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Journal of Virology, April 2007, p. 4091-4103, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02821-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enzymatic Activities of Human Cytomegalovirus Maturational Protease Assemblin and Its Precursor (pPR, pUL80a): Maximal Activity of pPR Requires Self-Interaction through Its Scaffolding Domain{triangledown}

Edward J. Brignole and Wade Gibson*

Virology Laboratories, The Department of Pharmacology & Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205

Received 20 December 2006/ Accepted 30 January 2007


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ABSTRACT
 
Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.


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INTRODUCTION
 
Human cytomegalovirus (HCMV) is a ubiquitous, opportunistic pathogen of the herpesvirus family that causes life-threatening illness in immunocompromised individuals (47, 52, 64). Identifying and characterizing antiviral targets is critical for the development of new therapeutic agents to treat infections and diseases caused by this virus (7, 17, 23, 40, 44, 75). One such target or potential set of targets to come from these efforts is the herpesvirus maturational protease (pPR, e.g., HCMV pUL80a) and its genetically related substrate proteins (Fig. 1A), including the precursor assembly protein (pAP, pUL80.5) (36, 37, 77, 78), which are essential for the production of infectious virus (19, 24, 58).


Figure 1
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  		FIG. 1. Stabilizing maturational protease pPR (pUL80a) against self-cleavage at I, C, R, and M sites. (A) Autoproteolytic cleavage sites and products of pPR. Internal (I), cryptic (C), release (R), maturational (M), and tail (T) sites are indicated by arrows; corresponding cleavage sequences, interrupted by a dash between the P1 and P1' residues (61), are below each. The proteolytic (dark) and scaffolding (light) domains of the precursor are indicated by shading. Fragment abbreviations are noted at their left-hand ends. The asterisk denotes the position of the ACD containing Leu382; the carboxyl-conserved domain (CCD) is the same sequence as C1 (62). N2 and C1 indicate amino and carboxyl sequences used to produce antipeptide antisera anti-N2 and anti-C1. Nuclear localization sequences 1 and 2 (55) are indicated by adjacent circles. (B) Expression of ICRM-pPR at 16°C, 37°C, and 42°C. ICRM-pPR-expressing bacteria were lysed (L) and separated into supernatant (S) and pellet (P) fractions at different times after inducing protein expression, as described in Results. Shown here is an image of the proteins following SDS-PAGE and protein staining with SYPRO-R. Molecular masses (kDa) of marker proteins (Mark 12) are shown at the left; the dot to the left of lane 4 indicates the position of ICRM-pPR. (C) pPR and fragments in bacteria expressing ICRM-pPR. ICRM-pPR, wild-type (Wt-pPR), and S132A-pPR were expressed, and equal amounts of pelleted bacteria from each culture were solubilized and subjected to SDS-PAGE (4 to 12% gradient gel), followed by protein staining with CBB (lanes 1 to 3) or by Western immunoassay using anti-pUL80a1-256 (lanes 4 to 6). Protein abbreviations to the right of the images are as for panel A; lines between lanes 3 and 4 indicate the same. Molecular masses (in kDa) of marker proteins are shown to the left of images.

Together, the UL80a proteins coordinate the process of capsid assembly and maturation to a form that is competent for DNA packaging (9, 25, 67). At the earliest stages of the process, pAP is required to translocate the major capsid protein (MCP; pUL86) into the nucleus and then to serve as an internal scaffold to direct its organization into the spherical procapsid shell (19, 39, 69, 70, 83). Once the procapsid is formed, the protease is required to sever pAP-MCP interactions, enabling elimination of the scaffolding proteins to make room for the viral DNA.

The 28-kDa protease is incorporated into the scaffolding structure as a 74-kDa precursor that includes the entire pAP sequence as its carboxyl end (Fig. 1A) (60). This fusion sequence ensures protease targeting to the interior of the capsid by enabling it to interact with pAP through its amino-conserved domain (ACD) and with MCP through its carboxyl-conserved domain. Activation of the protease has been proposed to result from a concentration increase during capsid assembly, based on the observed concentration-dependent dimerization required to activate purified assemblin (16, 42). However, the carboxyl end of free assemblin, which contributes to the dimer interface required for its activation, is constrained in pPR and may be unavailable for interaction or have different interactive properties (10, 71).

