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1 Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary
2 HIV Drug Resistant Program, National Cancer Institute at Frederick, MD, USA
3 Department of Biology, Georgia State University, Atlanta, GA, USA
Correspondence
József Tözsér
tozser{at}indi.biochem.dote.hu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, the human immunodeficiency virus type 1 (HIV-1) PR has proved to be a very important target for antiretroviral therapy of AIDS, and various PR inhibitors are in clinical use (reviewed by Barbaro et al., 2005). However, the continuing emergence of viral variants that are cross-resistant to the existing inhibitors of PR indicates that there is a continuous need for designing new, more effective, broad-spectrum PR inhibitors (Imamichi, 2004). Comparative studies of different PRs are expected to reveal the common features of their specificity, facilitating the design of such broad-spectrum inhibitors that may reduce the possibility of selection for viable mutants during therapy.
Moloney murine leukemia virus (MLV) has been one of the model retroviruses and its PR was one of the first to be purified from virus (Yoshinaka et al., 1985). Furthermore, the majority of the retroviral vectors are based on MLV (Thomas et al., 2003). In spite of the importance of MLV as a model system, only a few reports have dealt with cloning, expression, characterization and inhibition of its PR, as reviewed recently (Menéndez-Arias et al., 2004).
Retroviruses generally encode the PR within the pol open reading frame, while its synthesis is achieved by using various mechanisms. In the case of MLV, the PR is produced as part of the Gag–Pro–Pol polyprotein by in-frame suppression (readthrough) of the Gag terminator (Yoshinaka et al., 1985). In MLV, the transmembrane protein Pr15(E) is also processed to p12(E) and p2(E) by the PR (Crawford & Goff, 1985; Katoh et al., 1985).
Previously, we have cloned and expressed MLV PR and purified it to homogeneity (Fehér et al., 2004). Here, we describe the detailed characterization of the cloned enzyme and its comparison with the characteristics of viral-derived MLV PR, as well as HIV-1 PR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, 20 vols cold acetone (–70 °C) was added to purified virus suspension (1 ml) and, after 30 min incubation on ice, the suspension was centrifuged (8000 g, 20 min at 4 °C). The precipitate was dried under reduced pressure and then extracted with 2 ml 20 mM Tris (pH 7·2) containing 5 mM dithiothreitol (DTT), 1 M NaCl for 30 min at 4 °C, with occasional vortexing. The suspension was centrifuged again, and then the buffer of the supernatant was changed to 20 mM PIPES (pH 7·0) containing 1 mM EDTA, 100 mM NaCl, 10 % glycerol, 5 % ethylene glycol, 0·5 % Nonidet P-40 and 10 mM DTT, using Centricon YM-10 (Millipore) to increase the stability of the PR preparation. The active enzyme concentration of the solution was determined by active-site titration with the HPLC method (see below) using HIV-1 RT/IN substrate (IRKIL
FLDG, where arrow indicates the site of cleavage).
Purification of recombinant MLV (rMLV) PR.
The cloning, expression and purification of MLV PR with or without a C-terminal GGSIEGR sequence were described previously (Fehér et al., 2004). The two forms were found to be identical in kinetic assays (Fehér et al., 2004).
Oligopeptides and PR inhibitors.
Unmodified oligopeptides were synthesized and purified as described previously (Tözsér et al., 1991; Menéndez-Arias et al., 1993). Correct peptide concentration of stock solutions was determined by amino acid analysis with a Beckman 6300 amino acid analyser. Fluorescent substrate Arg-Glu(Edans)-Ser-Gln-Ala-Phe-Pro-Leu-Arg-Ala-Lys(Dabcyl)-Arg-OH, a modified version of the MLV p12/capsid cleavage site, was synthesized by Dr Ivo Blaha (Ferring Leciva). PR inhibitors used in AIDS therapy were a kind gift from Dr Bruce Korant (DuPont Experimental Station).
Spectroscopic assay.
