NH4)*S04 cut. This material was dialyzed against 50 mM Tris (pH 7.9, 4 “C) containing 1 mM dithiothreitol, 60 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol. The dialyzed proteins were applied to columns of DEAE-Sephacel and Q-Sepharose (Pharmacia, Montreal, Quebec, Canada), connected in series and equilibrated with the same buffer. The proteins in the

The abbreviations used are: HIV-1, human immunodeficiency virus-1; RT, reverse transcriptase; AZT, 3’-azido-3’-deoxythymidine.

9980

Monoclonal Antibody Inhibition of HIV-1 Reverse Transcriptase 9981

flow-through and wash were precipitated by the addition of (NH4)2S04 to 65% saturation. HIV-1 p66 RT was further purified by chromatography on CM-Trisacryl and Q-Sepharose essentially as described (Clark et al., 1990). The purified p66 RT was subjected to the denaturation/renaturation protocol described by Deibel et al. (1990) in order to maximize the specific activity of the enzyme preparation. Heterodimeric p51/p66 RT was prepared from p66 by treatment with purified HIV-1 protease (Medigenics, Omaha, NE). Recombinant HIV-1 p51/p66 RT prepared in this manner typically had a specific activity of 0.25-0.3 unit mg", where 1 unit equals 1 @mol of [3H]TMP incorporated into poly(rA).oligo(dT)/min in an assay for RNA-dependent DNA polymerase activity as described below. Unless otherwise indicated, RT polymerase activity was meas- ured in an assay consisting of 50 mM Tris (pH 7.8, 37 "c), 10 mM dithiothreitol, 60 mM KCl, 10 mM MgC12, 10 p M [3H]thymidine triphosphate (Du Pont), 0.1 unit/ml poly(rA) .oligo(dT) (Pharmacia) for RNA-dependent DNA polymerase activity measurements or 0.1 unit/ml activated calf thymus DNA (Sigma), plus 10 p M dNTPs for DNA-dependent DNA polymerase activity measurements. After incubation at 37 "C for varying times, the reaction was terminated by addition of 10% trichloroacetic acid containing 20 mM sodium pyro- phosphate. Samples were collected on glass fiber filters (Whatman 934AH), washed, and counted in a liquid scintillation spectrometer. RT-associated RNase H activity was assayed essentially as described (Hansen et al., 1988).

Determination of Potential Antigenic Regions of HIV-I R T Four computer algorithms were used to predict possible antigenic

regions of HIV-1 RT. Two involved hydrophilicity analysis as described by Hopp and Woods (1981) and Parker et al. (1986). Surface accessibility analysis was performed according to Janin (1979), and antigenicity analysis utilized the method of Welling et al. (1985). Synthetic peptides corresponding to those sequences of HIV-1 RT selected by all four methods were prepared and used for further study.

Preparation of Antibodies Murine Monoclonal Antibodies-HIV-1 p51/p66 RT (200 pg/ml in

phosphate-buffered saline, pH 7.4) was incubated with nitrocellulose paper (0.1 ml/cm2) in a humid atmosphere at room temperature for 1 h. Pieces of this nitrocellulose (0.5 cm2) were surgically implanted under the neck skin of BALB/c mice. Two weeks after implantation, the animals were boosted intraperitoneally with 25 pg of antigen in phosphate-buffered saline. Hybridomas were prepared, selected, and purified by standard procedures (Harlow and Lane, 1988). The monoclonal antibodies were purified as described (Bruck e t al., 1986) from mouse ascites by precipitation with (NH4),S04 followed by chromatography on DEAE-Affi-Gel Blue (Bio-Rad, Mississauga, Ontario, Canada).

Rabbit Monospecific Polyclonal Antibodies-Synthetic peptides comprising potentially antigenic sequences of HIV-1 RT were prepared with a CGG linker at the N terminus to facilitate conjugation to carrier (Research Genetics, Birmingham, AL). Peptides were conjugated to keyhole limpet hemocyanin using the Imject-TM Activated Immunogen Conjugation system according to the manufacturer's instructions (Pierce Chemical Co.). Monospecific anti-peptide antiserum was prepared in rabbits by standard methods (Harlow and Lane, 1988).