Once activated, pPR is autoproteolytically cleaved at four sites (Fig. 1A). The maturational (M) site appears to be cut first (31, 79), eliminating the carboxyl "tail" sequence and thereby severing interaction of pPR with MCP and the capsid shell. A second cleavage at the release (R) site separates the proteolytic domain, assemblin, from the scaffolding domain of pPR and is proposed to cause a rearrangement of assemblin resulting in its carboxyl end becoming buried to form part of its hydrophobic dimer interface (10, 71). The M and R sites are conserved among all herpesvirus homologs of pUL80a (26, 29, 79, 81), and their cleavage, with one known exception, is required for production of infectious virus (45, 53). The exception is a class of herpes simplex virus mutants with a noncleavable M site but having second-site compensating changes in their MCP sequence (18). Unlike most of its homologs, CMV pPR undergoes two additional cleavages, both within its assemblin domain (3, 11, 31). These are at the internal (I) and cryptic (C) sites and, although neither is absolutely essential for virus replication, when both are blocked virus production is reduced by nearly 90% (12, 38). Cleavage at the I site has little effect on the enzymatic properties of assemblin (27, 28, 51). Kaposi's sarcoma-associated herpesvirus also has a cleavage site within its assemblin domain, called the dimer disruption (D) site, which destroys the dimer interface when cleaved and inactivates the protease (50, 56).

Efforts to characterize the herpesvirus protease have focused on assemblin, which is comparatively short, soluble, and amenable to purification by standard methods (11, 28, 65). Enzymatic studies established that it is a unique example of allosteric activation through homodimerization (2, 16, 42, 57). Its dissociation constant is relatively high, approximately 1 µM, but can be decreased about 2 orders of magnitude by structure-enhancing (kosmotropic) salts, such as Na2SO4 (32, 76, 84). Assemblin is a serine protease (21, 30, 79), and structural studies revealed that it has a distinctive protein fold and a distinguishing Ser-His-His catalytic triad (13, 59, 63, 71). Residues near the carboxyl end of assemblin become ordered upon dimerization, in a way that may not be possible in pPR, inducing an active arrangement of oxyanion-stabilizing residues that are located in a loop adjacent to the dimer interface (1, 49). These findings suggest a mechanism whereby cleavage at the R site could alter protease activity (10, 71, 79).

In contrast to the abundance of information available for assemblin, comparatively little is known about the structure and enzymatic properties of its precursor, pPR. Its additional cleavage sites and tendency to adhere to membranes and chromatography medium under nondenaturing conditions have slowed progress (8). The precursor has not yet been crystallized, and only two studies—reaching opposite conclusions—have compared its enzymatic properties with those of assemblin. One used transfection-based assays and concluded that the two forms of the protease are equally active (31). The other used both a cell-based assay and an in vitro assay with purified pPR to conclude that its activity is significantly lower (~11-fold with peptide substrate; ~6-fold with protein substrate) than that of assemblin (82). A better understanding of the structure-function relationship of assemblin to its precursor is needed to determine how this enzyme is regulated and how it might be targeted and inhibited during virus replication.

To further characterize pPR, we have constructed a mutant stabilized against autoproteolysis at the I, C, R, and M sites and at a newly discovered fifth cleavage site, called the "tail" site (Fig. 1A). We have also developed methods to purify, renature, and assay pPR such that its enzymatic properties can be compared in vitro with those of assemblin. In the studies reported here we (i) use rate velocity sedimentation to identify pPR homo-oligomers and estimate their mass; (ii) show that urea-denatured pPR can be effectively folded into active enzyme either by a new gradient sedimentation method or by a two-step dialysis method; (iii) demonstrate that pPR and assemblin have equivalent enzymatic activities against a fluorogenic peptide substrate in vitro; and (iv) show that dimerization of pPR through its scaffolding domain is required for maximal enzymatic activity.


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MATERIALS AND METHODS
 
Plasmid construction. The following mutant forms of the HCMV protease precursor (pPR, pUL80a) and assemblin and simian CMV assembly protein precursor (pAP; UL80.5 homolog) were constructed, and the sequences were verified by standard procedures.

Wild-type pPR. The coding sequences for wild-type pPR (UL80a) and assemblin were PCR amplified from plasmids LM13R and LM12, respectively (27, 80) and cloned into the NdeI and HindIII sites of pET-17b (69663-3; Novagen, La Jolla, CA).

S132A-pPR and S132A-pPRHis. The S132A-pPR mutation inactivates pPR by replacing its Ser132 nucleophile. Using the pPR coding sequence from LM12 in pET-17b (see above), the catalytic serine was mutated to alanine (S132A, TGC->GCG) by cloning annealed mutagenic oligonucleotides into the ApaI and NgoMIV restriction sites, thereby also adding a silent XhoI marker sequence (CTCCAG->CTCGAG and destroying an adjacent NgoMIV site (GCCGGC->GCGGGC). The coding sequence for a six-His purification tag was added to the 3' end of UL80a by inserting annealed oligonucleotides between an XhoI restriction site in the final codon of UL80a and the downstream HindIII site. These two plasmids are called EB6R (S132A-pPR) and EB24 (S132A-pPRHis).