The chromogenic substrate Lys-Ala-Arg-Val-Nle-p-nitroPhe-Glu-Ala-Nle-amide (L6525; Sigma) was used. PR (70 nM) was assayed with 100–350 µM substrate in 50 mM MES, 100 mM Tris, 50 mM sodium acetate, 1 M NaCl at various pH values, and assayed over 10 min at 37 °C for the decrease in A310 on a Hitachi U-3000 spectrophotometer. Absorbances were converted to substrate concentration via a calibration curve. Michaelis–Menten curves and bell-shaped curves of pH-optimum studies were fitted by using SigmaPlot (SPSS Inc.).
HPLC-based PR assay.
The enzyme concentrations of MLV PR preparations were determined routinely by using the Bradford spectrophotometric method (Bio-Rad). The exact amount was calibrated by active-centre titration with DMP323 as an inhibitor using the HPLC method and HIV-1 MA/CA as substrate. The PR assays were initiated by mixing 5 µl PR, 10 µl 2x incubation buffer [0·5 M potassium phosphate buffer (pH 5·6) containing 10 % glycerol, 2 mM EDTA, 10 mM DTT, 4 M NaCl] and 5 µl 0·01–1·32 mM substrate. The range of substrate concentration was selected depending on the approximate Km values. The reaction mixture was incubated at 37 °C for 1 h and terminated by the addition of 180 µl 1 % trifluoroacetic acid (TFA). Separation of cleavage products by reversed-phase chromatography was performed as described previously (Tözsér et al., 1991). Cleavage products were identified by amino acid analysis and/or peptide sequencing. Kinetic parameters were determined by fitting the data obtained at <20 % substrate hydrolysis to the Michaelis–Menten equation by using the Fig. P program (Fig. P Software Corp.). The standard errors of the kinetic parameters were below 25 %.
Fluorescent assay for inhibition of the MLV PR.
For the inhibitor assays, a microtitre plate-reader assay using a fluorescent Dabcyl/Edans-tagged analogue of the p12/capsid substrate was used in 250 mM phosphate buffer (pH 5·6) containing 5 % glycerol, 1 mM EDTA, 5 mM DTT, 500 mM NaCl, 1 % DMSO, as described previously for HIV and human T-lymphotropic virus PRs (Bagossi et al., 2004). Ki values were calculated according to Williams & Morrison (1979).
Cloning, purification and cleavage of MLV Gag fragments.
To prepare the Gag_
1 construct, a region of the pRR88 plasmid harbouring an infectious MLV clone (Gorelick et al., 1988) was amplified by PCR carried out as described previously (Menéndez-Arias et al., 1992), by using primer MLVMAolig1: 5'-GCGCCGAGCTCAGAACCTCCTCGTTC-3', encoding the C-terminal sequence of MA and a SacI restriction site (underlined), together with MLVNColig2rc: 5'-GGCCAAGCTTCTGGTCATCTAGGGTCAGG-3', which contains a mutation to convert the stop codon of the Gag to Gln and a HindIII restriction site (both underlined). The Gag_2 construct was cloned by using MLVp12olig1: 5'-GCGCCGAGCTCTCTACGTGGGAGACG-3', encoding the C-terminal sequence of p12 and a SacI site (underlined), together with MLVNColig2rc. The amplified DNA fragments were cloned into the SacI and HindIII sites of pET-23b (Novagen). The proteins encoded by these plasmids are shown in Figs 4(a) and 3(a), respectively. Constructs were verified by DNA sequencing performed with an ABI Prism Dye Terminator Cycle Sequencing kit (Perkin-Elmer) and an Applied Biosystems model 373A sequencer. Ligation and transformation of BL21(DE3) cells were performed by using standard protocols (Sambrook et al., 1989). Protein expression was induced by the addition of 1 mM IPTG, the proteins were purified by using metal chelate-affinity chromatography as described in detail for the MLV PR (Fehér et al., 2004), followed by gel filtration on a Superdex-75 column (Amersham Pharmacia Biotech) that was equilibrated with 20 mM sodium phosphate buffer (pH 7·0), 150 mM NaCl and 5 mM 
