Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites Anita Fehe ´ r 1 , Irene T. Weber 2 ,Pe ´ ter Bagossi 1 ,Pe ´ ter Boross 1 , Bhuvaneshwari Mahalingam 2 , John M. Louis 3 , Terry D. Copeland 4 , Ivan Y. Torshin 5 , Robert W. Harrison 5 and Jo ´ zsef To¨ zse ´ r 1 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary; 2 Department of Biology, Georgia State University, Atlanta, GA, USA; 3 Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA; 4 NCI-Frederick, Frederick, MD, USA; 5 Department of Computer Science, Georgia State University, Atlanta, GA, USA The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibi- tors are now in clinical use. However, there is a rapid selec- tion of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 clea- vage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polypro- tein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural varia- tions or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural varia- tions showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Com- parison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases. Keywords: HIV-1 proteinase; Gag processing sites; oligo- peptide substrates; substrate specificity; molecular modeling. All replication competent retroviruses code for an aspartic proteinase (PR) whose function is critical for virion replication (reviewed in [1]). The HIV-1 PR has proved to be an excellent target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use (reviewed in [2]). However, as observed with reverse transcriptase inhibitors, resistant viruses rapidly emerge in PR inhibitor therapy. Moderate to high level of resistance (2- to 100-fold) to PR inhibitors has been observed both in vitro and in vivo, and has been attributed to the appearance of mutations in the PR gene. Many of these mutations are located in the substrate binding site of the PR, and these mutations have considerable impact on PR activity and specificity. Other resistant mutations alter residues outside of the substrate binding site. The compromised catalytic capability of the multiple drug resistant HIV-1 mutants is reflected by impaired processing of Gag precursors in PR-mutated virions [3,4] and by decreased in vitro catalytic efficiency of the PR towards peptides representing natural cleavage sites [5–8]. The development of high levels of resistance to PR inhibitors, possibly requiring multiple mutations in the PR, was therefore expected to be limited by the functional constraints of the enzyme, which must cleave all precursor cleavage sites during viral replication. Subsequently, a second locus was found to be involved in drug resistance to HIV PR inhibitors, both in vitro and in vivo,atthe nucleocapsid (NC)/p1 and p1/p6 cleavage sites [9–14]. Evolution of PR cleavage sites other than NC/p1 and p1/p6 in the internal (P2-P2¢) positions is limited, and mutations are rarely observed even upon drug treatment [12]. Cleavage at these sites appears to be a rate limiting step in polyprotein processing [9,11]. Peptides representing these sites have the lowest specificity constants (k cat /K m ) among all HIV-1 cleavage sites [15,16]. Furthermore, there is a significant sequence polymorphism at these sites, which also may have an impact on virion infectivity [17–19]. Natural polymorphism and resistant mutations occurring at these sites are shown in Fig. 1. Some of these amino acid substitutions are frequently detected, others have been found only in one clone including the P1 Asp, Ile and Lys substituted NC/p1 sites, which are not expected to be cleaved by the PR, based on previous extensive specificity studies [20]. If cleavage at this site is important for virus replication, as indicated by the mutations seen in resistance, Correspondence to J. To ¨ zse ´ r, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, PO Box 6, Hungary. Fax: + 36 52 314989, Tel.: + 36 52 416432, E-mail: tozser@indi.biochem.dote.hu Abbreviations:MA,matrixprotein;CA,capsidprotein;NC,nucleo- capsid protein; PR, proteinase. Enzyme: retropepsin (EC 3.4.23.16). Note: nomenclature of viral proteins is according to Leis et al. [50]. (Received 9 May 2002, revised 8 July 2002, accepted 11 July 2002) Eur. J. Biochem. 