©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of Internal Autocleavage on Kinetic Properties of the Human Cytomegalovirus Protease Catalytic Domain (*)

(Received for publication, August 1, 1994; and in revised form, November 28, 1994)

Donald R. O'Boyle II Karen Wager-Smith John T. Stevens III Steven P. Weinheimer (§)

From the Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 28-kilodalton (kDa) catalytic domain of the human cytomegalovirus (HCMV) protease undergoes autoproteolytic cleavage at an internal site (I site), yielding amino-terminal 15-kDa (N15) and carboxyl-terminal 13-kDa (C13) fragments. I site autocleavage has been postulated to inactivate the protease and provide a mechanism for the negative regulation of enzyme activity during viral infection. We purified recombinant enzymes to demonstrate I site autocleavage in vitro and used site-directed mutagenesis of the I site to stabilize the protease. No difference in the kinetic properties of wild type and stabilized mutant proteases were observed in an in vitro peptide cleavage assay. The consequences of I site cleavage on enzyme activity were investigated two ways. First, autodigestion of the wild type enzyme converted the intact protease to N15 and C13 autocleavage products without a corresponding loss in enzyme activity. Second, genetic constructs encoding the N15 and C13 autocleavage products were generated and expressed separately in Escherichia coli, and each fragment was purified. An active enzyme was reconstituted by refolding a mixture of the purified fragments in vitro to form a noncovalent complex. The kinetic properties of this complex were very similar to the wild type and stabilized enzymes under optimal reaction conditions. We concluded from these studies that I site cleavage does not inactivate the HCMV protease, in the absence of other virally induced factors, and that limited potential exists for the regulation of catalytic activity by I site cleavage.


INTRODUCTION

Herpesviruses encode a protease (1, 2) involved in capsid assembly which is essential for viral replication(3, 4, 5, 6, 7, 8, 9, 10) . The protease is expressed as a precursor protein, the carboxyl terminus of which is overlapped by a co-terminal gene encoding the capsid assembly protein (2, 11) . The amino acid sequence of the protease catalytic domain is highly conserved among the alpha, beta, and herpesvirus subgroups, and similar open reading frame structures have been observed for the cytomegalovirus (CMV, (^1)a beta herpesvirus) and herpes simplex virus type 1 (HSV-1, an alpha herpesvirus) proteases (12, 13, 14, 15) . The assembly protein is translated in the same open reading frame as the protease, resulting in sequence identity with the carboxyl terminus of the protease. The protease precursor undergoes autoproteolytic cleavages at two conserved sites, the release and maturation cleavage sites. The release cleavage site is upstream of the assembly protein domain and releases the protease catalytic domain from the amino terminus of the full-length precursor(2, 9, 16, 17, 18) . The maturation cleavage site is near the carboxyl terminus of the protease and is duplicated in the assembly protein which is also cleaved by the protease(1, 2) . The protease precursor and the viral assembly protein are currently the only known substrates for the herpesvirus proteases, suggesting that either the release or maturation site cleavages, or both, are essential for viral replication.

The CMV protease differs from the HSV-1 protease by virtue of a third autoproteolytic cleavage site near the center of its catalytic domain (17, 19) . This internal site, termed the I site, was mapped between amino acids Ala-143 and Ala-144 of the human CMV (HCMV) protease (17) and produces an amino-terminal fragment of 15 kDa(N15) and a carboxyl-terminal fragment of 13 kDa (C13)(17, 20) . An homologous site is also cleaved autocatalytically by the simian CMV protease(19) . Clear homologs of the I site are not apparent in the deduced amino acid sequences of proteases from alpha and herpesvirus. Moreover, the I site amino acid sequence exhibits only partial homology with the canonical release and maturation cleavage site sequences. These sites are highly conserved at the P4 to P1` positions and usually have alanine and serine residues at the P1 and P1` positions, respectively (2, 17, 21; using the nomenclature of Schechter and Berger(22) ). Hydrolysis at these sites is very sensitive to mutation of the conserved P4-P1` residues(23) . (^2)Similarly, mutations within P4-P1` of the I site prevent hydrolysis by the CMV proteases(19, 25) .