-mercaptoethanol. The yield of the purification procedure was 3·7 and 9·2 mg protein (l culture)–1 for the Gag_1 and Gag_2 constructs, respectively, as determined by the Bradford spectrophotometric method (Bio-Rad). Proteolysis of these purified proteins was performed in 250 mM phosphate buffer containing 1 mM EDTA (pH 5·6), using 0·6 µM and 1·2 µM Gag_1 and Gag_2 constructs, respectively, 20 nM MLV or 33 nM HIV-1 PR, and the samples were analysed by SDS-PAGE. Cleavage products were identified by immunoblot analysis using polyclonal antibodies against the CA and NC proteins, as well as against the hexahistidine tag.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Although the latter construct provided much higher yield, the two enzyme forms were found to be equivalent in specificity and could therefore be used interchangeably (Fehér et al., 2004). As shown in Fig. 1, the bacterially expressed PR with extra C-terminal residues was apparently homogeneous. Also, this recombinant PR was found to be 85 % active by comparing the protein content determined by amino acid analysis and number of active sites determined with DMP323, a potent inhibitor of the PR (data not shown). The processed, purified MLV PR migrated as a 16 kDa protein, with a somewhat higher molecular mass in SDS-PAGE than the viral PR, as expected from the small C-terminal extension. This purified rMLV PR was compared with the viral-derived PR by using four oligopeptide substrates in the HPLC system; their behaviour was identical within the experimental error of the measurements (Table 1).
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; Hyland et al., 1991), including the same chromogenic substrate that we used here for the MLV PR (Polgár et al., 1994). The pH-dependence curve of kinetic parameters can be affected by the ionizing groups of the substrate, like the P2' glutamic acid [notation is according to Schechter & Berger (1967)] of the chromogenic substrate (Polgár et al., 1994). The pH optimum of the rMLV PR was found to be 5·0 with this chromogenic substrate, substantially higher than the pH 4·0 optimal value determined for HIV-1 PR (Polgár et al., 1994), but similar to the optimal values determined for HIV-1 PR with substrates containing P2' residues other than glutamate (Boross et al., 1999). At its optimal pH (5·0), rMLV PR showed the highest kcat and the lowest Km values (Fig. 2). Unlike with rMLV PR, the kcat for HIV-1 PR showed a minimum, not maximum, value at the optimal pH, whilst the substrate with P2' Gln had a pH dependence similar to kcat, as described in our study for the rMLV PR by using the P2' Glu-containing analogue (Polgár et al., 1994).
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, 1992). The oligopeptides representing the MLV cleavage sites were tested previously by using MLV PR expressed as a glutathione S-transferase fusion protein under somewhat different conditions, but these peptides were not assayed as substrates of HIV-1 PR (Menéndez-Arias et al., 1993). Seven oligopeptides representing naturally occurring cleavage sites in the MLV Gag, Gag–Pro–Pol and Env polyproteins were tested as substrates for MLV and HIV-1 PRs (Table 2). All peptides were hydrolysed at the expected site by the MLV enzyme, as reported previously (Menéndez-Arias et al., 1993). Kinetic parameters were determined at high (2 M) salt concentration. High ionic strength was found to be optimal for MLV PR (Menéndez-Arias et al., 1993), similar to other retroviral PRs (Kotler et al., 1989; Wondrak et al., 1991; Tözsér et al., 1993). The peptides were also assayed as substrates of HIV-1 PR. Only three of them were substrates of this enzyme, but one of them was cleaved at a non-authentic site (Table 2).
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), although the kinetic parameters were typically lower than those obtained with the HIV-1 enzyme.