269, 4114–4120 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03105.x clones containing these Gag mutations should be replication defective. Mutations outside of the P4-P3¢ region have also been reported at these sites in PR inhibitor therapy [12,21]. However, those residues of a substrate are far from the substrate binding subsites and are not expected to alter the PR specificity directly, but they may alter the confor- mation of the substrate region within the polyprotein or may enhance viral fitness in a manner unrelated to the PR-mediated processing rates. Here we report kinetic studies using oligopeptides repre- senting cleavage sites with representative, frequently occur- ring mutations found as sequence polymorphisms and in drug resistance, with wild-type and five drug-resistant mutant PRs: M46L, V82A, I84V and L90M mutations that appeared together with the Gag mutations in vivo,in PR-inhibitor therapy [10,12]. V82S mutation was found in ritonavir therapy [22]. I84V was found to be in vitro selected against saquinavir and amprenavir [18], together with Gag mutations against two BILA inhibitors [23], and in patients treated with ritonavir [22] or indinavir [24]. Based on clinical studies, the resistant Gag mutations may occur early in PR inhibitor therapy, soon after the appearance of one (or a few) critical PR mutations, therefore we have characterized the effect of single protease mutations in response to Gag cleavage site mutations. A similar approach could be used to characterize the more complex inhibitor-resistant proteases harboring all mutations appearing in vitro or in vivo in the presence of PR inhibitors. MATERIALS AND METHODS Oligopeptide synthesis and characterization Oligopeptides were synthesized by standard 9-fluorenyl- methyloxycarbonyl chemistry on a model 430A automated peptide synthesizer (Applied Biosystems, Inc.). All peptides were synthesized with an amide end. Amino-acid compo- sition of the peptides was determined with a Beckman 6300 amino-acid analyzer. Stock solutions and dilutions were made in distilled water and the peptide concentrations were determined by amino-acid analysis. Construction and purification of HIV-1 PR mutants HIV-1 PR (HIVHXB2CG) having stabilizing substitutions (Q7K, L33I, L63I, C67A and C95A) was cloned into a pET vector, expressed in Escherichia coli and purified to homo- geneity as described [25]. The proteolytic activity of this enzyme, designated as wild-type, was indistinguishable from that of the native PR [25]. DNA derived from this clone was used as a template for generating the mutant enzymes by site directed mutagenesis. Mutations were confirmed by nucleic acid sequencing and protein mass spectrometry. The mutant enzymes were purified as described [26]. Enzyme assay with oligopeptide substrates The PR assays were initiated by the mixing of 5 lL(0.05– 8 l M ) purified wild-type or mutant HIV-1 PR with 10 lL 2x incubation buffer (0.5 M potassium phosphate buffer, pH 5.6, containing 10% glycerol, 2 m M EDTA, 10 m M dithiothreitol, 4 M NaCl) and 5 lL0.5–7 m M substrate. The reaction mixture was incubated at 37 °Cfor1hand terminated by the addition of 180 lL 1% trifluoroacetic acid. Substrates and the cleavage products were separated using a reversed-phase HPLC method described previously [15]. Kinetic parameters were determined by fitting the data obtained at less than 20% substrate hydrolysis to the Michaelis–Menten equation by using the FIG . P program (Fig. P Software Corp.). For the NC/p1 peptide series the specificity constants (k cat /K m ) were determined by using competitive assays [27] with substrate