All procedures involving animals were conducted according to protocols approved by the Facility and University Animal Care Com- mittees of the Sir Mortimer B. Davis-Jewish General Hospital and McGill University, both accredited by the Canadian Council on Animal Care.

Analysis of Antibody-RT Interactions ELISA procedures were carried out as described (Li et al., 1993)

with horseradish peroxidase-conjugated secondary antibody and 0- phenylenediamine as substrate. Western blot analysis was as described (Li et al., 1993). Inhibition of HIV-1 RT enzymatic activity was assessed by assay after incubation of the enzyme with varying molar ratios of purified antibody in 50 mM Tris (pH 7.9, 4 "C)

biology grade, Sigma) for 1 h at 37 "C and an additional 1 h at 4 "C. containing 0.1 M KC1 and 200 pg/ml bovine serum albumin (molecular

RESULTS

Znhibition of Reverse Transcriptase Activities by Monoclonal Antibodies-Three mice were immunized with native p51/p66

HIV-1 R T using the nitrocellulose implant method as described under "Materials and Methods." Fourteen stable hybridomas producing high affinity monoclonal IgG antibodies specific for HIV-1 reverse transcriptase were obtained. As has been generally found by us (Li et al., 1993) and by others (Hansen et al., 1988; Tisdale et al., 1988; Ferris et al., 1990; Ferns et al., 1991; Orvell et al., 1991; Restle et al., 1992; Szilvay et al., 1992) who have prepared anti-RT monoclonal antibodies, most of the RT-specific monoclonal antibodies had no effect on either the polymerase or the RNase H activities of the enzyme (data not shown). However, one of our antibodies, designated 1E8, significantly inhibited both RNA-dependent and DNA-dependent DNA polymerase activity of RT, but was without effect on the intrinsic RNase H activity of the enzyme. Inhibition of R T polymerase activity varied with the concentration of 1E8, with maximum inhibition noted at approximately equimolar concentrations of RT and 1E8 in the incubation mixture (Table I). Similar extents of inhibition of RT RNA-dependent DNA polymerase activity were noted when F(ab')2 fragments of 1E8 were used.

Kinetics of lE8 Znhibition of R T Polymerase Activity- Steady-state kinetic measurements of the RNA-dependent DNA polymerase activity of R T were carried out using poly(rA).oligo(dT) as primer/template and [3H]TTP as deoxynucleoside triphosphate substrate. In the absence of 1E8, our preparations of recombinant p51/p66 HIV-1 R T gave an apparent K, for TTP of 4.2 f 0.5 p ~ , and for poly(rA). oligo(dT) of 0.2 f 0.03 AZGo unit ml-', values similar to those reported for HIV-1 R T from viral lysates (Cheng et al., 1987), as well as to whose noted for other preparations of recombinant HIV-1 R T (Reardon and Miller, 1990; Hizi et al., 1991).

In the presence of constant TTP concentration, inhibition of R T polymerase activity by 1E8 was noncompetitive with respect to the template/primer poly(rA) .oligo(dT) (Fig. Dl). In contrast, the antibody acted as a competitive inhibitor with respect to the deoxynucleoside triphosphate substrate TTP (Fig. 1B). The competitive nature with respect to TTP of 1E8 inhibition of R T polymerase activity is also demonstrated by experiments illustrated in Fig. 2. In the presence of constant template/primer concentration, the extent of 1E8 inhibition of R T polymerase activity decreased with increasing concentration of TTP in the incubation. In contrast, at any given concentration of TTP, the inhibition by 1E8 was unaffected

TABLE I Effect of IE8 on enzymatic activities of HIV-I reverse transcriptase

HIV-1 p55/p66 RT (2.5 pg ml" final concentration) was mixed with intact 1E8 or the F(ab'I2 fragment of 1E8 at the indicated final concentrations in 100 p1 of 50 mM Tris-HC1, pH 7.5 (4 "C) containing 0.1 M KC1 and 200 pg ml" bovine serum albumin. The mixtures were incubated at 37 "C for 1 h, then at 4 "C for an additional 30 min. Aliquots (25 pl) were withdrawn and assayed for the various RT activities as described under "Materials and Methods."

