©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Transient Precursor of the HIV-1 Protease
ISOLATION, CHARACTERIZATION, AND KINETICS OF MATURATION (*)

(Received for publication, September 7, 1995; and in revised form, December 8, 1995)

Ewald M. Wondrak (1) Nashaat T. Nashed (2) Martin T. Haber (2) Donald M. Jerina (2) John M. Louis (1)(§)

From the  (1)Molecular Mechanisms of Development Section, Laboratory of Cellular and Developmental Biology, and the (2)Section on Oxidation Mechanisms, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-DeltaTF-PR-DeltaPol, which contains the protease linked to short native flanking sequences (DeltaTF and DeltaPol) fused to the maltose binding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina, D. M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP-DeltaTF-PR-DeltaPol protein leads to the formation of the PR-DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands. We now report the purification and characterization of the PR-DeltaPol intermediate and the kinetics of its processing to the mature protease. Unlike the MBP-DeltaTF-PR-DeltaPol precursor, PR-DeltaPol has proteolytic activity similar to that of the mature enzyme at pH 5.0. The pH rate profile for k/K is similar to that of the mature protease above pH 4.0. Although the PR-DeltaPol is more sensitive than the mature protease toward denaturing reagents, both the enzymatic activity and the intrinsic fluorescence of PR-DeltaPol are linearly dependent on the protein concentration, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteolytic reaction at the N terminus of the protease, the proteolytic reaction at the C terminus of the protease is second order in protein concentration. These results are discussed in terms of a mechanism in which the C-terminally located DeltaPol peptide chains are cleaved intermolecularly to release the mature protease.


INTRODUCTION

Limited proteolysis by specific proteases is an established post-translational mechanism by which cellular and viral precursor proteins are converted to functional species(1, 2, 3) . Retroviruses, including the human immunodeficiency virus type 1 (HIV-1), (^1)encode a protease that is a member of the family of aspartic acid proteases(1, 2, 3) . The HIV-1 protease is translated as a part of the Gag-Pol polyprotein flanked by the transframe (TF, also termed p6*) and reverse transcriptase proteins at its N and C termini, respectively(1, 2, 3, 4) . The protease is responsible for its own maturation and for the release of structural and active functional proteins required for viral replication(1, 2, 3) . The mature protease is a symmetrical homodimer whose active site is formed along the interface between the two subunits with each subunit contributing one of the two catalytically important aspartic acid residues(5) . Partial inhibition of the protease in infected cells(6) , premature processing of viral polyproteins(7) , or overexpression of the Gag-Pol polyprotein relative to the Gag precursor (8) results in aberrant virus assembly and formation of non-infectious particles. In addition, activation of the protease is initiated on the membrane of infected cells(9) . These results suggest that temporal and spatial regulation of protease activity during the viral life cycle is crucial for proper assembly and maturation of the viral polyproteins to produce infectious particles.

Since studies of the autoprocessing reaction of the Gag-Pol polyprotein are complicated by the presence of multiple cleavage sites(1) , we developed a model protease precursor that contains only two cleavage sites. This model polyprotein (MBP-DeltaTF-PR-DeltaPol) was constructed by the addition of 19 residues of the native reverse transcriptase sequence (DeltaPol) to the C terminus of the protease domain and 12 residues of the native transframe region (DeltaTF) to its N terminus, which was additionally fused to the maltose binding protein (MBP) of Escherichia coli(10) . Specific mutational analyses of the two cleavage sites in the model polyprotein showed that the N-terminal cleavage precedes the C-terminal cleavage and that the N-terminal cleavage site is more sensitive to mutations(11) . These studies also showed that blocking the N-terminal cleavage further restricts cleavage at the C terminus of the protease(10) . Investigation of the autoprocessing of the model polyprotein led to the mechanism summarized in step 1 of Fig. 1(12) . Initially, the protease domain of the model polyprotein forms a dimer with an active site that is capable of binding known substrates and inhibitors of the mature protease. The dimeric polyprotein, which has low intrinsic catalytic activity, is converted to the mature protease in two steps. The first step includes intramolecular cleavage at the N terminus of the protease to produce the PR-DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands in the second step. In this study, we describe (i) purification of the transient PR-DeltaPol, (ii) characterization of the catalytic activity of PR-DeltaPol and its stability toward denaturing reagents in comparison with the mature protease, and (iii) kinetics of proteolytic processing of PR-DeltaPol to mature protease. Finally, the results presented are discussed in terms of a mechanism in which the hydrolysis of the DeltaPol sequence, step 2 (Fig. 1), occurs via an intermolecular process.