KTGVL-fl-VVQPK [28], while for the p1/p6 peptide series, it was determined under pseudo first order conditions using 0.02–0.15 m M concentration range, in which the activity was a linear function of the substrate concentration. The standard error for kinetic values was below 20%. Active site titration The active amount of enzyme used in the assays was determined by active site titration using the potent HIV-1 PR inhibitor DMP-323 for the wild-type PR and for the M46L, V82A, I84V, L90M mutants, while the V82S mutant was titrated using amprenavir. Active site titrations were performed by using the HPLC method [15], except 0.2 lL aliquot of the inhibitor (0–10 l M ) in dimethylsulfoxide was added to the reaction mixture. The inhibition curves were determined at three substrate concentrations, and the active enzyme concentration was determined using the DYNAFIT program [29]. The standard error for the enzyme concen- trations was below 20%. Molecular modeling All models were built from the high resolution crystal struc- ture (PDB entry 1fgc) of HIV-1 PR-inhibitor complex [26] by altering the appropriate residues of enzyme and Fig. 1. Sequence around the NC/p1 and p1/p6 cleavage sites in HIV-1. The sequence of HIV-1 IIIB , a member of the B subtype, is shown. The P4-P3¢ region of the cleavage site sequences are marked with an upper line, these residues are expected to bind in the S4-S3¢ substrate binding subsites of the enzyme. Residues appearing in other HIV-1 virus iso- lates [18] are indicated under the sequence of HIV-1 IIIB , while residues appearing only in drug resistance are in bold [9–13]. Mutations underlinedintheFigurealsoappearedindrugresistanceonapar- ticular natural sequence background, however, the same residues have also been found in other natural sequences [18], therefore these mutations are considered to be due to sequence polymorphism, as they can be observed without the selective pressure caused by the PR inhibitors. Ó FEBS 2002 Studies on mutant HIV-1 processing sites (Eur. J. Biochem. 269) 4115 inhibitor. All water molecules in the crystal structure were used. A proton was placed between the carboxylate oxygens of catalytic aspartates D25 and D25¢, as described previ- ously [30]. Minimization with AMMP included short runs of molecular dynamics as described previously [31]. Analysis of hydrophobic interactions was performed with INTG [32]. RESULTS AND DISCUSSION Kinetic parameters for wild-type and mutant HIV-1 proteinase-catalyzed hydrolysis of matrix/capsid Gag cleavage site substrate Previous specificity studies based on the substrate repre- senting the matrix/capsid (MA/CA) cleavage site of HIV-1 suggested that this site is evolutionarily optimized for efficient hydrolysis, as most of the substitutions in the sequence resulted in less efficient cleavage, and none of the substitutions resulted in substantially improved hydrolysis [33–36]. We have tested the mutant HIV-1 proteinases on this substrate (Table 1). The K m values were increased for all mutants to varying degrees. This is in good agreement with previous studies showing that binding site mutants tend to increase K m values [5]. However, the k cat values for the mutants were also improved, in some cases (e.g. I84V) dramatically, yielding higher specificity constants (k cat /K m ) for these enzymes (Table 1). Crystal structures of the inhibitor bound HIV-1 PR with the studied mutations have been reported previously [8,26,37–39]. Met 46 forms part of the flap and Leu at this position could reduce its mobility, as suggested for the Ile mutant from molecular dynamics simulations [40]. PR with M46L mutation alters both K m and k cat for the HIV-1 MA/CA substrate, but yields a specificity constant similar to that of the wild-type enzyme (Table 1). V82 mutations to Ala or Ser increase the size of the internal substrate binding subsites, particularly S1 and S1¢,result- ing in larger K i values