Vol. 36, No. 7

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, p. 1441-1446

0066-4804/92/071441-06$02.00/0 Copyright © 1992, American Society for Microbiology

Identification of the Human Immunodeficiency Virus Reverse Transcriptase Residues That Contribute to the Activity of Diverse Nonnucleoside Inhibitors JON H. CONDRA,* EMILIO A. EMINI, LEAH GOTLIB, DONALD J. GRAHAM, ABNER J. SCHLABACH, JILL A. WOLFGANG, RICHARD J. COLONNO,t AND VINOD V. SARDANA Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486-0004 Received 26 March 1992/Accepted 8 May 1992

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds. These include pyridinone derivatives, tetrahydroimadazo[4,5,1-j,kJ[1,41-benzodiazepin-2(lH)-one and -thione, and BI-RG-587 (nevirapine). The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates. Despite a high degree of similarity between the 1IV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors. Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities. The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined. Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188. The region defined by residues 101 to 106 was found to functionaliy interact with the domain from 155 to 217. In addition, the functional equivalence of the three inhibitor groups was shown.

Upon infection of susceptible cells by human immunodeficiency virus type 1 (HIV-1), the viral reverse transcriptase (RT) catalyzes the synthesis of a double-stranded DNA copy of the viral RNA genome. This reverse transcription is essential for viral infectivity, as inhibition of RT blocks viral replication. Thus, this enzyme has been a major focus in anti-HIV drug development and is the target of the nucleoside analogs 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), which are the only currently approved drugs for anti-HIV therapy. These drugs appear to act by mediating premature chain termination of nascent DNA strands (reviewed in reference 17). Neither of these drugs is absolutely HIV specific, and treatment is associated with toxicity that limits their long-term use in the clinic. A separate pharmacologic class of nonnucleoside inhibitors was recently identified and consists of several distinct structural classes of compounds. These are shown in Fig. 1 and include L-697,639, a member of the pyridinone class of inhibitors (7, 23); BI-RG-587 (nevirapine), one of several inhibitory dipyridodiazepinones (16); and R82150 and R82913, tetrahydroimadazo[4,5,1-j,k][1,4]-benzodiazepin-2 (1H)-one and -thione (TIBO) compounds (19). Inhibition of RT activity by these compounds is characteristic of a slowly binding, reversible inhibitor and is noncompetitive with respect to template-primer and nucleotide substrates (6, 7, 16, 29, 30). In contrast to the nucleoside analogs, these compounds are highly specific for HIV-1 RT (RT1), and little or no inhibitory activity is observed against a variety of other viral or cellular polymerases, including the HIV-2 RT (RT2) (6, 7, 16, 29). A major concern in any antiviral drug development effort *

is the potential emergence of drug-resistant virus variants. Resistance to AZT or ddI accompanies patient treatment with these drugs (2, 10, 11, 20, 22, 26). Similarly, drugresistant variants of HIV-1 have been selected by virus growth in cell culture in the presence of either the pyridinone inhibitors (18) or BI-RG-587 (21). These studies demonstrated that variants resistant to inhibition by the pyridinone compounds are equivalently resistant to inhibition by the TIBO compounds and BI-RG-587, thereby suggesting the functional similarity of these diverse classes. The development of clinical resistance to one structural member of this group would likely engender resistance to the others. It was therefore of interest to define the structural basis for sensitivity of RT1 to the nonnucleoside inhibitors and to define the basis for potential resistance. We exploited the total insensitivity of RT2 to inhibition by these compounds and constructed a series of molecular chimeras of RT1 and RT2. These chimeras, along with several site-directed mutants, were used to map the RT residues essential for the activity of the nonnucleoside inhibitors. These results provide insight into the basis of RT inhibition by the compounds and firmly establish their functional equivalence.