Proteolytic cleavage of protease zymogens can result in dramatically increased enzyme activity(26) . I site cleavage does not increase the catalytic activity of HCMV protease since mutation of the I site to prevent its cleavage did not reduce enzyme activity(19, 25) , so the protease precursor does not appear to be a zymogen. Instead, the occurrence of a cleavage site near the center of the CMV protease catalytic domain has led to the proposal that it inactivates the protease and could provide a mechanism for regulating enzyme activity during infection: the original I site designation stands for `` inactivation site''(17, 20) . Deletion mutagenesis has demonstrated that amino acid sequences near the amino terminus and carboxyl terminus of both the HSV-1 and HCMV protease catalytic domains are required for enzyme activity(17, 27, 2) . Thus, if I site cleavage causes dissociation of the resulting N15 and C13 products, then an inactive protease would be the product of this cleavage(20) . During purification of the HCMV protease catalytic domain from a recombinant bacterial source, however, I site cleavage products were found to co-fractionate with the intact HCMV catalytic domain through a variety of chromatographic separations even after a cycle of denaturation using 7 M urea(20, 24) . (^3)This suggested that I site cleavage does not cause the dissociation of the N15 and C13 products and raised the possibility that the complexed fragments might retain enzymatic activity. This report investigates the in vitro catalytic and kinetic properties of purified HCMV proteases that are either resistant to I site cleavage or that consist entirely of a noncovalent complex of I site cleavage products. By comparison to the properties of wild type HCMV protease, the effect of internal cleavage was determined.


MATERIALS AND METHODS

I Site Mutagenesis and Construction of N15 and C13 Fusion Proteins

Substitutions of amino acids 143 and 144 of the HCMV UL80 protease were introduced into pGST-HCMV-256 using the polymerase chain reaction.^3 Oligonucleotide primers spanning codons 139-148 were used, incorporating base substitutions for Ser (TCC), Thr (ACC), Val (GTC), Asn (AAC), or nonsense (TAG) codons at position 143 and Thr (ACG) or Val (GTG) codons at position 144 and which contained the adjacent AatII restriction enzyme site. The second polymerase chain reaction primer overlapped codons 1 and 2 of the protease, and polymerase chain reaction reaction conditions were according to the Perkin Elmer Cetus instructions except that 1 µg of template was used for seven cycles. Polymerase chain reaction products were gel-purified and digested with restriction enzymes AatII and SacII, which span the I site, for reconstruction of the mutated proteases. The vector was linearized with AatII followed by complete digestion with SacII. Each of the predicted mutations was confirmed by DNA sequencing. The stop codon at amino acid 143 was used to generate the amino-terminal 15-kDa product fused to GST (GST-N15). To generate the carboxyl-terminal 13-kDa product fused to GST (GST-C13), two complementary oligonucleotides were obtained: 5`-GATCCTCGCTTTCGGGCTCGGAAACCACGCCGTTCAAAACAC-3` and 5`-GTGTTTGAACGGCGTGGTTTCCGAGCCCGAAAGCGAG-3`. These sequences were designed to substitute Gly-Ser-Pro (from thrombin cleavage site) for Ala-144 and Thr-145, with BamHI and PmlI restriction enzyme overhangs for cloning. After annealing, these oligonucleotides were ligated to the parental pGST-CMV-256 vector digested with BamHI and PmlI. All purified oligonucleotides were purchased from Genosys Biotechnologies, Inc.