RT activity (% no antibody control) Antibody

concentration DNA polymerase

RNA-dependent DNA-dependent RNase H

d m l "

0 100 100 100 0.125 82 79 100 0.5 63 55 100 2.5 45 38 100 5.0 48 41 100

0.8 73 ND ND 1.5 59 ND ND

1E8

F(ab'),

9982 Monoclonal Antibody Inhibition of HIV-1 Reverse Transcriptase

I

11s (mu)” (pM1-l

FIG. 1. Steady state kinetics of 1ES inhibition of HIV-1 RT RNA-dependent DNA polymerase activity. p51/p66 RT (2.5 pg/ ml) was preincubated for 30 min at 37 “C in the absence (0) or the presence of 1E8 at 0.125 pg/ml (0) or 2.5 pg/ml (A). Aliquots of the mixtures were then assayed for RT RNA-dependent DNA polymerase activity using poly(rA). oligo(dT) as template/primer and [3H]TTP as deoxynucleoside triphosphate substrate under standard conditions as described under “Materials and Methods.” A, double-reciprocal plot of dTMP incorporation as a function of poly(rA). oligo(dT) concentration. TTP was held constant at 10 p ~ ) . B, double-reciprocal plot of TMP incorporation as a function of TTP concentration. Poly(rA). oligo(dT) was held constant a t 0.2 unit/ml.

2o t 0 ‘ I

I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Ab Concentration (pg/mL)

FIG. 2. Effect of TTP concentration on the inhibition of HIV-1 RT RNA-dependent DNA polymerase activity by 1E8. p51/p66 RT (2.5 pg/ml) was incubated with the indicated concentrations of 1E8 for 15 min at 37 “C. Aliquots were then added to polymerase assays containing 0.2 unit/ml poly(rA). oligo(dT) and [3H]TTP at a final concentration of 1 pM (O), 10 pM (e), or 40 pM (A). Samples were incubated at 37 “C for 30 min, and the incorporation of TMP into template/primer determined as described under “Materials and Methods.”

by changes in template/primer concentration (data not shown).

These kinetic data strongly suggest that 1E8 interacts with a region of RT involved in the binding of substrate deoxynucleoside triphosphates. However, the currently accepted kinetic mechanism of RT polymerase activity is complex (Ma- jumdar et ul., 1988; Kedar et ul., 1990; Reardon, 1992). The interpretation of steady state kinetic data for the enzyme is not straightforward since the polymerization reaction com- prises several steps. Therefore, in order to obtain more direct evidence for the co-identity of the region involved in the binding of 1E8 and of nucleoside triphosphates, the ability of dNTPs to prevent the binding of 1E8 to RT was studied. When RT was preincubated in the absence of template/ primer, then exposed to immobilized 1E8 in the presence of dNTP, the extent of 1E8 binding to RT was essentially

TABLE I1 Effect of dNTP and primerltemplate on solution binding of 1E8 to

HIV-1 RT RT (2.5 pg ml” final concentration) was preincubated in the

absence or the presence of primer/template in 50 pl of 50 mM Tris, pH 7.5 (37 “c), containing 0.15 M NaCl and 10 mM MgCl, for 15 min at 37 “ C . This solution was then mixed with 50 pl of a 50% mixture of 1E8 immobilized on goat anti-mouse Ig agarose (+1E8), or normal mouse IgG immobilized on goat anti-mouse agarose (-1E8), in the same buffer containing dNTP. Incubation was continued for 10 min at 37 “ C , the samples centrifuged for 1 min at 12000 X g, and aliquots of the supernatants assayed for RT RNA-dependent DNA polymerase activity as described under “Materials and Methods.”