MATERIALS AND METHODS Cloning of HIV RT. The cloning and expression of the HIV-1 (NY-5 strain) RT gene in Escherichia coli have been described previously (1). The vector originally used was a modified version (13) of pKK223-3 (3). In order to reduce basal expression levels of RT from the tac promoter of the vector, the original plasmid was modified to express the E. coli lac repressor. To accomplish this, a 1.2-kbp fragment containing the lacd gene and its promoter was amplified by polymerase chain reaction (PCR) from plasmid pMAL-C (Pharmacia) by incorporating BspEI sites into the 5' ends of

Corresponding author.

t Present address: Bristol Myers-Squibb Research Institute, Princeton, NJ 08543-4000. 1441

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CONDRA ET AL.

RT1 IC50 (AM) c

N

L-697,639

0.047 ± 0.004 H

BI-RG-587 (Nevirapine)

0.175 ± 0.014

R82150

0.126 ± 0.017

(TIBO)

HNt

H

R82913

(Cl-TIBO)

ci

N

0.381 ± 0.024

#H 3

FIG. 1. Structures of HIV-1-specific nonnucleoside inhibitors. Chemical names: L-697,639, 3-{[(4,7-dimethyl-1,3-benzoazol-2-yl)

methyl]amino-5-ethyl-6-methyl-pyridin-2(1H)one; BI-RG-587, 5,11dihydro-11-(cyclopropyl)-6-methyl-6H-dipyrido-[3,2-B:2',3'-E] [1,4] diazepin-6-one; R82150, 4,5,6,7-tetrahydro-5(S)-methyl-6-(3-methyl2-butenyl)imidazo[4,5,1-j,k][1,4]benzodiazepin-2(1H)-thione; R82913, 4,5,6,7-tetrahydro-5(S)-methyl-6-(3-methyl-2-butenyl)-9-chloroimidazo[4,5,1-j,k][1,4]benzodiazepin-2(1H)-thione. IC50s for wild-type recombinant RT1 are shown and are represented as means standard errors from multiple determinations.

primers, digesting the amplified fragment with BspEI, cloning it into the unique SgrAI site of the original expression vector. The resulting plasmid, pKK-lacI, was used to express all subsequent RT constructs. The RT1 gene was recloned into pKK-lacI as an EcoRI-HindIII fragment, to yield pRT1-lacI. The complete coding sequence of the HIV-2 (ROD strain) RT p64 was cloned by PCR amplification from XHIV-2RoD (obtained from the NIH AIDS Research & Reference Program) by using the 5' and 3' primers CCGATAACCTGCA TAGAATTCATGCCAGTCGCCAAAGTAG and AGATCT AAGCYIACAACACTTlGTCTGATACC, respectively. Because the RT2 gene contains an internal EcoRI site, a BspMI site (ACCTGCN4 0) was positioned 4 nucleotides 5' of an EcoRI cleavage site (G I AATTC) in the 5' PCR primer. This allowed BspMI digestion to generate an EcoRI terminus without cleavage of RT2's internal EcoRI site and simultaneously removed the extraneous bases from the RT gene fragment. Immediately following the EcoRI site, an ATG initiation codon was followed by the first Pro codon of the mature RT gene. At the 3' end, a termination codon and HindIII site were inserted by the PCR primer. The PCR product was digested with BspMI and HindIII and cloned into the EcoRI-HindIII sites of pKK-lacI to yield plasmid pRT2-lacI. The resulting RT2 plasmid contained an initiation codon followed by the N-terminal proline codon of p64 the and