ICRM-pPR and ICRM-pPRHis. The mutant pPRs ICRM-pPR and ICRM-pPRHis are stabilized against autoproteolytic cleavage by four point mutations that result in cleavage-inhibiting amino acid changes at the I, C, R, and M sites (Fig. 1A). They were made from different plasmids containing UL80 sequences by using PCR, site-directed mutagenesis, mutagenic primers, and subcloning (8). Standard procedures were used for all steps, mutations were confirmed in construction intermediates, and the final two UL80a mutant sequences were verified in their entirety and called ICRM-pPR (EB68) and ICRM-pPRHis (EB48). The specific amino acid substitutions and their codon changes and silent marker restriction sites added to simplify cloning were as follows: I site (A143V, GCC->GTC, NruI marker site), C site (A209V, GCG->GTG, MluI marker site destroyed), R site (S257V, AGC->GTG, BtsI marker site), and M site (A643V, GCC->GTC, HpaI marker site).

L382A/ICRM-pPR and L382A/ICRM-pPRHis. The L382A/ICRM-pPR mutation of ICRM-pPR (L382A/ICRM-pPR) interferes with self-interaction of pPR through the ACD (39, 83). The L382A mutation (L47A in the pAP amino acid sequence of UL80.5) was subcloned in a BspMI restriction fragment from the UL80.5 sequence of a yeast two-hybrid expression plasmid (83) into the corresponding BspMI sites of the ICRM-pPR (EB68) and ICRM-pPRHis (EB48) expression plasmids. These plasmids are called L382A/ICRM-pPR (EB69) and L382A/ICRM-pPRHis (EB49).

ICRMT-pPRHis and L382A/ICRMT-pPRHis. The ICRMT-pPRHis and L382A/ICRMT-pPRHis mutants are blocked at the four known cleavage sites and the newly discovered T site reported here. The codon for Ala676 was mutated to encode Gln (GCC->CAG; silent PvuII marker site) by using QuikChange mutagenesis (200518; Stratagene, La Jolla, CA) with ICRM-pPRHis and L382A/ICRM-pPRHis as templates to make ICRMT-pPRHis (EB87) and L382A/ICRMT-pPRHis (EB88).

IC-assemblin and IC-assemblinHis. The IC-assemblin and IC-assemblinHis mutants of assemblin are blocked for I and C site cleavage. ICRM-pPRHis was cleaved with SpeI to leave the 5' sequence for the I and C sites but remove the rest of the UL80a sequence. The 3' end of assemblin was replaced by cloning a SpeI fragment containing the wild-type 3' end from an H63A-assemblin expression plasmid (46), resulting in IC-assemblin (EB66). PCR amplification was used to add the coding sequence for amino-terminal Met-six-His to the 5' end of the IC-assemblin expression plasmid, and the resulting DNA fragment was cloned into the NdeI and HindIII restriction sites of pET-17b to create IC-assemblinHis (EB84).

pAPHis. The pAPHis plasmid encodes the assembly protein precursor (pAP, pAPNG.5) (78) of simian CMV (strain Colburn) with a tandem six-His and MHWHWH purification tag (65) at its amino terminus. The APNG.5 open reading frame with 5'-proximal tag coding sequence was subcloned from plasmid AW37 into the NdeI and HindIII restrictions sites of pAC28 (33), adding the distal six-His tag coding sequence to its 5' end.

Protein expression in Escherichia coli. Cultures inoculated with bacteria from overnight colonies of freshly transformed BL21(DE3)pLysS E. coli cells (70232-3; Novagen) were grown to an optical density at 600 nm of ~0.6 in 100 ml or 1 liter of 2x YT medium. Protein expression was induced with isopropyl-beta-D-thiogalactopyranoside (1 mM; 15529-019; Invitrogen, Carlsbad, CA) at an optical density at 600 nm of ~0.6, and the cells were collected by centrifugation (6,000 x g, 10 min, 4°C) after overnight growth at 16°C for soluble protein or 3 to 5 h at 37°C for insoluble protein. Proteins were prepared under nondenaturing or denaturing conditions as described below.

Preparation of nondenatured protease. Bacterial pellets from cultures grown overnight at 16°C were suspended in nondenaturing lysis buffer (NLB; 50 mM sodium phosphate buffer, 300 mM NaCl, pH 8.0; 30 ml per pellet of 1-liter culture or 10 ml per pellet of 100-ml culture) and passed twice at ~12,000 lb/in2 through a French pressure cell (American Instrument Co., Silver Spring, MD) (22, 41, 74) chilled to 4°C. Inhibitors (1697498; Roche, Indianapolis, IN) added during cell lysis to prevent degradation by bacterial proteases were without detected effect on viral protease activity, as measured using crude lysate preparations of protease with a fluorogenic peptide substrate and with purified pAP substrate (see below) (8).