Addition RT polymerase

supernatant activity in

Incubation 1 Incubation 2 -1E8 +1E8

Experiment 1‘ None None 127.5 27 (21%)b None Poly(rA). (dT)12-18 142 22 (16%) None 1 pM TTP 123.5 26 (21%) None 10 p M TTP 125 24 (19%) None 40 pM TTP 122 37 (30%) Poly(rA). (dT)1p-18 1 p~ TTP 126 42 (33%) Poly(rA). (dT)lz.18 10 p~ TTP 135 61 (45%) Poly(rA). (dT)lz.la 40 p~ TTP 134 94.5 (70%) None 40 pM dGTP 120 24 (20%) Poly(rA). (dT)12-18 40 GM dGTP 124.5 23.5 (19%)

Poly(rC). (dG)12-18 40 p~ TTP 24.5 3.5 (14%) Poly(rC). (dG),z.ls 40 p~ dGTP 22.5 14.5 (64%)

Experiment 2‘

Final assays included 10 p~ [3H]TTP and 0.2 unit ml” poly(rA).

RT activity remaining in the supernatant, reported as percent of

Final assays included 10 p~ [3H]dGTP and 0.2 unit ml” poly(rC).

(dT)r-m.

the -1E8 control.

(dG)12-18.

identical to that seen in the absence of dNTP (Table 11). Similarly, template/primer alone did not affect 1E8-RT interaction. However, when RT was first incubated with template/primer, then exposed to immobilized 1E8 in the presence of dNTP, a significant decrease in the amount of RT bound to the antibody was noted. This decrease in 1E8-RT binding was directly correlated with the concentration of dNTP in the secondary incubation (Table 11).

Interestingly, the ability of dNTP to inhibit binding of 1E8 to RT was a function of the identity of the template/primer

Monoclonal Antibody Inhibition of HIV-1 Reverse Transcriptase 9983

used in the preincubation. When poly(rA) . oligo(dT) was present, only TTP could significantly affect the binding of 1E8 to RT. Neither dGTP (Table 11) nor dCTP or dATP (data not shown) had any effect on this binding. Similarly, with poly(rC) .oligo(dG) as template/primer, only dGTP had any effect on the ability of 1E8 to bind to the enzyme.

Determination of the Epitope Recognized by 1E8 on HIV-1 RT-lE8 bound to the p51/p66 RT heterodimer in solution and to both p51 and p66 subunits of RT on Western blots (data not shown) and is therefore considered to recognize a linear surface epitope of the enzyme. The HIV-1 RT sequence was analyzed by four different computer algorithms in order to identify potentially antigenic epitopes. Two of these methods assessed hydrophilicity parameters (Hopp and Woods, 1981; Parker et al., 1986) since antigenic epitopes are generally located within hydrophilic regions on the surface of proteins. Another method (Janin, 1979) analyzed surface accessibility probabilities of residues in the HIV-1 RT sequences. The final algorithm, which is based on known epitope structures of other proteins (Welling et al., 1985), analyzed the probability of RT residues residing within antigenic epitopes. Although the prediction results differed somewhat among the four algorithms, seven potentially antigenic epitopes were defined by all four methods. Peptides with sequences corresponding to the predicted epitopes were synthesized and tested for reactivity with 1E8 by ELISA. As shown in Fig. 3,1E8 reacted strongly with a peptide with the sequence KKDSTKWRK, corresponding to residues 65-73 in the HIV-1 RT sequence, but had essentially no reactivity with the other peptides.

In order to confirm that RT residues 65-73 comprised the epitope recognized by 1E8, monospecific rabbit antiserum to ~ e p t i d e ~ ~ - ~ ~ was prepared and tested for its ability to compete for the binding of 1E8 to RT. ELISA plates coated with p51/ p66 RT were preincubated with rabbit antiserum to peptide65- 73, then subjected to normal ELISA analysis using the murine monoclonal 1E8. As shown in Fig. 4, the anti-peptide serum significantly blocked the ability of 1E8 to bind to RT. The

1.4 I 1

-.- C 1 2 3 4 5 6 7

Peptide Antigen FIG. 3. Reactivity of 1E8 with synthetic peptides corre-

sponding to antigenic regions of HIV-1 RT. Peptides with sequences corresponding to computer-determined antigenic regions of HIV-1 RT were synthesized with CGG linkers at the N terminus. These peptides were coupled to ovalbumin carrier as described under "Materials and Methods." Reactivity of 1E8 with the peptide-carrier

peptides were as follows: 1 , CGGKKDSTKWRK, 2, CGGELNKR- conjugates was assessed by ELISA. The sequences of the synthetic

TQD; 3, CGGAGLKKKKSVT, 4, CGGLDEDFRKYT; 5, CGGFKK- QNPDI; 6, CGGQHRTKLEELR 7, CGGTPDKKHQKE. C, control ovalbumin alone.