normally derived by processing by HIV protease. The complete insert sequence was verified by DNA sequencing. Molecular cloning manipulations other than those described below were carried out by standard procedures (15). Oligonucleotide-directed mutagenesis. Point mutations and block substitutions involving fewer than 16 codons were generated by oligonucleotide-directed mutagenesis of plasmid DNA by a modification of the gapped-duplex method, as described previously (5). Construction of chimeric RT genes. RT1/RT2 chimeric genes were constructed by a novel, general method for gene fusion, which allows the joining of any DNAs in a predetermined way, irrespective of sequence homology or the locations of restriction sites. The method relies on the fact that class IIS restriction enzymes, e.g., BspMI (ACCTGCN4 i / TGGACGN8 t ), recognize nonpalindromic sequences and make staggered cuts in DNA a fixed distance away from these sequences (reviewed in reference 28). Recognition and cleavage sequences for a class IIS enzyme that does not normally cut the target DNA, e.g., BspMI (ACCT GCN4 1 N4), are inserted near the 5' ends of PCR primers used to amplify subfragments of RT1 and RT2. Following the cleavage sites, the remaining primer sequences determine the priming specificity by standard base pairing. By positioning the BspMI sites a suitable distance 5' of the desired cleavage sequences (5 to 8 bases for BspMI) and by choosing the bases within the cleavage sites to be compatible with their intended ligation partners (which may be prepared the same way or by conventional restriction enzyme digestion), any desired compatible fragments can be created. Digestion of these molecules with BspMI simultaneously removes the BspMI sites and creates the desired cohesive ends for fragment ligation, yielding a seamless junction. By this method, nonpalindromic cohesive ends can be generated, preventing self-ligation of the fragments and allowing the ligation of multiple fragments into a unique, predetermined structure. To construct RT1/RT2 chimeras, the desired subfragments of RT1 and RT2 genes were recovered from pRT1-lacI and pRT2-lacI by PCR amplification. The 5' termini of the N-terminal coding fragments were engineered to contain an EcoRI cloning site and initiation codon as described above for RT2 cloning. The C-terminal coding fragment contained a stop codon and a HindIII site for cloning at its 3' end. Nonpalindromic cohesive termini for joining the RT fragments were created at the desired recombination points as described above. Following PCR amplification, the products were digested with BspMI and HindIII and then cloned by multifragment ligation reactions into the EcoRI and HindIII sites of pKK-lacI. Complete coding regions of all chimeras were verified by DNA sequencing. Expression and purification of recombinant RTs. Expression was performed in E. coli AB1899 (8) (from the E. coli Genetic Stock Center) or BL21 (27) (from Novagen). To minimize potential selection against expression, all cultures were inoculated with colonies from fresh transformants of plasmid DNA on the morning of the experiment. Cultures (500 ml) were grown in shake flasks at 37°C in LB broth (15) containing 100 p.g of ampicillin per ml to an A6. of 0.5. Isopropyl-,-D-thiogalactopyranoside (IPTG) was then added to 1 mM, and the culture was shaken for 2 h at 25°C for induction. Cells were harvested by centrifugation, and pellets were frozen at -70°C until used for purification. Expression was verified by Coomassie blue staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels (9), by Western