for inhibitors and increased K m values for the substrates. These mutants were also less efficient on other Gag cleavage site substrates [5,8,41]. The I84V mutation also influences the internal ligand binding sites [38], consistent with the observed improvement in the hydrolysis of the MA/CA substrate, as reported previ- ously for a CA/p2 substrate [42]. Others reported decreased catalytic efficiency for this mutant [5,43]. However, PR with I84V mutation did not substantially alter Gag processing and did not affect virion replication [23]. Leu 90 has no direct contact with the ligands, however, it is close to the dimer interface of the PR, and Met at this position substantially decreased protease stability under low ionic strength conditions [8,44]. However, this effect is not seen in the kinetics of the MA/CA hydrolysis. In the high ionic strength of the assays the dimerization is strengthened as compared to low ionic strength conditions [45]. Kinetic parameters for wild-type HIV-1 proteinase- catalyzed hydrolysis of NC/p1 and p1/p6 cleavage site substrates The oligopeptides were chosen to represent the naturally occurring NC/p1 and p1/p6 cleavage sites in HIV-1 HXB2 ,a representative isolate of the B subtype [18]. The kinetic parameters were determined for hydrolysis of the oligopep- tides by the wild-type HIV-1 PR (Table 2). The two peptides had low specificity constants (k cat /K m ), as compared to peptides representing other Gag and Gag-Pol cleavage sites tested under identical conditions (Table 1, [15]), in good agreement with the hypothesis that cleavage at these sites might be the rate limiting step of Gag processing [9]. Others also reported that peptides representing these cleavage sites are not efficient substrates of PR [7,16]. Although the specificity constants for the two substrates were similar, the K m and k cat values differed remarkably: the NC/p1 peptide exhibited low K m and k cat values, while the p1/p6 cleavage site showed both higher K m and k cat values (Table 2). Increasing the concentration of the NC/p1 substrate above the K m resulted in a decreased velocity, suggesting the possibility of increased nonproductive binding at the substrate binding site. Nevertheless, the specificity constants determined with a competition assay for the NC/p1 substrate, as well as under pseudo first order conditions for the p1/p6 substrate were in good agreement with the values calculated from the Michaelis–Menten curve (Table 2). Processing of peptides representing NC/p1 cleavage site sequences by wild-type and mutant HIV-1 proteinases Oligopeptides including both natural sequence polymor- phisms and resistant mutations of the NC/p1 cleavage site Table 1. Kinetic parameters for the hydrolysis of the oligopeptide rep- resenting the matrix/capsid cleavage site of HIV-1 (VSQNY-fl-PIVQ). Enzyme K m (m M ) k cat (s )1 ) k cat /K m (m M )1 Æs )1 ) Wild-type 0.15 6.9 46.0 M46L 0.56 18.4 32.9 V82S 1.34 13.4 10.0 V82A 0.42 7.0 16.7 I84V 1.02 93.5 92.0 L90M 0.64 33.0 51.6 Table 2. Processing of peptides representing wild-type HIV-1 Gag NC/p1 and p1/p6 cleavage sites by wild-type HIV-1 proteinase. Site Substrate sequence K m (m M ) k cat (s )1 ) k cat /K m a (m M )1 Æs )1 ) k cat /K m b (m M )1 Æs )1 ) 1. NC/p1 ERQAN-fl-FLGKI 0.17 0.15 0.9 1.0 7. p1/p6 RPGNF-fl-LQSRP 1.20 0.98 0.8 0.8 a Values calculated from the individual K m and k cat values. b Value determined for peptide 1 using a competitive assay with substrate KTGVL-fl-VVQPK [28] and under pseudo first order reaction condition for peptide 7 using a substrate concentration range (0.02–0.15 m M ) in which the activity was a linear function of the substrate concentration. 4116 A. Fehe ´ r et al. (Eur. J. Biochem. 