1.2,

1 .o 1

E 0.8

- 9 0.6

m r

0 e Y) 0.4 s

0.2

0.0 I "- l o - 1 10 -2 10 -3 C N

Antiserum FIG. 4. Competitive ELISA analysis of the ability of anti-

~ept ide '~"~ serum to prevent binding of 1E8 to HIV-1 RT. Plates coated with p51/p66 RT were incubated with buffer alone ( N ) , undiluted preimmune rabbit serum ( C ) , or the indicated dilutions of anti-~eptide"'~' serum for 1 h at 37 "C. Unbound material was removed by extensive washing with phosphate-buffered saline containing 0.5% Tween 20, and the plates then incubated with 1E8 (4 pl/ml) for 1 h at 37 "C. After washing as above, the extent of binding of 1E8 was assessed by the addition of peroxidase-conjugated goat anti-mouse IgG and the appropriate chromogenic substrate.

L 10 -4 10 -3 10 -2 10 -1

Antiserum Dilution

:nhibition of RT RNA-deDendent DNA Dolvmerase I activity by anti-~eptide~""~ serum.p5l/p66 RT (2.5 &ml) was incubated with the indicated dilutions of antiserum for 1 h at 37 "C. Aliquots of the incubation mixtures were then assayed for RNA- dependent DNA polymerase Activity in assays containing 0.2 unit/ ml poly(rA). oligo(dT) and 10 p~ [3H]TTP as described under "Ma- terials and Methods."

extent of this competition was proportional to the concentration of anti-peptide serum used in the preincubation, suggest- ing that both the anti-peptide serum and 1E8 compete for the same epitope on HIV-1 RT.

The a n t i - ~ e p t i d e ~ ~ - ~ ~ serum was also tested for its effect on RT activities. As noted with 1E8, the anti-peptide serum had no effect on RT RNase H activity (data not shown) but demonstrated a significant inhibitory effect on RT RNA- dependent DNA polymerase activity. This inhibition was proportional to the concentration of the anti-peptide serum used (Fig. 5), under the maximum inhibition noted was essen-

9984 Monoclonal Antibody Inhibition of HIV-I Reverse Transcriptase

tially identical to that given by 1E8 (compare Fig. 5 with Table I). As observed with 1E8, the extent of inhibition of R T polymerase activity by a n t i - ~ e p t i d e ~ ~ - ~ ~ serum was a function of the concentration of TTP used in the incubation and assay (data not shown).

DISCUSSION

HIV-1 reverse transcriptase is a logical target for the development of new specific antiviral agents, since there are few eukaryotic cell analogs for this enzyme. The development of effective inhibitors requires detailed knowledge of the func- tionally important domains of RT. Immunochemical ap- proaches employing monoclonal antibodies provide a useful method for the study of protein structure-function relation- ships (Cotton, 1985; Friguet et al., 1989; Goldberg, 1991). A number of groups have described monoclonal antibodies prepared against HIV-1 RT (Hansen et al., 1988 Tisdale et al., 1988 Ferris et al., 1990; Ferns et al., 1991; Orvell et al., 1991; Li et al., 1993; Restle et al., 1992; Szilvay et al., 1992). Although most of these antibodies have been ineffective in neutralizing the enzymatic activities of RT, a few monoclonal antibodies that inhibit RT polymerase activity have been noted (Ferns et al., 1991; Orvell et al., 1991; Restle et al., 1992; Szilvay et al., 1992). These polymerase-neutralizing antibodies recognize epitopes predominantly within residues 160-300 of HIV-1 RT, in the middle third of the enzyme sequence. In most cases, detailed characterizations of this antibody-mediated inhibition of RT polymerase activity were not carried out.