Expression, Purification, and Analysis of HCMV Protease

GST fusion proteins of I site mutants were isolated from freshly transformed BL21 cells (Novagen) plated on LB-agarose plates containing 50 µg/ml ampicillin. Cultures were grown in LB/ampicillin broth (29) at 37 °C to an OD of 0.5, at which time isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.1 mM, then growth was continued for 3 h. Cell pellets were resuspended in 20 ml of lysis buffer (50 mM Tris, pH 8.0, 2 mM DTT, 5 mM EDTA, 25 mM NaCl, 1 mg/ml lysozyme) per liter of cell culture, using a Dounce homogenizer. Triton X-100 was added to 1%, and the resulting suspension was sonicated. Lysates were then centrifuged at 27,000 times g for 30 min at 4 °C, and the fusion proteins were found invariably in the insoluble pellets, which were resuspended in 20 ml of denaturing buffer (7 M urea, 50 mM Tris, pH 8.0, 10 mM DTT) on ice using a Dounce homogenizer. This solution was dialyzed against renaturing buffer (50 mM Tris, pH 8.0, 0.1 mM EDTA, 1 mM DTT, 25 mM NaCl) using 1000 volumes for a minimum of 8 h, 4 °C, sufficient to permit affinity purification. The dialyzed sample was clarified at 26,000 times g, and the supernatant was mixed with previously hydrated and washed glutathione (GSH)-agarose beads (Sigma, 330 mg dry weight per liter of culture) for 20 min at 4 °C with gentle rotation. After affinity binding, the beads were washed 5 times with 10 volumes of thrombin buffer (100 mM Tris, pH 8.8, 100 mM NaCl). For stability tests and initial enzymatic assays, I site mutants were eluted with buffer containing 5 mM glutathione (GSH). The intact fusion protein samples were dialyzed extensively versus renaturing buffer before being tested. The wild-type preparation included 43-kDa (GST-N15) and 13-kDa (C13) bands resulting from I site cleavage when analyzed by sodium dodecyl sulfate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE). (The N15 band was absent since the GST domain is still fused to the amino terminus of the protease.)

To purify the intact catalytic domain, and N15 and C13 fragments, fusion proteins were not eluted with GST. Instead, one-tenth volume of thrombin buffer was added to create a dense slurry of GSH agarose beads + fusion protein. The bound fusion protein concentration was determined using Bradford protein assay solution (30, Bio-Rad) by removing a sample of the slurry. Human thrombin (Sigma) was then added to 0.1% (w/w) by stirring with the pipette tip, and the mixture was placed at 30 °C for 10 min to cleave the fusion protein while bound to GSH-agarose. The cleaved protein was eluted five times with 1 volume of thrombin buffer, then mixed with 0.5 ml of hydrated benzamidine-agarose beads (Sigma) at 4 °C for 20 min to selectively bind and remove contaminating thrombin. Beads were completely removed by centrifugation, and ammonium sulfate was added to 60% saturation. After overnight incubation at 4 °C, the resulting precipitate was collected by centrifugation and resuspended in, and dialyzed against, denaturing buffer (3000-Da cutoff, Spectrum Medical Industries). Proteins were separated by chromatography on a Mono Q anion exchange column (Pharmacia Biotech Inc.) eluted with a gradient of 0 to 1 M NaCl. Under these conditions, C13 was recovered in the flow-through, N15 eluted at 180 mM NaCl, and the full-length protease eluted at 170 mM NaCl. Polyclonal rabbit antisera raised against the GST-HCMV protease fusion protein recognized both N15 and C13 in Western immunoblots (data not shown) after affinity purification and thrombin elution. Direct amino-terminal amino acid sequencing of HPLC-purified C13 fragment confirmed the predicted cleavage site (data not shown). Fractions of interest were pooled following SDS-PAGE and placed in a Centriprep 10 (Amicon) concentrator until the volume was reduced to 1% bed volume for gel filtration chromatography through Superose 12 equilibrated in 2 M NaSCN, 50 mM Tris, pH 8.0, 10 mM DTT(20) . The resulting peaks were examined by SDS-PAGE, and fractions containing the desired proteins were pooled. To reactivate the enzyme, the protease was placed into 100 volumes of renaturing buffer for 4 h followed by 1000 volumes of 2 times concentrated renaturing buffer for 16 h, followed by the addition of glycerin to 50% and an additional 16 h of dialysis. Aliquots were removed and placed at -80 °C at a concentration of 0.1 to 3 mg/ml until use. Purity estimates ranged between 80 and 95% as determined using Coomassie-stained samples separated by SDS-PAGE. The intact protease was >90% pure as determined by mass spectroscopy (data not shown).