269) Ó FEBS 2002 sequences were assayed as substrates of HIV-1 PR using competitive assays (Table 3). Natural variations of this cleavage site (peptides 2 and 3) exhibited either decreased or increased specificity constants with the wild-type PR (Table 3). The mutated residues in this sequence are in the outer regions of the cleavage site sequence recognized by the PR, and the respective S4 and S3 binding sites could accept a variety of residues [33,34,46]. When compared to peptides having the same natural sequence background (peptides 4 and1,5and2,6and3),theAlatoValmutationatP2,seen in resistance, increased the specificity constant by two to tenfold. As the Ala to Val mutation had a varying effect on the specificity constant depending on the peptide sequence, this result further supports the view that substrate binding subsites of the HIV-1 PR do not act independently of each other [35,46]. Based on molecular modeling, the P2 Val fits much better than Ala in the S2 binding site due to more favorable van der Waals contacts with Val32, Ile47 and Ile84 (not shown). P2 Val also shifts the sequence toward a type 2 consensus sequence, where beta-branched residues were found to be the best at this position [46]. P3 Arg is often disordered in HIV-1 PR-inhibitor crystal structures, however, when it is ordered it interacts with Arg8 and Glu21 through water molecules and makes hydrophobic interactions with Phe53, Pro81 and Val82 [26]. P3 Arg could interact favorably with Asp29 and P1 Asn, which may contribute to its beneficial effect in specificity. The 20-fold increased specificity con- stant obtained for the doubly substituted peptide is within the range of good Gag cleavage site substrates [15]. The same substrate set was also tested with the mutant PRs. M46L, V82S and V82A mutants gave lower specificity constants for each substrate as compared to the wild-type enzyme. These enzymes were also less efficient on the MA/ CA cleavage site (Table 1). Similar to wild-type PR, the resistant mutation of P2 Ala to Val always increased the specificity constant when compared to peptides with the same natural sequence. While the combined P3 Arg and P2 Val mutations (peptide 6) provided the best combination for the M46L mutant, the P2 Ala to Val mutation alone provided the best specificity constant (peptide 4) for the V82 mutants (Table 3). In an in vivo study using indinavir, the V82A mutation arose with the P2 Ala to Val mutation, without the appearance of P3 Arg mutation [10]. In these mutants the positive effect of P3 Arg is offset due to the loss of favorable hydrophobic interaction with the smaller Ala or Ser side chain. These results agree with the observations that the naturally existing sequence variation in the virus at this cleavage site may be important for developing resistance [19]. The I84V mutant gave somewhat better specificity con- stant with the wild-type substrate and with some of its variants. This enzyme was also more efficient than the wild- type PR on the MA/CA substrate. The L90M mutant was also more efficient than the wild-type enzyme on the substrates. As seen with the wild-type enzyme and M46L, the P2 Val mutation in the respective natural sequence background always increased the specificity constant for these mutants. Processing of peptides representing p1/p6 cleavage site sequences by wild-type and mutant HIV-1 proteinases Although previous studies indicated that the p1/p6 cleavage site is rather conserved and the p2/NC cleavage site is the most variable [17], analysis of various HIV isolates in the database suggested that substantial sequence polymorphism occurs at this site, especially at positions close to the site of cleavage (see Fig. 1). To our knowledge, the effect of these variations on the susceptibility towards PR cleavage has not been reported yet. Strikingly, there are several variants at P1 and P1¢ positions, which are important determinants of specificity [34,46]. However, the natural variations we examined did not substantially change the specificity constants for the wild-type PR, except for the P1 Leu substitution (Table 4), which