Cleavage of recombinant Gag fragments by rMLV PR
To further characterize the specificity of MLV PR, MLV Gag fragments containing subsets of cleavage sites were expressed in bacteria, purified to homogeneity and used as substrate for rMLV PR. Unlike HIV-1 Gag fragments, which can be expressed only with a very low yield (Erickson-Viitanen et al., 1989; Luban & Goff, 1991; our unpublished results), a high level of expression was achieved for these proteins. The PR-cleavage assays were performed in low ionic strength, which is the typical condition for this type of assay. As higher ionic strength, similar to the observations for other retroviral PRs, was found to enhance MLV PR activity (Menéndez-Arias et al., 1993), the kinetic parameters for the peptides representing the cleavage sites incorporated into these Gag fragments were also assayed under identical conditions (Table 2, values in parentheses). All of the peptides were hydrolysed less efficiently at lower salt concentration, with four- to 13-fold-lower specificity constants. Due to the substantially elevated Km values, we were unable to determine the individual kcat and Km values for these substrates. A similar Km effect of the ionic strength was reported previously for HIV-1 PR (Wondrak et al., 1991). Time-dependent cleavage of the Gag fragments is shown in Figs 3(b) and 4(b), where the linear structure of the expressed proteins is also provided [Figs 3(a) and 4(a)]. The time-dependent cleavage of the shorter Gag fragment expressed from the Gag_2 plasmid suggested that the first processing occurs either at the p12/CA site, by generating a 40 kDa protein, or at the CA/NC site, generating a 33 kDa protein (Fig. 3), whilst the final mature CA was obtained by processing at the alternating site of each intermediary fragment (Fig. 3). Based on these results, the cleavage rate at the p12/CA and CA/NC sites is comparable at the protein level, even if the peptide corresponding to the p12/CA cleavage site showed much lower specificity constants, independent of the ionic strength of the assay (Table 2). At these sites, the rate of hydrolysis at the protein level may also be a function of steric effects. Gel-filtration experiments suggested that both Gag fragments that we have studied were oligomers under the assay conditions (unpublished data) and that oligomerization might hinder the accessibility of some cleavage sites.
By using the larger construct Gag_1 (Fig. 4), again, multiple intermediate forms appeared even within a short time of incubation, indicating that the rate of cleavage at the MA/p12 site is comparable to the rate of cleavage at the other sites. These results suggest that processing at the MLV cleavage sites occurs at a similar rate, as also could be inferred from the relatively small range of the specificity constants, in sharp contrast to the findings about HIV-1 PR (Tözsér et al., 1992). The same fusion proteins were also tested as substrates for HIV-1 PR. In good agreement with the lack of cleavage of the CA/NC and NC/PR sites (Table 2), only the 40 kDa CA–NC–C-terminal-extended protein was observed even after 2 h incubation, in the absence of PR inhibitor [Figs 3(c) and 4(c)].
Inhibition profile of rMLV PR
We have designed a fluorescent substrate for the MLV PR, which was based on the p12/CA cleavage site of MLV. Although this cleavage site was not the most efficient, we have selected it as it contains P1' Pro and, therefore, it is expected to be more selective for retroviral PRs than cleavage-site peptides containing other residues at P1' (Tözsér et al., 1992). Inhibition profiling was performed at moderate ionic strength (500 mM NaCl), as higher ionic strength is not compatible with the assay (Bagossi et al., 2004). All of the tested compounds inhibited HIV-1 PR, with Ki values of <1 nM in the fluorescent assay using another fluorescent substrate based on the MA/CA cleavage site of HIV-1 (Bagossi et al., 2004). These HIV-1 PR inhibitors included the first clinical drugs in this class, saquinavir, indinavir and ritonavir, and the more recently developed drugs nelfinavir and amprenavir. Amprenavir was the best inhibitor of rMLV PR under this moderate ionic strength, with the lowest Ki value of 20 nM (Table 3). DMP323 was also a relatively good inhibitor of the enzyme; furthermore, at high ionic strength, as was possible in the HPLC-based assay (but prohibitive for the fluorescent detection), it was even suitable for active-site titration (with a Ki of 0·8 nM). However, saquinavir was the least effective inhibitor of rMLV PR. Although indinavir showed a Ki value of 0·21 µM for rMLV PR, when tested against MLV in an in vitro cell-based assay, indinavir had no appreciable effect in the absence of reverse transcriptase inhibitors (Powell et al., 1999). In a previous study, KH-164, a statine-based inhibitor (Lai et al., 1993), was found to be the best of those tested for MLV PR (Menéndez-Arias et al., 1993); in our set, it was one of the weakest inhibitors of MLV PR (Table 3). These data suggest that later-developed HIV-1 PR inhibitors, such as amprenavir, not only work much better on that PR, but may also be more potent inhibitors of other retroviral PRs.