HPLC Peptide Assay

The peptide substrates (designated mP4-P6` and mP4-P8`) were based on the maturation site sequence of HCMV UL80 and were synthesized by the Dept. of Oncology and Peptide Chemistry, PRI, Princeton NJ (C. Mapelli and J. Tsao). The recognition sequences were VVNA-SCRLAT for mP4-P6` and VVNA-SCRLATAS for mP4-P8`, the dash denoting the scissile bond.

Relative rates of I site mutants were measured using 2.2 µM enzyme, 10 mM DTT, 100 mM NaCl, 0.1 mM EDTA, 50 mM Tricine, pH 8.0, 200 µM peptide, and 50 µg/ml BSA. For kinetic studies, initial reaction rates were measured using mixtures (20) containing 120 nM enzyme, 100 mM MOPS, pH 7.2, 30% glycerol, and 200 µM peptide substrate. After incubating reactions at 30 °C, 180 µl of 8 M guanidine HCl, 10 mM DTT was added to terminate the reaction, and 200 µl was injected onto an HPLC C4 column which was eluted with a 0 to 100% acetonitrile gradient in 0.1% trifluoroacetic acid. The absorbance at 215 nm was recorded, and peaks corresponding to the peptide products were quantitated as described elsewhere(31) . Product peaks were confirmed by mass spectroscopy and peptide sequencing (data not shown). Assay results used for quantitative analysis resulted in less than 20% cleavage of the peptide. To determine kinetic constants, the substrate concentration was varied from 4 mM to 0.016 mM. V(max), the maximal velocity, and K(m), the Michaelis constant, were determined by fitting initial rate data to the equation [v = V(max) times S/(K(m) + S)], using the KinetAsystII program for nonlinear regression, where v is the measured initial rate and S is the substrate concentration. Because it was not possible to titrate the active site of the HCMV protease, the exact proportion of each enzyme preparation that was active could not be determined, so the kinetic constants reported here should be considered estimations of their true values.

Autocleavage Reaction Conditions

GST fusion proteins of I site mutants were eluted from GSH-agarose and incubated under assay conditions for up to 96 h. Samples were removed at time points during the incubation and analyzed using SDS-PAGE. Coomassie-stained SDS-PAGE gels were scanned using a laser densitometer (LKB Ultroscan) to quantitate the accumulation of I site cleavage products. The time of 50% cleavage was interpolated from these data points for the less stable samples and extrapolated for the more stable samples (those exhibiting less than 50% autocleavage at 96 h). Ratios of these values with that for wild type protease were compared to determine the relative stability ranking (Fig. 1).


Figure 1: I site mutations stabilize the HCMV protease. Left panel, fusion proteins of wild type and mutant proteases bound to GSH-agarose were separated in SDS-PAGE and stained with Coomassie Blue. Lane designations include the wild type single-letter amino acid code preceding its codon number followed by the substituted amino acid single-letter code (e.g. A143S, serine substituted for alanine 143). Intact GST-HCMV protease (GST-HCMV Prt.) and GST fused to the cleaved amino-terminal fragment (GST-N15) are indicated. Right panel, internal autocleavage of each mutant was monitored during in vitro autocleavage reactions to determine the stability of each mutant relative to wild type (see ``Materials and Methods''). The -fold stabilization relative to wild type protease for each I site mutant is shown along with the P1 and P1` amino acids.



The HCMV protease catalytic domain was incubated at 8 µM (wild-type and A143T/A144T) in 100 mM MOPS, pH 7.2, 30% glycerol for 0 to 96 hr. Samples were removed at time points, examined by SDS-PAGE, and assayed by peptide cleavage. Autocleavage was quantitated by laser densitometry of a Coomassie-stained SDS-PAGE gel. According to these measurements, 89% of the purified wild type protease sample was present initially as the intact 28-kDa species.

Refolding and Assay of Recombinant Fragments

Purified recombinant N15 and C13 fragments in denaturing buffer were mixed in weight to weight (w/w) ratios of 3.3 to 0.16 (C13/N15), corresponding to approximate molar ratios of 4:1 to 1:4. The mixtures and individual fragments were placed in dialysis tubing and subjected to the reactivation protocol described above for affinity purification. Following the final dialysis, a protein measurement was made and enzyme activity was quantitated using the peptide cleavage assay. Native gel electrophoresis employed 10-20% polyacrylamide gels stained with Coomassie Blue.