Figure 1: Proposed mechanism for the autoprocessing of HIV-1 protease from the model polyprotein MBP-DeltaTF-PR-DeltaPol (12) . The MBP and protease are denoted as large hatched ovals and small closed ovals, respectively. Lines represent DeltaTF and DeltaPol sequences that flank the protease domain. The protease catalyzes the hydrolysis at its N terminus from a dimeric MBP-DeltaTF-PR-DeltaPol via an intramolecular mechanism (12) to release the intermediate PR-DeltaPol (step 1). Results presented in this paper show that the conversion of the PR-DeltaPol to the mature protease occurs via an intermolecular process (step 2).




MATERIALS AND METHODS

Buffers employed in this study were buffer A (50 mM Tris-HCl at pH 8.2, 25 mM NaCl, 1 mM EDTA, and 0.1 M DTT), buffer B (100 mM sodium acetate at pH 5.1, 1 mM DTT, 1 mM EDTA, 0.05% Triton X-100, 1.6% Me(2)SO, and 0.8 µM pepstatin A), buffer C (50 mM Tris-HCl at pH 8.2, 5 M urea, 5 mM DTT, 5 mM EDTA, and 0.5% Tween 20), and buffer D (100 mM sodium acetate at pH 5.0, 1 mM DTT, 1 mM EDTA, and 0.05% reduced Triton X-100). Unless otherwise indicated, all incubations, assays, and measurements were carried out at 25 °C.

Isolation and Purification of the Transient PR-DeltaPol

The model polyprotein MBP-DeltaTF-PR-DeltaPol was purified as described previously(10, 12) . MBP-DeltaTF-PR-DeltaPol was concentrated by acid precipitation. Typically, trifluoroacetic acid and NaCl were added to a solution of approximately 0.5 mg/ml MBP-DeltaTF-PR-DeltaPol in 50 mM Tris-HCl buffer, pH 8.0, containing 25 mM NaCl, 1 mM DTT, and 1 mM EDTA to give final concentrations of 1% trifluoroacetic acid and 0.5 M NaCl. The solution was placed in an ice bath for 10 min, and the precipitated protein was collected by centrifugation at 15,000 times g for 30 min at 4 °C. The pellet was suspended in buffer A to give a protein concentration of 4 mg/ml. After incubation for 30 min, an equal volume of 10 M urea solution was added to solubilize the protein. The solution of denatured protein was incubated for 10 min before adding 10 volumes of buffer B to initiate the autoprocessing reaction. After 2 h of incubation, the reaction was terminated by adding approximately 10 volumes of ice-cold methanol and incubating on ice for 30 min. Precipitated proteins were collected by centrifugation at 20,000 times g for 30 min at 4 °C. The pellet was dissolved in 1 ml of buffer C and dialyzed overnight against 1 liter of buffer C at 4 °C. The dialyzed protein solution was loaded on a gel filtration column (Superose 12 HR, 1 times 30 cm; Pharmacia (Sweden)), and the column was eluted with buffer C at a flow rate of 0.5 ml/min. Protein elution was monitored at 280 nm, and 1-ml fractions were collected. Aliquots of each fraction were analyzed for protease activity (see below) and by SDS-PAGE on 10-20% gradient Tris-Tricine gels (NOVEX, CA). The 13.2-kDa PR-DeltaPol (160-200 µg/ml) was stored at -80 °C in 100-µl aliquots.