In the present report, we have extensively characterized a murine monoclonal antibody (1E8) that very effectively in- hibits the RNA-dependent and the DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase. Maximal inhibition was obtained at an approximately equimolar ratio of IgG to p51/p66 R T heterodimer. Unlike the previously described R T polymerase-neutralizing antibodies, 1E8 recognizes a linear epitope mapped to residues 65-73 of HIV-1 RT, very near the N terminus of the enzyme. Since 1E8 binds to R T in solution with good affinity, this region is presumably on the surface of the protein. These residues form a portion of the “finger” subdomain of RT, part of the polymerase binding cleft, and have been proposed to be important for the binding of primer (Kohlstaedt et al., 1992). Our data imply that these residues may also play a role in the binding of deoxynucleoside triphosphate substrate. Inhibition of R T polymerase activity by 1E8 was competitive with respect to dNTP substrate, implying that both dNTP and 1E8 bind to similar regions of RT. In contrast, the antibody-mediated inhibition was noncompetitive with respect to template/ primer. In addition, template/primer was unable to inhibit the binding of 1E8 to RT.

Although 1E8 bound both to free RT and to the RT- template/primer complex with apparently equal affinity, dra- matic decreases in the binding of the antibody to the enzyme were noted upon addition of dNTP to the RT-template/ primer complex. This inhibition was proportional to the concentration of dNTP. However, dNTP alone was rather ineffective in inhibiting the binding of 1E8. DNA synthesis by HIV-1 R T occurs by an Ordered Bi Bi mechanism, in which the binding of template/primer is followed by the binding of dNTP to form the RT-template/primer-dNTP ternary complex (Majumdar et al., 1988; Kedar et al., 1990; Reardon, 1992). If the dNTP is complementary to the nucleotide residue of the template, the ternary complex undergoes a conformational change to form the catalytically competent complex. Studies using measurements of R T fluorescence to study dNTP interaction with the enzyme have indicated that the

relative affinity of the RT-template/primer complex for the binding of dNTP is not affected by the identity of the complementary nucleotide in the bound template/primer (Painter et al., 1991). These investigators have concluded that Watson- Crick base pairing is thus not important in the initial binding of dNTP. Our data show that only the dNTP complementary to the template nucleotide was effective in preventing the association of the antibody with the enzyme-template/primer complex. I t is thus possible that the observed inhibition of 1E8 binding to the RT-template/primer complex by dNTP complementary to the template may result from a conformational change in the enzyme ternary complex, subsequent to the initial dNTP binding reaction, which results in the formation of the catalytically active complex in which the epitope recognized by 1E8 is no longer accessible to the antibody. In this case, the inhibition of R T polymerase activity by 1E8 might be due in part to the inability of the antibody-RT complex to undergo the necessary conformational change required for catalysis.

Previous studies using monoclonal antibodies have concluded that residues 200-230 form part of the dNTP binding site (Restle et al., 1992). Additionally, chemical modification studies imply tha t LyP3 (Basu et al., 1989) and Arg277 (Mitch- ell and Cooperman, 1992) may also be active site residues. These regions may seem somewhat distant from residues 65- 73, the putative dNTP binding region identified in the present report. However, molecular modeling studies (Lindborg, 1992; Narasimhan and Maggiora, 1992) and crystallographic analysis (Kohlstaedt et al., 1992) indicate that these two different regions may be closely associated in the tertiary conformation of the enzyme. Both regions of the enzyme may thus play a role in the formation of a binding pocket for dNTP substrate.

Finally, it is interesting to note that two mutations correlated with resistance to AZT (3’-azido-3’-deoxythymidine) have been identified in the epitope recognized by 1E8 (Larder et al., 1989; Larder and Kemp, 1989). This observation is consistent with our assumption that residues 65-73 are important for the binding of dNTPs. The mechanism by which these mutations (Asp67 --., Asn; Lys70 + Arg) lead to resistance to AZT is still unclear. A recent steady state study revealed no significant differences in the inhibition by AZT triphosphate of drug-resistant R T compared to the wild type enzyme (Lacey et al., 1992). More comprehensive investigation of the binding events involved in the formation of the ternary complex and its isomerization to a catalytically active species may yield information concerning the mechanism of drug resistance.

Acknowledgment-We thank S. Fraiberg for assistance in the preparation of the manuscript.

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