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MAPPING OF HIV RT DRUG RESISTANCE DETERMINANTS

blotting (immunoblotting) using monoclonal or polyclonal antibodies specific for RT, and by RT assays of cell lysates. Frozen pellets from 500-ml cultures were suspended in 18 ml of 50 mM Tris-HCI (pH 7.8)-4 mM EDTA-50 mM KCI-5% (vol/vol) glycerol-50 p,g of soybean trypsin inhibitor per ml-1 ,ug of aprotinin per ml-1 ,g of leupeptin per ml. Just before cell lysis, phenylmethylsulfonyl fluoride was added to a 2 mM final concentration, and then cells were lTsed by four passages through a Stansted Press at 90 lb/in with 5- to 10-lb/in2 back pressure. Dithiothreitol was added to a 1 mM final concentration, and an equal volume of DE-52 cellulose (Whatman), previously equilibrated in 50 mM Tris-HCI (pH 7.8)-50 mM KCI-4 mM EDTA-1 mM dithiothreitol, was mixed with the cell lysate. The resin was centrifuged 30 min at 10,000 x g, and the supernatant was recovered and retreated with DE-52 as described above. Some of the chimeric RTs bound to the DE-52 resin under these conditions. When this occurred, the RT was recovered by back extracting the resin with the above buffer containing 150 mM KCI. The supernatant was concentrated to 1 to 2 ml and exchanged into buffer A (20 mM NaPi [pH 6.5 or 7.5], 4 mM EDTA, 1 mM dithiothreitol) in a Centriprep-30 apparatus (Amicon). Concentrated samples were injected onto a Mono-S fast-protein liquid chromatography column (0.5 by 5 cm; Pharmacia), and the enzyme was eluted at 1 ml/min with a 30-min linear 0 to 500 mM KCI gradient in buffer A. The RT activity eluted at approximately 150 to 200 mM KCI. Column fractions were assayed for RT activity and analyzed by SDS-polyacrylamide gel electrophoresis. Final enzyme purities were 70 to 85%. RT assays. L-697,639 (7, 23), BI-RG-587 (16), and R82150 (19) were synthesized in-house. The TIBO derivative R82913 (19) was purchased from PharmaTech International, Inc., West Orange, N.J. Enzyme assays were carried out in the presence or absence of-inhibitors as previously described (7) by using the rC-dG template-primer system, measuring incorporation of [3H]dGTP into acid-precipitable material. Suitable quantities of RT to incorporate 10 to 20 pmol of nucleotide were used for each assay. The RT activities of all recombinant enzymes were shown to be sensitive to 100 ,uM phosphonoformic acid. Inhibitors were dissolved in dimethyl sulfoxide and added to the assays in desired concentrations, maintaining the final dimethyl sulfoxide concentration at 6,000 >6,000 >6,000 >6,000 >6,000 42.8 (+5.9)

1.0 > 1,700 >1,700 >1,700 >1,700 >1,700 >1,700

1.0 >790 >790 >790 >790 >790 66.9 (+1.8)

a IC50s relative to RT1 were determined by incorporation of [3H]dGTP into acid-precipitable material, using an rC-dG template-primer. Data are reported as the geometric means (± geometric standard errors) of multiple determinations and were calculated as described in Materials and Methods. IC50s for the highly resistant RTs could not be determined because of solubility limits for the compounds.

acquire drug resistance through amino acid changes K to N at position 103 (K103N) and/or Y to C at position 181 (Y181C) (18). The effects of the single mutations were more than additive in the double mutant, K103N/Y181C. In addition, these mutations conferred cross-resistance to L-697, 639, TIBO compounds, and BI-RG-587, suggesting that these residues interact and lie in or near the binding site(s) for these compounds. To examine the role of these residues in drug resistance, site-directed mutants in these regions were constructed. A mutant of RT1 containing the RT2 residue at position 181 (RT1 Y1811) showed full drug resistance (Table 1). However, substituting the RT1 Y-181 residue into an RT2 background (RT2 I181Y) did not detectably reduce the RT2 drug resistance. This suggests that I-181 is partially responsible for the HIV-2 enzyme's resistance to the inhibitors but that additional residues of RT2 contribute significantly to the phenotype. Because a K103N mutation in RT1 has also been shown to confer viral resistance to these compounds (18), we wished to explore the potential involvement of this region in the drug resistance exhibited by RT2. Although both RT1 and RT2 have a lysine at this position, the two enzymes differ in five of the six neighboring residues from positions 101 to 106. In order to determine whether this region, together with I-181, could account for the drug resistance of RT2, we constructed a site-directed mutant of RT2 containing a block substitution of RT1 residues 101 to 106 and RT1 residue Y-181. As shown in Table 1, this mutant (RT2 [101-106]RT1/ I181Y) was also fully resistant to all three drugs and indistinguishable from RT2. Therefore, the region from 101 to 106 and residue 181 do not alone explain the drug resistance of RT2. If the determinants of drug sensitivity and resistance map to a defined region(s) of these molecules (as opposed to global conformational differences between the enzymes), it should be possible to map the contributing regions in a systematic way by constructing molecular RT1/RT2 chimeras and correlating the sequences with the resulting drug sensitivity phenotypes. Although both enzymes share about 66% amino acid sequence identity, the two genes share few restriction sites that would be useful as recombination points to create chimeric molecules. Therefore, we developed a general strategy that permits joining of virtually any combination of DNA sequences in a predetermined way. The technique exploits the cleavage properties of class IIS restriction enzymes and is described in Materials and Methods.