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). At 5 µM concentration, a complete absence of the p30 protein was observed. A similar pattern was observed by using anti-NC antibody (data not shown). The 54 kDa band observed in the presence of a high concentration of inhibitors is predicted to be due to proteolytic degradation by cellular PRs; a band with similar mobility was also observed previously in cells producing PR-defective virions (Oshima et al., 2004).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The pH optimum of the individual kinetic parameters for the recombinant enzyme was also determined. Whilst detailed studies on the pH optimum of HIV-1 PR have been performed previously, no such study has been reported for other retroviral PRs, including MLV PR. The type of amino acid at P2' has been found to be critical in determining the specificity and especially the pH dependence of retroviral PRs (Polgár et al., 1994; Boross et al., 1999). Retroviral cleavage sites are classified into two groups: type 1 cleavage sites have aromatic residues and Pro, whilst type 2 cleavage sites have hydrophobic residues (excluding Pro) at the site of cleavage (Pettit et al., 1991). It has been established that, in type 2 cleavage-site substrates, P2' Glu is preferred by the PR of HIV-1 (and HIV-2), but it is not preferred by the MLV PR (and that of Equine infectious anemia virus; Boross et al., 1999). The pH optimum for type 2 cleavage sites and the P2' specificity of primate lentiviral PRs appear to be exceptions, probably due to the presence of two Asp residues (Asp29' and Asp30') in the S2' binding site. One of these aspartates, Asp30', was shown to share a proton with P2' Glu (Weber et al., 1997). The residues corresponding to these aspartates in MLV PR are Gln36' and His37', respectively, and, based on molecular modelling, these residues are not expected to provide hydrogen-bond interactions with P2' Glu, as seen in HIV-1 PR (Boross et al., 1999).
The specificity of MLV PR was also assayed and compared with the specificity of HIV-1 PR. The two PRs showed substantial differences in their specificity. Both the original (Menéndez-Arias et al., 1994) and the improved (Boross et al., 1999) molecular models for MLV PR suggested that almost all of the residues forming the subsites are different from those forming HIV-1 PR subsites. Whilst MLV PR was able to cleave most of the HIV-1 cleavage sites, more than half of the MLV PR-cleavage sites were not substrates of HIV-1 PR. Most of the MLV cleavage sites have Leu–(Val, Ala)–Leu at the P2 and P1 positions, and these were relatively good substrates for the MLV PR, but were not hydrolysed by HIV-1 PR. Although, in most cases, their own enzyme hydrolysed the peptides better, the MLV p12/CA cleavage-site peptide was a substantially better substrate for HIV-1 PR than for MLV PR, whilst the HIV-1 ‘in p6’ site peptide was a substantially better substrate for the MLV PR (Table 2). All substrates showed low Km values and moderate catalytic (kcat and kcat/Km) values. The range of specificity constants (kcat/Km) for MLV PR (1·7–15·0 mM–1 s–1) is narrower than that measured for HIV-1 PR (0·02–202 mM–1 s–1), but they are in a similar catalytic range, unlike those measured for the avian myeloblastosis virus (AMV) PR, which were substantially lower (Tözsér et al., 1996). This is in good agreement with the relative amounts of the PR in the virions. Whilst the PRs of MLV and HIV-1 are produced by different mechanisms (suppression of translational termination and –1 frameshifting, respectively), the amount of PR is about 5–10 % of Gag in both cases (Shehu-Xhilaga et al., 2001). In contrast, the PR of AMV is encoded in the gag gene and therefore it is synthesized equimolarly with the structural Gag proteins. Indeed, a recent study demonstrated that replacement of the readthrough mechanism in MLV with HIV-1-like frameshifting resulted in infectious virions (Brunelle et al., 2003). Strikingly, the MLV p12E/p2E cleavage-site peptide was cleaved by the HIV-1 PR one residue upstream from the residue where the cleavage was observed with MLV PR, at Ala–Leu instead of the MLV PR-cleavage site Leu–Val. It is very rare among the retroviral PRs that, if they cleave a heterologous site, they would cleave it between residues differing from those observed in a homologous system (unpublished data). The MLV Env was found to be cleaved by HIV-1 PR in a cell-culture study, but the cleavage site was not determined (Kiernan & Freed, 1998). One residue shift at the site of cleavage is not expected to cause a functional defect, in good agreement with the in vivo data (Kiernan & Freed, 1998).