RESULTS AND DISCUSSION

Stabilization of HCMV Protease by Site-directed Mutagenesis

The HCMV protease catalytic domain was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified from insoluble inclusion bodies (see ``Materials and Methods'').^3 After affinity purification on glutathioneagarose, two contaminants were observed of the size expected from internal autocleavage of the protease at a previously mapped site between Ala-143 and Ala-144(17) , the I site. I site autocleavage of HCMV protease has been documented in HCMV-infected tissue culture cells, transfected cells, reticulocyte lysates, and E. coli(2, 17, 20) . It has been proposed that I site cleavage leads to inactivation of the HCMV protease, although direct demonstration of this has been lacking. To further investigate the role of I site cleavage in HCMV protease function, we first introduced mutations at the I site to prevent its cleavage and to stabilize the protease.

If I site cleavage inactivates the protease, then elimination of the cleavage site would be predicted to produce an enzyme with greater catalytic stability, as long as the mutations introduced would not significantly alter the catalytic or structural properties of the enzyme. Previous studies have shown that amino acids at the P4-P1` positions of canonical release and maturation cleavage sites are critical for hydrolysis by herpesvirus proteases(23) , and that the P1 and P1` positions are the least tolerant of amino acid substitutions (23) .^2 Although the HCMV I site is not highly homologous at each of these positions, its P3 valine and P1 alanine residues are identical with the HCMV maturation site sequence, and the P1` alanine conforms to the apparent requirement for small amino acid side chains (Ser, Ala, Gly) at P1`.^2 Mutations were introduced at the P1 and P1` positions of the I site in the GST-HCMV protease fusion protein. Constructs were generated with the substitutions A143S, A143V, A143N, A144T, A144V, and A143T + A144T (where each pair of single letter codes represent the wild type alanine and substituted amino acids of the indicated codon, respectively). The mutations were selected for their inhibitory phenotype in an E. coli co-expression assay^2 and were biased toward conservative amino acid substitutions to limit the possible disruption of secondary structure. All the substitutions except for A143S introduced amino acid side chains that were larger than the P1 and P1` alanine residues, with varying degrees of structural similarity.

The effect of each mutation was evaluated initially by comparing the stabilities of the mutant fusion proteins expressed in E. coli after affinity purification. High expression levels were observed for all five mutants upon expression in E. coli, and each mutation stabilized the enzyme against I site cleavage (Fig. 1). The efficiency of I site autocleavage over a time course of incubation varied among the mutants with A143T/A144T < A143V < A143N < A144V < A143S < A144T < wild type (Fig. 1). It was not immediately clear, however, whether these mutations reduced I site cleavage by eliminating recognition of the I site, the desired result, or by inadvertantly inactivating the protease. To distinguish between these possibilities, the catalytic activity of each mutant enzyme was measured in vitro using affinity-purified fusion proteins in a quantitative HPLC-based peptide cleavage assay (Fig. 2) and in a recombinant fusion peptide cleavage assay (data not shown). Both assays showed that each mutant catalyzed the hydrolysis of a peptide substrate at a rate comparable to the wild type enzyme (Fig. 2). The various I site mutations, therefore, did not have any effect on enzyme activity, but instead prevented recognition of the I site.


Figure 2: I site mutants retain enzyme activity. Initial reaction rates were measured for each I site mutant and wild type HCMV fusion proteases using the mP4-P6` peptide substrate. The percent conversion of substrate to product was an average of two determinations.



Kinetic Properties of an HCMV Protease I Site Mutant

To determine whether mutations at the I site altered the kinetic properties of HCMV protease, initial reaction rates were measured and compared for hydrolysis of a synthetic peptide substrate (mP4-P8`) by the wild type protease and the most stable I site mutant, A143T/A144T. Rates were measured over a range of substrate concentrations in the presence and absence of glycerol (see ``Materials and Methods''). It has been reported that the addition of glycerol to peptide cleavage assays of HCMV protease enhances the reaction rate for peptide hydrolysis(20) . This comparison was added as an additional test of the effect of I site mutations on kinetic properties of the enzyme. In the absence of glycerol, K(m) and k values for the wild type protease were 500 ± 40 µM and 0.06 ± 0.01 min, respectively, versus 560 ± 20 µM and 0.07 ± 0.01 min, respectively, for the A143T/A144T mutant. In the presence of 30% glycerol, these values were 440 ± 80 µM and 1.6 min for the wild type protease and 440 ± 90 µM and 2.0 min for the mutant protease (Table 1). Thus, mutation of the I site did not alter kinetic properties of the HCMV protease in either the absence or presence of glycerol.