provided a very inefficient processing. This result raises the question of whether viral proteins having this mutation could be processed at this site and whether viruses harboring this mutation could be replication competent. The only sequence reported to have this residue (C.BR92BR025) is not from a full-length clone [18]. In contrast to the natural variations, the P1¢ Phe substitution, which is seen only in resistant virus, was a substantially better substrate for the wild-type enzyme. Differences in free energy of enzyme–substrate inter- action can be related to kinetic data by the equation DG ¼ –RTln(k cat /K m ) from the transition state theory. The logarithmic value of the specificity constant showed a strong correlation with the volume of the P1¢ residue (correlation coefficient R ¼ 0.90) and the number of hydrophobic contacts the side chain formed with residues of the S1¢ subsite (R ¼ 0.98). The fit of various P1¢ residues into the S1¢ binding site of HIV-1 PR is shown in Fig. 2. The results Table 3. Processing of peptides representing wild-type and mutant HIV-1 Gag NC/p1 cleavage sites by HIV-1 proteinase. Ratios of specificity constants for substituted over wild-type substrate are shown in parentheses. Substrate sequence a Type of mutation b k cat /K m (m M )1 Æs )1 ) Wild-type M46L V82S V82A I84V L90M 1. ERQAN-fl-FLGKI (–) 1.0 (1.0) 0.3 (1.0) 0.4 (1.0) 0.7 (1.0) 1.6 (1.0) 2.5 (1.0) 2. EGQAN-fl-FLGKI (P) 0.4 (0.4) 0.2 (0.8) 0.2 (0.5) 0.03 (0.04) 0.4 (0.3) 1.0 (0.4) 3. ERRAN-fl-FLGKI (P) 1.7 (1.7) 0.6 (2.0) 0.3 (0.8) 0.5 (0.7) 3.2 (2.0) 4.2 (1.7) 4. ERQ VN-fl-FLGKI (R) 2.6 (2.6) 2.4 (8.0) 1.7 (4.3) 2.1 (3.0) 5.9 (3.7) 9.6 (3.8) 5. EGQVN-fl-FLGKI (R) 0.8 (0.8) 0.7 (2.3) 0.4 (1.0) 0.7 (1.0) 2.8 (1.8) 4.1 (1.6) 6. ERRVN-fl-FLGKI (R) 20.1 (20.1) 11.7 (39.0) 0.8 (2.0) 1.1 (0.9) 7.9 (4.9) 45.4 (18.2) a Substituted residues are in bold. b The type of mutation: natural polymorphism (P), mutation appearing only in drug resistance (R, see Fig. 1). If two mutations occurred and one of them was occurring only in drug resistance, the cleavage site was considered as R type. Ó FEBS 2002 Studies on mutant HIV-1 processing sites (Eur. J. Biochem. 269) 4117 suggest that the maximization of the van der Waals interactions of P1¢ with S1¢ residues may be the most important feature determining the efficiency of cleavage. Similar effects were observed for P2 substitution in the NC/ p1 site. The specificity changes obtained with the mutants for the substituted peptides tend to be similar to those obtained for the wild-type enzyme, but some exceptions were also observed. For example, M46L preferred P1Y and P1¢F substantially more than the wild-type enzyme, while the same substrate mutations were much less preferred by the V82S mutant. For all mutant PRs the P1¢F substitution provided the best substrate within the series. Similar results were reported for P1¢F substitution in the CA/p2 cleavage site [42]. CONCLUSIONS HIV-1 grown in cultured cells in the presence of PR inhibitors produces multiple PR mutants of lower suscep- tibility to inhibitors. Furthermore, mutants selected with one inhibitor are often cross-resistant to other inhibitors (reviewed in [47]). The number of mutations increases with the time of therapy. Schock et al. [6] proposed that mutations located in the binding cleft of the enzyme can Table 4. Processing of peptides representing wild-type and mutant HIV-1 p1/p6 Gag cleavage sites by HIV-1 proteinase. Ratios of specificity constants for substituted over wild-type substrate are shown in parentheses. Substrate sequence a Type of mutation b k cat /K m (m M )1 Æs )1 ) Wild-type M46L V82S V82A I84V L90M 7. RPGNF-fl-LQSRP (–) 0.8 (1.0) 0.6 (1.0) 1.2 (1.0) 1.2 (1.0) 1.2 (1.0) 2.1 (1.0) 8. RPGNY-fl-LQSRP (P) 1.3 (1.6) 2.6 (4.3) 0.9 (0.8) 2.2 (1.8) 1.2 (1.0) 0.6 (0.3) 9. RPGNL-fl-LQSRP (P) 0.01 (0.01) 0.05 (0.08) 0.03 (0.03) 0.09 (0.08) < 0.07 (< 0.01) 0.07 (0.03) 