ANTIMICROB. AGENTS CHEMOTHER.

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777

1 1 1 I I I

m

.M I

I

I

I

I

m-

RT1 RT2 100A 128A 155A 217A 410A 100B 155B 217B 410B 100A/217B/A 100B/217A/B

100A/106B/155A/217B/A 100B/106A1155B/217A/B 170B/185A/B 175B/19OA/B

100B/106A/175B/190A/B FIG. 2. Representation of chimeric RT1/RT2 constructs. Open areas correspond to HIV-1 (NY-5) RT sequences, and shaded areas represent HIV-2ROD RT sequences. Numbers represent amino acid residues where recombination points between RT1 and RT2 sequences are located. Chimeras ending with A have RT1 sequences in the N-terminal segment, and those ending with B start with RT2 sequences. For example, chimera 175B/190A/B consists of RT2 residues 1 to 175, RT1 residues 176 to 190, and RT2 residues 191 to 560.

By this method, a family of RT1/RT2 chimeras was constructed for expression in E. coli, as shown in Fig. 2. The N-terminal boundary of RT2's drug resistance determinants was localized by comparing a series of chimeras with N-ter-

minal RT1 sequences and C-terminal RT2 sequences (Aseries chimeras; see legend to Fig. 2 for nomenclature). As noted in Table 2, chimeras 100A (i.e., RT1 residues 1 to 100, followed by RT2), 128A, and 155A remained fully drug resistant, while sensitivity was observed in chimeras 217A and 410A. This indicates that the N-terminal boundary of RT2's drug resistance maps between amino acid residues 155 and 217. Similarly, the C-terminal boundary of the residues conferring drug resistance was localized by comparing a series of chimeras containing various lengths of N-terminal RT2 sequences fused to C-terminal RT1 sequences (the B series). Chimeras lOOB and 155B remained fully drug sensitive, while 217B and 410B were as resistant as RT2 (Fig. 2; Table 2). This places the C-terminal boundary of the resistance determinants also between amino acid residues 155 and 217. To verify these boundaries further, two reciprocal doublerecombinant chimeras were constructed with recombination points at codons 155 and 217. However, these chimeras (155A/217B/A and 155B/217A/B, not shown) had such low enzymatic activities that their drug resistance phenotypes could not be determined. We reasoned that this low activity might result from the loss of a critical interaction between the domain from 155 to 217 and additional residues of the enzyme. We further reasoned that the latter might be near residue 103, given the influence of both this residue (18) and the domain from 155 to 217 upon sensitivity to the inhibitors. Accordingly, reciprocal constructs containing block substitutions of the interval from 101 to 217, chimeras 100A/ 217B/A and 10OB/217A/B (Fig. 2), were constructed and found to be fully active (data not shown). In these recombinants, the drug sensitivity phenotypes correlated with the parental segments from 101 to 217 (Table 2). Further, we confirmed that the region surrounding residue 103 was indeed critical for enzymatic activity, since introducing the homologous regions from 101 to 106 into the 155/217 chimeras, generating 10OA/106B/155A/217B/A and 10OB/106A/ 155B/217A/B (Fig. 2), also restored RT activity (data not

TABLE 2. Drug resistance phenotypes of RT1/RT2 chimeras Relative IC50b of:

Enzymea RT1 RT2 100A 100B 128A

155A 155B 217A 217B 410A 410B

100A/217B/A 100B/217A/B

100A/106B/155A/217B/A 100B/106AI1SSB/217A/B 106A/180B/181A/217B/A

170B/185A/B 175B/19OA/B

100B/106A/175B/19OA/B

L-697,639

BI-RG-587

1.0 >6,000 >6,000 0.81 (±0.17) >6,000 >6,000 1.33 (±0.18) 6.99 (±2.63) >6,000 1.79 (±0.85) >6,000 >6,000 1.96 (±0.21) >6,000 1.84 (±0.38) >6,000 >6,000 13.0 (±3.95) 6.81 (±0.50)