Since mutation of the I site did not alter the kinetic properties of the HCMV protease, the A143T/A144T mutant was an ideal candidate to use in determining the effect of I site cleavage on the stability of enzyme activity. I site cleavage is not efficient under conditions used for the quantitative analysis of peptide hydrolysis (i.e. less than 20% conversion of substrate), however, so although that mutant had normal kinetic properties, these results failed to indicate whether activity of the mutant enzyme was more stable than the wild type protease or whether I site cleavage might have a negative effect on enzyme activity.

The Effect of in Vitro Autodigestion on HCMV Protease Activity

To address the effect of I site cleavage on stability of HCMV protease activity, two approaches were used. First, an autocleavage experiment was carried out using the wild type and mutant proteases. The purified catalytic domains were incubated for up to 96 h under standard reaction conditions, and the outcome was determined using SDS-PAGE analysis and peptide cleavage assays. Coomassie-stained SDS-PAGE gels were scanned using a laser densitometer to quantitate the amount of intact 28-kDa protease that was converted over time to N15 and C13 (Fig. 3). After the 96-hr incubation, 42% of the 28-kDa species present at time zero remained intact with 58% converted to N15 and C13 fragments, while 100% of the mutant enzyme, A143T/A144T, remained intact (Fig. 3). The effect of autocleavage on activity was measured using a quantitative peptide cleavage assay. In this experiment, 81% of the enzyme activity of wild type protease remained after the 96-h incubation, and no reduction in activity was observed for the mutant enzyme which remained full-length (Fig. 3). Thus, an approximate 60% reduction of intact catalytic domain resulted in an approximate 20% loss of enzyme activity. That is, the amount of activity after autocleavage was much greater than would be expected of an inactivating cleavage event. A similar finding has been reported by others(24) . The loss of 20% of the wild type enzyme activity in these experiments suggested either that partial activity was lost as a result of I site cleavage or that the in vitro stability of the otherwise active enzyme might be reduced. Taken together, the data suggest that the effect of autocleavage is not to fully inactivate the protease but could tend to make the cleaved enzyme more labile.


Figure 3: I site autocleavage does not eliminate HCMV protease activity. Top panel, protease samples taken before (0 h) and after (96 h) autodigestion were separated by SDS-PAGE and Coomassie-stained. Autoproteolytic cleavage products of the wild type catalytic domain are indicated (15 kDa and 13 kDa). Bottom panel, the percentage of intact protease (28-kDa protein) that remained after 96 h was quantitated by laser densitometry. Aliquots from each sample were tested for enzyme activity to determine catalytic stability (see ``Materials and Methods'').



Reconstitution of a Bipartite Enzyme Complex

If I site cleavage is not an inactivating cleavage, then a complex of the N15 and C13 autocleavage products must retain enzyme activity. Previous observations have indicated that a complex of the N15 and C13 proteolytic fragments co-fractionated with the intact HCMV protease during a variety of chromatographic separations, even after a cycle of denaturation and renaturation(20, 24) .^3 This indicated that the I site cleavage products possessed a strong affinity for each other, and, due to the constant presence of reducing agent, further suggested the formation of a noncovalent complex of the N15 and C13 fragments. (The native enzyme has 5 cysteines and no disulfide bonds (20) .) Due to the similarity in physical properties between this complex and the uncleaved protease, it appeared that the complex might resemble the native protease with a nick introduced at the I site.