Purification of the Mature Protease

MBP-DeltaTF-PR-DeltaPol was precipitated as described above and dissolved to a final concentration of 0.25 mg/ml in buffer containing 50 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5% Nonidet P-40, and 8 M urea. Processing was carried out to completion by dialyzing the protein solution against 3 times 1 liter of 50 mM Mes buffer, pH 6.5, containing 100 mM NaCl, 1 mM EDTA, and 1 mM DTT for 4-6 h(10) . The processed protein containing the released mature protease was precipitated with 10 volumes of ice-cold methanol and further processed as described above.

Assays and Kinetics

The spectrophotometric assay for protease activity was carried out as described previously using peptide I, Lys-Ala-Arg-Val-Nle-Phe(NO(2))-Glu-Ala-Nle-NH(2) (California Peptide Research, CA) as the substrate(12) . In a typical assay, 10 µl of the protein in buffer C was added to 80 µl of buffer D in a 100-µl spectrophotometer cell. Reaction was initiated by addition of 10 µl of a 3.5 mM solution of peptide I in water and monitored by following the decrease in absorption at 310 nm (Delta = 1800). The active site concentrations of the mature protease and PR-DeltaPol were determined by titration with a transition-state analog (compound 3 in (13) ) as described previously for the mature protease(13) . Kinetic parameters and inhibition constants for the mature protease and PR-DeltaPol were determined in buffer D containing 100 mM NaCl using peptide I and the inhibitor, Arg-Val-Leu-(r)Phe-Glu-Ala-Nle-NH(2) (Bachem Bioscience Inc., PA). In all cases, data were collected at substrate concentrations above and below K(m). Sodium acetate and formate buffers containing 100 mM NaCl and the same additives as in buffer D were used to obtain the pH rate profile. The final volume of the assay was 100 µl containing 0.2 µM of protein, 0.5 M urea, and varying concentration of peptide I. The kinetic parameters, K(m) and k, were obtained by fitting the Michaelis-Menten equation to initial rates. Inhibition constants were obtained by fitting the equation: K`(m) = K(m) (1 + [I]/K(i)) to the observed K`(m) and the inhibitor concentrations.

At low enzyme concentrations, a fluorometric assay was used(14) . In a typical measurement, 135 µl of buffer D, which was 9 µM in peptide II, 4-(4-dimethylaminophenylazo)benzoyl--aminobutyryl-Ser-Gln-AsnTyr-Pro-Ile-Val-Gln-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid (Bachem Bioscience Inc., PA), was placed in a microfluorescence cell (Hellma Cells Inc., NY). Reaction was initiated by adding 15 µl of the protein solution in buffer C and monitored by following the increase in fluorescence at 490 nm using an excitation wavelength of 340 nm.

Kinetics of Conversion of PR-DeltaPol to Mature Protease

A solution of PR-DeltaPol in buffer C was diluted 10-fold in buffer D. Aliquots of 20 µl were drawn at various time intervals, and the reaction was terminated by addition of an equal volume of 2 times Laemmli sample buffer(10) . Samples were subjected to SDS-PAGE on 10-20% gradient gels. Electrophoresis was performed until the pre-stained 6-kDa marker protein (SeeBlue; NOVEX, CA) reached the bottom of the gel. Under these conditions, the PR-DeltaPol (13.2 kDa) and the mature protease (11 kDa) were well separated. Proteins transferred onto nitrocellulose membranes were immunoblotted using HIV-1 PR-specific antibodies and I-labeled protein A(10) . Radioactive bands were visualized on a Phosphor screen (PhosphorImager, Molecular Dynamics), and the band intensities were quantified by using the computer program Image Quant V 3.3 (Molecular Dynamics, CA).

Protein Fluorescence

Intrinsic protein fluorescence was measured in 150 µl of 100 mM acetate buffer, pH 5.0, which was 0.5 M in urea, 5 mM in EDTA, 5 mM in DTT, and contained 0.05% reduced Triton X-100 in a microfluorescence cell. The excitation wavelength was 280 nm with a slit width of 10 nm, and the emission wavelength was 345 nm with a slit width of 20 nm. For the acid denaturation experiment, fluorescence of a 250 nM solution of protein was measured between pH 2.5 and 5.0 in 50 mM formate and acetate buffers containing 2.5 mM DTT using an excitation wavelength of 280 nm with a slit width of 2.5 nm and an emission wavelength of 348 nm with a slit width of 20 nm.