1.0 >1,700 >1,700 1.14 (±0.20) >1,700 >1,700 1.33 (±0.41) 11.0 (±0.8) >1,700 0.61 >1,700 >1,700 1.52 (±0.51) >1,700 2.51 (±0.41) >1,700 >1,700 86.6 (±23.2) 39.3 (±6.85)

R82150

1.0

>2,380 >2,380 0.93 (±0.06) >2,380 >2,380 1.25 39.0 (±0.10) >2,380 3.26 (±0.93) >2,380 >2,380 7.43 (±1.66) >2,380 3.73 (±1.37) >2,380 >2,380 28.2 (±4.30) 33.4 (±4.59)

a See legend to Fig. 2 for nomenclature. b IC50s relative to RT1 were determined by incorporation of [3H]dGTP into acid-precipitable material, using an rC-dG template-primer. Data are reported as the geometric means (+ geometric standard errors) of multiple determinations and were calculated as described in Materials and Methods. IC50s for the highly resistant chimeras could not be determined because of solubility limits for the compounds.

VOL. 36, 1992

MAPPING OF HIV RT DRUG RESISTANCE DETERMINANTS

shown). As predicted, the drug phenotype tracked with the segments from 101 to 106 and 155 to 217 (Table 2). In an attempt to further define the residues within the region from 155 to 217 that contribute to drug sensitivity, smaller chimeric substitutions near residue 181 were constructed. Substitution of RT1 residues 171 to 185 into an RT2 background (chimera 170B/185A/B) did not sensitive the enzyme to these inhibitors (Fig. 2; Table 2). However, introducing RT1 residues 176 to 190 into RT2 (chimera 175B/19OA/B) significantly sensitized the enzyme to all three inhibitor classes. This chimeric enzyme was only 13- to 87-fold less sensitive than RT1 (Fig. 2; Table 2). The residual resistance of the chimera was not overcome by the addition of the RT1 region from 100 to 106; the drug responses of chimera 100B/106A/175B/190A/B were not significantly different from those of 175B/190A/B (Fig. 2; Table 2). Finally, recent photoaffinity labeling experiments identified Y-181 as the major RT1 residue interacting with BI-RG70, an analog of BI-RG-587 (4). In addition, Y-188 was also found to be weakly labeled, suggesting that it also interacts with the drug. Accordingly, we constructed a site-directed double mutant of RT2 containing the RT1 residues at positions 181 and 188 (RT2 I181Y/L188Y). This mutant expressed a sensitive phenotype with respect to inhibition by L-697,639 and by a TIBO compound but not by BI-RG-587 (Table 1). In contrast, the single mutants RT2 I181Y and RT2 L188Y were fully resistant to the three compounds (Table 1).

DISCUSSION The three structurally diverse classes of nonnucleoside inhibitors used in this study all have significant clinical potential for the control of HIV-1 infection. Yet this potential may be limited by the emergence of resistant virus variants. Such variants in fact have been derived in cell culture by selection with the pyridinone compounds and with BI-RG-587 (18, 21). These variants exhibit cross-resistance to inhibition by the different nonnucleoside classes. Similarly, RT2 is fully resistant to all the inhibitors and may be considered a prototypic resistant variant. An understanding of the structural basis for RT sensitivity and resistance to the nonnucleoside inhibitors is critical for monitoring the potential development of clinical resistance. In addition, novel insights into RT structure and function may be obtained. Hence, chimeras between the sensitive RT1 and resistant RT2 enzymes were prepared along with several site-specific mutant enzymes. The relative sensitivities of the mutants to each of the three nonnucleoside inhibitor classes were determined. In this way, RT residues that are essential for the activity of the inhibitors were identified. The minimal sequence identified in this study that confers sensitivity to inhibition is delineated by amino acid residues 176 to 190. Substitution of this region from RT1 into RT2 results in a chimeric enzyme that is somewhat sensitive to inhibition by all three compound classes. The analysis of site-specific mutants within this segment further showed that amino acid residues 181 and 188 were primarily responsible for the activity of the pyridinone and TIBO compounds. Substitution of tyrosine at both these positions within RT2 sensitized the enzyme to inhibition by L-697,639 and TIBO compounds; however, sensitization to inhibition by BI-RG587 was not seen. It is possible that BI-RG-587 binding requires an enzyme contact(s) in addition to Y-181 and Y-188 that is not required for binding the TIBO or pyridinone inhibitors. Thus, in view of the clear structural differences