To generate a properly folded complex of N15 and C13 in the absence of any possible contamination with intact protease, GST fusion proteins were constructed for the independent expression and affinity purification of N15 and C13 (see ``Materials and Methods''). The purified fragments were then used to reconstitute an active enzyme complex by refolding a mixture of the denatured fragments to permit their interaction during renaturation (see ``Materials and Methods''). Mixtures of the two fragments were combined in 7 M urea at weight ratios of 0.16-3.3 (C13/N15) to determine the optimal ratio for their combination. Each sample was examined by Coomassie-stained SDS-PAGE for comparison with the wild type composition of C13 versus N15 (Fig. 4A). To check for reconstitution of an active protease, each dialyzed mixture was tested for hydrolysis of a maturation site peptide substrate (Fig. 4B). The refolded mixtures of N15 and C13 fragments clearly possessed significant enzyme activity. A 1:1 complex of N15 bullet C13, as exists in the native enzyme, would constitute a C13/N15 mass ratio of 0.86, and the staining intensity of N15 and C13 in the wild type sample was intermediate between fragments combined in C13/N15 mass ratios of 0.66 and 1.3. The highest activity was achieved using a mixture combined with a ratio of 0.66, the closest ratio to 0.86 that was tested. This indicated that optimal activity would be achieved with a 1:1 complex and that both fragments were fully active, or that a similar proportion of each was active without interference from the remaining inactive portion of either fragment. No enzyme activity was detectable when either fragment was refolded alone or if the fragments were mixed after being refolded separately (data not shown), and similar results were obtained with N15 and C13 isolated during purification of the wild type protease (data not shown). Together, these results indicate that N15 and C13 formed a noncovalent complex that very closely resembles the active protease catalytic domain.


Figure 4: Reconstitution of bipartite HCMV protease. Top panel, recombinant N15 and C13 fragments were mixed in 7 M urea at weight ratios (w/w) of 3.3 to 0.16, renatured, and then separated by SDS-PAGE, Coomassie-stained, and compared to fragments generated during preparation of the wild type HCMV protease (far right lane). Staining intensities of the N15 and C13 fragments from the wild type preparation are intermediate to that of mixtures with ratios of 1.3 and 0.66. Bottom panel, equal amounts of each sample were assayed by peptide cleavage (% hydrolysis of mP4-P8`) to measure the relative efficiency of enzyme reconstitution in each of the mixtures. A 1 to 1 complex of the fragments corresponds to a C13/N15 ratio of 0.86 (arrow).



To determine the extent of similarity between the bipartite N15bulletC13 protease and the wild type and stabilized forms of HCMV protease, a 1:1 complex was reconstituted in vitro and examined by native gel electrophoresis and enzyme activity assays. A sample of each protease preparation was run in parallel through native and denaturing polyacrylamide gels (Fig. 5, A and B). Migration of the intact enzyme was revealed by examination of the A143T/A144T sample which contains a single stained protein band in both systems. The wild type protease co-migrates with this band in both systems, as expected since this material is almost entirely intact. The bulk of the enzyme reconstituted using the N15 and C13 fragments migrates as a complex with slightly faster mobility than the intact enzyme in the native gel (Fig. 5B). Faster migration of the complex might be caused by conformational changes resulting from internal autocleavage. Alternatively, modification of the fragments by E. coli enzymes after the new termini are exposed might alter their migration.


Figure 5: Native and denaturing gel electrophoresis. Aliquots of wild type protease, the stabilized A143T/A144T mutant, and a reconstituted 1 to 1 complex of N15 and C13 (N15bulletC13) were examined in parallel by native (left) and denaturing (right) gel electrophoresis (see ``Materials and Methods'').