To complement the specificity studies performed with oligopeptide substrates, recombinant proteins containing MLV Gag fragments with authentic cleavage sites were also tested as substrates of the two enzymes. The cleavage sites in these proteins were utilized with a similar efficiency, although the kinetic parameters measured at identical conditions suggested that CA/NC should be the most efficient. Other studies also suggested that this cleavage site has the highest specificity constant among the Gag sites (Menéndez-Arias et al., 1993). Analysis of results obtained with a bacterial expression system in which Gag–Pro fusion protein was expressed (Cannon et al., 1998) also suggests that CA/NC cleavage may occur first. However, studies of processing within the virus suggested that another cleavage site, the p12/CA site, was first to be cleaved (Naso et al., 1979; Campbell et al., 2002). Furthermore, a recent study indicated that mutation of the p12/CA site to a non-cleavable sequence also made the CA/NC site somewhat inefficient, implying that the CA/NC cleavage-site accessibility is probably dependent on cleavage at p12/CA (Oshima et al., 2004). It should be noted that substantial amounts of dimers and oligomers of Gag protein fragments were detected in solution by gel filtration (our unpublished results). The altered cleavage of these Gag protein fragments with HIV-1 PR also suggested a substantial difference in specificity of the two enzymes. Virion-derived Gazdar MLV Pr65gag was also assayed previously as a substrate of HIV-1 PR (Bu et al., 1989). Whilst this Gag protein was processed properly by MLV PR (Yoshinaka & Luftig, 1982), overnight incubation with bacterial extract containing HIV-1 PR resulted in an altered proteolytic profile and indicated the lack of cleavage at the CA/NC site (Bu et al., 1989). Furthermore, when HIV-1 PR sequence was introduced in place of MLV PR in an infectious viral genome, non-infectious chimeric particles were obtained and, although some mature CA protein was detected, most of it remained in partially processed precursor forms (Kohl et al., 1991).
We have tested several HIV-1 PR inhibitors on the MLV PR. Only limited information has been reported so far on the inhibition of MLV PR by using kinetic assays (Menéndez-Arias et al., 1993), cell-culture studies (Black et al., 1993; Powell et al., 1999) or in vivo studies (Lai et al., 1993; Black et al., 1996). Whilst none of the previously tested PR inhibitors showed IC50 values lower than 60 nM at high ionic strength (3 M NaCl), which substantially facilitates the PR–ligand interaction (Menéndez-Arias et al., 1993), amprenavir exhibited a 20 nM Ki value at low ionic strength. To demonstrate its potency, we tested the effect of amprenavir in a cell-culture system. Amprenavir was able to block the formation of mature capsid protein completely at 5 µM concentration. Therefore, amprenavir appears to be the most suitable inhibitor among the currently utilized therapeutic HIV-1 PR inhibitors for future in vivo inhibition studies on the MLV system. Utilization of the MLV system may contribute to our understanding of PR-related events, such as the development of antiviral-drug resistance against PR inhibitors, for which currently only primate lentiviral data are available.
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ACKNOWLEDGEMENTS |
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Received 26 July 2005;
accepted 19 January 2006.
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, kcat/Km (mM–1 s–1); 
, Km (mM); 
, kcat (s–1x102).