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between the three classes of compounds (Fig. 1), they may exhibit subtle differences in their interactions with the various amino acid residues of a common binding site. While this manuscript was in preparation, Shih et al. (25) reported the results of similar experiments in which the tyrosine replacements at 181 and 188 yielded a minimal sensitization of the RT2 enzyme to BI-RG-587. As in our study, a higher degree of sensitization was noted upon replacement of the region from 176 to 190 from RT1. In a separate study, Shaharabany and Hizi (24) have also employed RT1/RT2 chimeras to map TIBO compound sensitivity to the region from 93 to 226. Taken together, these results suggest that the three classes of nonnucleoside inhibitors probably interact directly with the amino acid residues at 181 and 188. This supports the results of photoaffinity studies performed with BI-RG-70 (4) that yielded specific labeling of these residues. However, other residues and/or domains within the RT also influence the activity of the inhibitors, and the degree of this influence upon the three inhibitor structural classes is apparently different. Residues 101 to 106 may constitute such a region. Nunberg et al. (18) reported that an alteration at position 103 resulted in a 10-fold loss of viral sensitivity to the pyridinone inhibitors. Similarly, we noted that the expression of functional RT required an appropriate interaction between the regions from 101 to 106 and 155 to 217. Chimeric enzymes that possess only the latter segment within the heterologous background were found to be inactive. This is consistent with the reported loss of enzymatic activity in other RT1/ RT2 chimeras with recombination points at residue 126 or 143 (24). Also, previous studies have shown that aspartic acid residues 110, 185, and 186 are essential for RT activity but not for primer-template binding, and mutations at residue 110, 113, 114, or 183 can affect both nucleotide and phosphonoformate binding to RT (12, 14). We therefore suggest that residues within both regions contributing to PPi exchange and/or nucleotide binding may also interact with the nonnucleoside inhibitors. As a result, alterations within either region will probably contribute to clinically significant resistance development. It will be important to carefully analyze these sequences in primary HIV-1 isolates from patients undergoing treatment with any of the nonnucleoside inhibitor classes. Finally, the results of our studies demonstrate the general functional equivalence of these structurally diverse compounds and predict that the derivation of clinically resistant variants during patient therapy with one compound may result in the loss of clinical activity for all of the nonnucleoside inhibitors. ACKNOWLEDGMENTS We thank Don Lineberger and Bill Long for assistance in protein purifications; Jacob Hoffman, Stan Rooney, and John Wai for providing inhibitors; Tim Schofield for performing statistical analyses; MaryJo Zaborowski for computer sequence analyses; Jules Shafer, Mark Goldman, David Olsen, Katharine Holloway, and Andrew Stern for helpful discussions; and Dolores Wilson for aid in preparation of the manuscript. REFERENCES 1. Azzolina, B. A., N. D. Behrens, B. Chang, M. E. Dahlgren, H. George, J. G. Menke, D. L. Linemeyer, and D. L. Hupe. 1990. Cloning, expression in Escherichia coli, and purification of HIV-1 reverse transcriptase. FASEB J. 4:A2253. 2. Boucher, A. B., M. Tersmette, J. M. A. Lange, P. Kellam, R. E. Y. deGoede, J. W. Mulder, G. Darby, J. Goudsmit, and B. A. Larder. 1990. Zidovudine sensitivity of human immuno-

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Identification of the human immunodeficiency virus reverse transcriptase residues that contribute to the activity of diverse nonnucleoside inhibitors.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside c...
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