RESULTS

Isolation and Purification of the Transient Intermediate PR-DeltaPol

The time course for the autoprocessing of MBP-DeltaTF-PR-DeltaPol (52 kDa) to mature protease in buffer D at pH 5.0 shows that maximum accumulation of the intermediate PR-DeltaPol (13.2 kDa) occurs after 45 min(12) . At this time, >30% of the full-length precursor is converted to the mature protease. Thus, initial experiments were aimed to increase the amount of PR-DeltaPol relative to the mature protease. In the presence of 0.8 µM pepstatin A, an inhibitor of aspartic acid proteases(12) , the autoprocessing of MBP-DeltaTF-PR-DeltaPol is significantly retarded, and about 25% of the starting material is converted to PR-DeltaPol in 2.5 h. Analysis of the reaction mixture by SDS-PAGE and Coomassie staining showed that the accumulation of PR-DeltaPol is not accompanied by a significant amount of the mature protease under these conditions. The reaction was terminated by the addition of 10 volumes of ice-cold methanol to precipitate the proteins. Proteins were recovered and subjected to gel filtration chromatography under denaturing conditions to prevent further processing of the PR-DeltaPol to the mature protease (Fig. 2). Following the elution of the large molecular weight proteins MBP-DeltaTF-PR-DeltaPol and MBP-DeltaTF, the PR-DeltaPol eluted separately in fraction 8 (Fig. 2). The major enzymatic activity peak coincided with the PR-DeltaPol absorbance peak. Using this procedure, 200-300 µg (15-18% yield) of PR-DeltaPol with high enzymatic activity was recovered from 5-6 mg of the starting material. Quantitation by dye-binding assay and SDS-PAGE followed by densitometry indicated that the purified PR-DeltaPol (13.2 kDa) fusion protein was >90% pure (Fig. 2B, fraction 8).


Figure 2: Purification of PR-DeltaPol by gel filtration chromatography. Processing of the protease from the purified MBP-DeltaTF-PR-DeltaPol was initiated by renaturation of the protein in the presence of pepstatin A. After 2.5 h, the reaction was terminated by precipitation, and proteins were collected by centrifugation and fractionated under denaturing conditions. A, elution monitored for changes in absorbance at 280 nm (closed circles) and protease activity (open circles). B, fractions 3-8 were subjected to SDS-PAGE on Tris-Tricine gels, and proteins were visualized by Coomassie staining. The MBP-DeltaTF-PR-DeltaPol, MBP-DeltaTF, and PR-DeltaPol are indicated as 52-, 38-, and 13.2-kDa proteins, respectively. The mature protease elutes in fractions 9 and 10 under the same conditions.



Characterization of the Enzymatic Activity of PR-DeltaPol

Similar to the mature protease, PR-DeltaPol catalyzes the hydrolysis of peptide I between Nle and (NO(2))Phe, and its catalytic activity is competitively inhibited by known inhibitors of the mature protease. Saturation kinetics were observed for the PR-DeltaPol-catalyzed hydrolysis of peptide I. Table 1summarizes the kinetic parameters for PR-DeltaPol and PR-catalyzed hydrolyses of peptide I and inhibition constants for the hydrolytic reactions with the inhibitor. The pH rate profile for k/K(m) for PR-DeltaPol-catalyzed hydrolysis of peptide I is shown in Fig. 3. The kinetic measurements below pH 4.0 were not included in the study due to acid denaturation of the PR-DeltaPol (see below).




Figure 3: pH dependence of k/K for PR-DeltaPol-catalyzed hydrolysis of peptide I in Mes, acetate, and formate buffers at 25 °C.