10. RPGNF-fl-PQSRP (P) 0.3 (0.4) 0.3 (0.5) 0.06 (0.05) 0.2 (0.2) 0.8 (0.7) 0.5 (0.2) 11. RPGNF-fl-VQSRP (P) 0.8 (1.0) 0.5 (0.8) 0.7 (0.6) 1.2 (1.0) 1.2 (1.0) 2.1 (1.0) 12. RPRNF-fl-LQSRP (P) 0.5 (0.6) 0.4 (0.7) 0.6 (0.5) 0.7 (0.6) 0.8 (0.7) 1.3 (0.6) 13. RQGNF-fl-LQSRP (P) 0.3 (0.4) 0.05 (0.1) 0.3 (0.3) 0.4 (0.3) 0.4 (0.3) 0.4 (0.2) 14. RPGNF-fl-FQSRP (R) 7.6 (9.5) 8.7 (14.5) 2.2 (1.8) 11.0 (9.2) 8.3 (6.9) 22.3 (10.6) a Substituted residues are in bold. b Type of mutation: natural polymorphism (P), mutation appearing in drug resistance (R). Fig. 2. Fitting of various P1¢ residues in the p1/p6¢ substrate sequence into the S1¢ binding site of HIV-1 PR. Space filling models of the P1¢ residue of peptides 7, 10, 11 and 14 (Table 4) are shown occupying the S1¢ binding site of wild-type HIV-1 PR. 4118 A. Fehe ´ r et al. (Eur. J. Biochem. 269) Ó FEBS 2002 lead to the development of drug resistance by increasing K i of the inhibitors at the expense of impaired proteinase function, while non-active-site mutations may act by enhancing the catalytic efficiency. However, reduced cata- lytic efficiency has been reported for nonactive site muta- tions L90M and N88D [8], and both increases and decreases in catalytic efficiency have been observed for active site mutants of HIV-1 PR [8,42]. This variation is confirmed by our results: the active site mutant I84V enzyme had higher specificity constant than the wild-type PR, while the nonactive-site mutations M46L and L90M did not sub- stantially improve the catalytic efficiency of the PR, and even resulted in reduced activity on some substrates. Resistant mutants of HIV-1 PR must possess sufficient proteolytic activity (k cat /K m ) to support viral replication by correctly cleaving Gag and Gag-Pol precursors. The order of Gag cleavage is important for infectivity [48]. The most efficiently cleaved sites are not mutated in resistance, and the catalytically compromised mutant HIV-1 PRs can still cleave these substrates sufficiently for viral replication. In fact, it may be difficult to improve the cleavage by mutation of these sites, as they appear to be optimized for rapid hydrolysis (for example the MA/CA or CA/p2 cleavage sites [34,36]). On the other hand, the NC/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis by wild-type PR, as substan- tially improved specificity constants were obtained with resistant cleavage site mutations. Therefore, it is possible to increase the cleavage rate by mutation at these sites when the PR activity is diminished due to the accumulation of PR mutations. The P2 Val and P1¢ Phe residues appearing in drug resistance at these sites were the most frequent residues in efficiently cleaved substrates selected by screening a phage display library [49]. The observed improvement in hydrolysis of the substrates harboring these mutations was more pronounced for the wild-type enzyme and nonactive-site mutants (L90M and M46L) than for the active-site mutants. Consequently, mutation of the rate limiting cleavage sites, NC/p1 and p1/p6, can partly compensate for the reduced catalytic activity of resistant PR mutants. ACKNOWLEDGEMENTS We thank Dr Bruce Korant (DuPont Pharmaceuticals) for providing DMP-323 and amprenavir for the active site titrations, Szilvia Peto ¨ for technical assistance and Suzanne Specht for peptide synthesis and amino-acid analysis. Research sponsored in part by the Hungarian Science and Research Fund (OTKA T 30092; F34479), by the United States Public Health Service Grant GM62920, by the National Cancer Institute, DHHS under contract with ABL, by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of NIH and by AIDS FIRCA Grant TW01001. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. REFERENCES 1. Oroszlan, S. & Luftig, R.B. (1990) Retroviral proteinases. Curr. Top. Microbiol. Immunol. 157, 153–185. 2. Swanstrom, R. & Eron, J. 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