Initial hydrolysis rates were measured in the peptide cleavage assay using the reconstituted N15bulletC13 enzyme complex with various concentrations of a maturation site peptide substrate, mP4-P8`, in the presence and absence of 30% glycerol. The derived constants were then compared with those from the wild type and stabilized HCMV proteases described above (Table 1). K(m) and k for the N15bulletC13 enzyme complex measured in the presence of glycerol were 380 µM and 2 min, respectively (Table 2), essentially identical with that of the wild type and stabilized forms of the protease and within the range of experimental error for all three proteases. From this we concluded that the refolded complex is essentially identical with the native catalytic domain after complete processing at the I site, and that I site cleavage of the HCMV protease had no effect on its catalytic properties. The K(m) and k constants derived for the N15bulletC13 complex measured in the absence of glycerol, were 1.45 ± 61 mM and 0.04 ± .01 min, respectively (Table 2). In this case, I site cleavage resulted in about a 3-fold reduction in the affinity of this enzyme for the mP4-P8` substrate relative to the other proteases, with only a slight reduction in k. By comparing the ratio k/K(m), applicable under conditions of limiting substrate, activity of the N15bulletC13 complex was reduced by a factor of 4- to 5-fold relative to the wild type and stabilized forms of the protease in the absence of glycerol ( Table 1and Table 2). Nevertheless, the N15 bullet C13 complex still hydrolyzed the peptide substrate at nearly 60% of the wild type rate, indicating that the active conformation of HCMV protease was not significantly altered by I site cleavage.



Conclusion

I site cleavage has been proposed to inactivate the HCMV protease and to provide a mechanism for regulating enzyme activity during cytomegalovirus infection(17, 20) . The results reported here rule out the possibility that this cleavage inactivates the HCMV protease and indicate that I site cleavage does not measurably alter the kinetic properties of the catalytic domain. This situation is not unlike the autoproteolytic cleavage of neotrypsinogen which results in a two-chain complex that can subsequently be fully activated by trypsin. There is no measurable effect of internal autocleavage by trypsinogen, as the activated two-chain enzyme has kinetic properties very similar to the activated one-chain enzyme(28) . A slight reduction in catalytic rate of the bipartite N15bulletC13 complex was observed under suboptimal conditions, however, suggesting that alteration of the HCMV protease catalytic properties by I site cleavage might be possible. Alternatively, I site cleavage could simply destabilize the enzyme to permit more rapid inactivation by external factors.

To be sure in the case of HCMV protease, additional factors present during HCMV infection could either enhance or diminish the effects of internal cleavage on the HCMV protease, but it is not clear whether such factors exist or or how they might act. Interactions between the HCMV protease and other proteins during capsid assembly could stabilize the active conformation of the protease, perhaps like the in vitro effect of glycerol, in which case essentially no effect of I site cleavage on enzyme activity would result. If, on the other hand, I site cleavage is inhibitory, then interactions with other proteins might further destabilize the bipartite N15bulletC13 enzyme, achieving more significant levels of negative regulation then achieved by cleavage alone in vitro. Our results suggest, however, that the possibilities for negative regulation might be limited. The best way to address these questions would be to introduce I site mutations back into the CMV chromosome and to measure their effects on both protease activity and viral replication. Another aspect of protease function that has not been addressed in HCMV is whether or not the catalytic domain functions during assembly by participating in specific interactions that are important for the structural integrity of capsids, and whether or not I site cleavage might effect some unidentified function of the protease that would not be reflected in enzyme activity measurements. The findings reported here do not rule out the existence of a mechanism to regulate protease activity during infection, but they clearly indicate that I site cleavage alone can not account for a major change in activity levels.


FOOTNOTES

*
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 and reprint requests should be addressed. Tel.: 203-284-6599.

(^1)
The abbreviations used are: CMV, cytomegalovirus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; N15, amino-terminal fragment of 15 kDa; C13, carboxyl-terminal fragment of 13 kDa; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
I. Deckman and P. J. McCann III, unpublished results.

(^3)
S. P. Weinheimer, J. T. Stevens III, D. R. O'Boyle II, C. L. DiIanni, M. G. Cordingley, and R. J. Colonno, manuscript in preparation.

(^4)
G. Yamanaka and S. Weinheimer, unpublished results.


ACKNOWLEDGEMENTS

We gratefully acknowledge Claudio Mapelli and Jonglin Tsao for synthetic peptides. Linda Matusick-Kumar and Ingrid Deckman provided the HSV-1 fusion substrate plasmid, and Fenyong Liu and Bernard Roizman provided pRB4090. Greg Yamanaka, Min Gao, and, especially, Carolyn DiIanni provided helpful discussion and advice during the completion of these studies. We thank Rich Colonno for his advice and enthusiastic support of this work.


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