Comparison of Conformational Stability of PR-DeltaPol and Mature Protease

The x-ray structure of the mature protease suggests that a major part of the intermonomeric interaction occurs between the terminal residues of the two subunits(5) . Since the protease dimerizes as a polyprotein(12) , urea and acid denaturation were used to assess the influence of the sequence at the C terminus (DeltaPol) on the stability of the enzymatically active form of the PR-DeltaPol in comparison with the mature protease. Both proteins undergo reversible urea and acid denaturation. Fig. 4shows the proteolytic activity of PR-DeltaPol and the mature protease as a function of urea concentration. The urea concentrations leading to 50% loss in enzymatic activity of PR-DeltaPol and protease are 1.45 and 1.85 M, respectively.


Figure 4: Urea denaturation curves for PR-DeltaPol (open circles) and protease (filled circles) as determined by measuring changes in enzymatic activity in buffer D without Triton X-100 at 25 °C.



The intrinsic fluorescence of the PR-DeltaPol and the mature protease displays a sigmoidal decrease in intensity with the lowering of pH. The fluorescence changes occur over a narrow pH range. For PR-DeltaPol, the range is 0.4 pH unit with an inflection at pH 3.7, whereas for the mature protease the changes occur over a range of 0.9 pH unit with an inflection point at pH 2.9. Fluorescence spectra obtained at pH 2.5 and >4 for both proteins are identical to those of the urea-denatured and enzymatically active proteins, respectively. Similar fluorescence changes are observed on acid denaturation of staphylococcal nuclease (15) . Thus, the pH-dependent fluorescence change of PR-DeltaPol and mature protease is due to protein denaturation.

Autoprocessing of PR-DeltaPol Fusion Protein

Upon 10-fold dilution of the protein in buffer D, pH 5.0, PR-DeltaPol undergoes a time-dependent reaction to produce the 11-kDa mature protease. Fig. 5shows the time course for the disappearance of the PR-DeltaPol and the concomitant appearance of the mature protease upon cleavage of the C-terminal strands. The identity of the mature protease and PR-DeltaPol were confirmed by comigration with previously characterized proteins(12) . Initial rates for the conversion of the PR-DeltaPol to mature protease were measured by quantifying the disappearance of the PR-DeltaPol fusion protein and the appearance of the mature protease after SDS-PAGE and immunoblotting. Incubation times ranged from 2 to 10.5 min at a protein concentration of 476 nM and from 11 to 62 min at 68 nM. Under these conditions the observed conversion of the PR-DeltaPol to the mature protease was leq25%, and the band intensity of the blotted protein was linearly dependent on the protein concentration (see inset in Fig. 6). The linear relationship between initial rates and the square of the protein concentration shown in Fig. 6indicates that the reaction is second order in protein concentration. The slope of the line is the second-order rate constant for the reaction, 675 ± 30 M s.


Figure 5: Time course of the conversion of PR-DeltaPol to protease monitored by immunoblotting analysis. Aliquots were drawn at the indicated times, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with a protease specific antibody.




Figure 6: Kinetics of the conversion of PR-DeltaPol to protease at pH 5.0 in buffer C at 25 °C. The reactions were monitored by immunoblotting, and band intensities corresponding to PR-DeltaPol and protease were quantified by densitometry. Reactions were monitored to leq25% conversion, and initial rates were estimated from plots of conversion versus time. Band intensities are linearly dependent on the amount of protein blotted as shown in inset.



The Dimeric Nature of PR-DeltaPol

Since only the dimeric form of PR-DeltaPol is enzymatically active, the monomer-dimer equilibrium of the enzyme can be examined by measuring the proteolytic activity and the intrinsic fluorescence of the PR-DeltaPol as a function of the protein concentration. Because of its higher sensitivity, the fluorogenic substrate for HIV-1 protease (peptide II) is used at a concentration of 9.6 µM, which is well below its K(m) value (15) so that no substantial fraction of the protein is present as the enzyme-substrate complex. Conversion of a substantial fraction of the enzyme to enzyme-substrate complex would shift the equilibrium in favor of the dimeric form and would thus alter the apparent association constant(3) . The initial rates of hydrolysis of peptide II and the intrinsic protein fluorescence were measured at PR-DeltaPol concentrations from 10 to 245 nM. In both experiments, the measured activity and fluorescence are linearly proportional to the protein concentration, and the lines intersect the origin. Thus, the PR-DeltaPol is largely in the dimeric form >10 nM.


DISCUSSION

We have developed a procedure to purify the protein intermediate PR-DeltaPol (see Fig. 1) to near homogeneity and examined the mechanism of the C-terminal proteolytic cleavage of HIV-1 protease from MBP-DeltaTF-PR-DeltaPol. The method described, which requires addition of pepstatin A in the autoprocessing reaction, allows the isolation in 18% yield of the PR-DeltaPol formed during the autoprocessing of MBP-DeltaTF-PR-DeltaPol and substantially increases the accumulation of PR-DeltaPol relative to mature protease. In addition, since PR-DeltaPol and the mature protease were separable by gel filtration chromatography under denaturing conditions, the purified PR-DeltaPol was shown to contain very little mature protease.

Both protease and PR-DeltaPol fold and dimerize instantaneously with concomitant appearance of enzymatic activity after the denatured proteins are diluted from 5 M urea with 10 volumes of acetate buffer at pH 5.0. The proteolytic activity of the PR-DeltaPol is nearly indistinguishable from that of the mature protease at pH 5.0 (Table 1) as is the pH rate profile for k/K(m) for the PR-DeltaPol-catalyzed hydrolysis of peptide I above pH 4.0. The pK(a) of about 5.2 determined from the pH rate profile of PR-DeltaPol is in agreement with the pK(a) of 4.8 obtained for the mature protease(16) . These results clearly demonstrate that the addition of C-terminal flanking sequences to the protease does not distort the active site of the enzyme nor alter the environment of the catalytic groups.

Unlike the cleavage of the protease at its N terminus, which is an intramolecular reaction of the dimeric fusion protein and follows first-order kinetics(12) , the cleavage at its C terminus is second order in protein concentration (Fig. 6). Since PR-DeltaPol is predominantly in the dimeric form at a concentration geq10 nM (see below), the most reasonable explanation for the second-order kinetics is that the cleavage of DeltaPol occurs via an intermolecular mechanism (Fig. 1). In such a mechanism, a PR-DeltaPol dimer catalyzes the hydrolysis of the C-terminal DeltaPol sequences of another dimer with an observed second-order rate constant of 675 ± 30 M s (100 mM acetate, pH 5.0, at 25 °C), which corresponds to k/K(m) for this reaction.

The above mechanism is somewhat unexpected since the structure of the dimeric mature protease shows that the C-terminal strand of one subunit is involved in a hydrogen bond network with the N- and C-terminal strands of the other subunit (see below). Therefore, the C-terminal strands of a dimeric PR-DeltaPol may not be easily accessible to bind to another enzyme dimer and that should be reflected in the observed second-order rate constant. The k/K(m) for the HIV-1 protease-catalyzed hydrolysis of a peptide substrate spanning the C-terminal cleavage site is 24,000 M s in 0.2 M phosphate buffer, pH 5.6, and 2 M NaCl at 37 °C(17) . It is unlikely that the large (40-fold) difference in k/K(m) for this substrate relative to cleavage of PR-DeltaPol results only from differences in the experimental conditions. The value of k/K(m) increases with a higher salt concentration (18, 19) and temperature and decreases with a higher pH(17) . Thus, the large difference in k/K(m) must be due to the inaccessibility of the C-terminal sequence in the dimeric structure of PR-DeltaPol.

Attachment of short native sequences to the N or the C termini of the protease domain of HIV-1 has little effect, if any, on the proteolytic activity, whereas non-native and/or long flanking sequences decrease the enzymatic activity. Tang and co-workers (20) compared the proteolytic activity of a mutant protease (A28S) with and without 25 amino acids of the native TF region linked at its N terminus. These two proteins were shown to have similar enzymatic activities. Sequence alignment of various retroviral proteases show that several of these enzymes have short sequences that extend beyond the HIV-1 protease (21) . These results are consistent with the observed catalytic activity of PR-DeltaPol. In contrast, the attachment of the non-native 14-kDa protein A sequence at the N terminus of HIV-1 protease causes a 10-fold decrease in catalytic activity(22) . MBP-DeltaTF-PR-DeltaPol, which contains the 38-kDa MBP domain, has at least 600-fold lower catalytic activity than the mature protease(12) . Qualitatively, these results indicate that the type of sequence and/or its length have an influence on the proteolytic activity.

The major difference observed between PR-DeltaPol and the mature protease is in their conformational stabilities. PR-DeltaPol is more sensitive toward urea and acid denaturation than the mature protease. These results can be explained based on the three-dimensional structure of the mature protease. The major interactions between the two subunits of the mature protease occur in the region of the N- and C-terminal strands. The two C-terminal strands form an antiparallel beta-sheet, and the N terminus of each subunit interacts with the C terminus of the other subunit to extend the antiparallel beta-sheet(5) . Each positively charged N terminus is anchored to the beta-sheet by an electrostatic interaction with the negatively charged carboxylate group of the C terminus(23) . Since the dimeric form of PR-DeltaPol lacks this electrostatic interaction, it is expected to be less stable than the mature protease in urea. It should be pointed out that the DeltaPol sequences may provide some additional interactions between the two subunits to partially compensate for the absence of electrostatic interactions.

Although the addition of the DeltaPol sequence at the C terminus of the protease destabilizes PR-DeltaPol, the results presented in this study show no evidence that it alters the monomer-dimer equilibrium. Above 10 nM, both the rate of PR-DeltaPol-catalyzed hydrolysis of peptide II and the measured intrinsic protein fluorescence are linearly dependent on the protein concentration. Due to the sensitivity limits of the protease assay using peptide II and the intrinsic fluorescence measurement, the monomer-dimer equilibrium of PR-DeltaPol could not be assessed below 10 nM. Since enzymatic activity requires dimer formation, these results indicate that PR-DeltaPol is predominantly dimeric under our experimental conditions.

In vitro studies of the autoprocessing of HIV-1 protease from the model polyprotein MBP-DeltaTF-PR-DeltaPol provide a plausible mechanism for the maturation of the viral Gag-Pol polyprotein similar to that in Fig. 1. Our previous results suggested that the dimeric Gag-Pol polyprotein has low intrinsic proteolytic activity relative to that of the mature enzyme and that the cleavage at the N terminus of the protease domain occurs via an intramolecular mechanism(12) . This mechanism for the separation of the structural and functional proteins is consistent with proteolytic steps involved in the processing of other viral polyproteins(2) . The low proteolytic activity of the dimeric Gag-Pol may be necessary to prevent the separation of the functional domain that contains the protease, reverse transcriptase, and integrase until the assembly of the viral particle is complete. On the other hand, the Pol product resulting from the initial cleavage may possess enzymatic activity comparable to that of the mature protease, which allows it to carry out the remainder of the processing events leading to the release of the mature proteins via intermolecular reactions.


FOOTNOTES

*
This work was supported by grants from the Intramural AIDS Targeted Program of the Office of the Director of the National Institutes of Health (to J. M. L. and D. M. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 6, Rm. B1-16, NIDDK, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8958; Fax: 301-496-5239.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; MBP, maltose binding protein; DTT, dithiothreitol; Mes, 4-morpholineethanesulfonic acid; PR, HIV-1 protease; DeltaPol, 19 amino acids of the reverse transcriptase sequence; DeltaTF, 12 amino acids of the transframe protein sequence; MBP-DeltaTF-PR-DeltaPol, the polyprotein containing DeltaTF and DeltaPol sequences at the N and C termini of HIV-1 protease, respectively, fused to the MBP; peptide I, Lys-Ala-Arg-Val-Nle-Phe(NO(2))-Glu-Ala-Nle-NH(2); Nle, norleucine; peptide II, 4-(4-dimethylaminophenylazo)benzoyl--aminobutyryl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid; inhibitor, Arg-Val-Leu-(r)Phe-Glu-Ala-Nle-NH(2), where (r) denotes a reduced peptide bond; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine.


ACKNOWLEDGEMENTS

-We are thankful to Dr. A. R. Kimmel for support and comments, to Dr. J. M. Sayer for helpful criticisms, and to Dr. D. Grobelny for providing the transition-state analog.


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