Resulting lysates were clarified by ultracentrifugation at 100,000 x g (28,500 rpm in a Beckman SW41 rotor) for 30 min at 4°C, and the supernatant was subjected to rate-velocity sedimentation through glycerol gradients (5 to 25% [vol/vol] glycerol in NLB containing 50 mM dithiothreitol [DTT]) at 200,000 x g (40,000 rpm in a Beckman SW41 rotor) for 20 h at 4°C. Gradients were collected into 0.5-ml fractions as described before (4); small portions of each fraction were combined 3:1 with 4x Laemmli protein sample buffer (4x is 8% sodium dodecyl sulfate [SDS], 40% ß-mercaptoethanol, 40% glycerol, 100 mM Tris, pH 7.0, and 0.04% bromophenol blue), and all samples were stored at –80°C.

Preparation of denatured protease from inclusions. Protease-containing inclusions were prepared from lysates of bacteria that had been grown at 37°C for 3 to 5 h, essentially as described before (11). Cells were lysed by French press, and inclusions were collected by centrifugation at 7,700 x g for 10 min and washed once in 1% acetic acid and once in deionized water. Inclusions were suspended in water, rapidly frozen, and stored at –80°C.

In preparation to purify the proteases by immobilized metal affinity chromatography (IMAC), inclusions were collected by centrifugation and dissolved in 5 to 20 ml (according to pellet size from the 100-ml or 1-liter culture) of IMAC binding buffer (100 mM sodium phosphate, 10 mM Tris, pH 8.0) containing 8 M urea (IBB/8 M) and 10 mM DTT. Insoluble material was cleared by ultracentrifugation at 100,000 x g as described above, and the resulting supernatant was aliquoted and stored at –80°C.

Six-His-tagged protease was selected from the denatured, clarified inclusion preparations by IMAC, using Ni2+-Sepharose 6 Fast Flow resin (17-5318-01; GE Healthcare, Piscataway, NJ). Columns containing a 2-ml bed volume of resin were equilibrated with IBB/8 M. Clarified protease preparations (3 ml pPR, 1.5 ml assemblin) were diluted to 10 ml with IBB/8 M and passed twice through the column by gravity flow. The column was then washed twice with 5 ml IBB/8 M containing 25 mM imidazole (I-2399; Sigma, St. Louis, MO) and twice with 5 ml of the same buffer containing 2 M instead of 8 M urea (IBB/2 M). Proteins were eluted in IBB/2 M containing a final imidazole concentration of 300 mM (IEB) and collected in 1-ml fractions. The two fractions containing the highest concentration of protease were combined and used immediately or frozen at –80°C.

Refolding denatured protease. Urea was removed by either rate-velocity sedimentation or by dialysis. Renaturation by rate-velocity sedimentation in glycerol gradients was done as described above for soluble protease, with the following differences. The material layered onto the gradient was IMAC-selected protein in IEB (see above). The gradients were 10 to 25%, instead of 5 to 25%, glycerol to allow overlaying the denser, urea-containing sample, and centrifugation was 16 to 20 h. Fractions collected from the gradients and determined to contain the highest concentrations of protease were combined and stored at –80°C.

Renaturation by dialysis was done in Slide-a-Lyzer cassettes (66425; Pierce, Rockford, IL) immersed overnight at 4°C in 250 ml NLB (see above) containing 1 M arginine and 1 mM DTT. The first dialysis buffer was replaced for an additional 3 to 5 h at 4°C with 250 ml of refolding buffer (RB; NLB containing 15% glycerol and 50 mM DTT) to approximate conditions from which gradient-refolded enzymes were recovered. RB used for the preparations shown below in Fig. 6A and B also contained 0.1 mM EDTA.


Figure 6
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  		FIG. 6. Proteolytic activity of renatured proteases. (A) Kinetics of substrate cleavage in the presence of Na2SO4. Preparations renatured by dialysis (Fig. 4, lanes 5, 10, 15, and 20) or by gradient sedimentation (Fig. 4, lanes 4, 9, 14, and 19) were combined with fluorogenic substrate and assayed as described in Materials and Methods. Shown here are the initial reaction rates of the four protease preparations in activation buffer containing 0.5 M Na2SO4. Standard errors (bars) were calculated for measurements obtained from triplicate reactions. Abbreviations in all panels indicate IC-assemblinHis (Asbln), ICRM-pPRHis (ICRM), L382A/ICRM-pPRHis (L382A), and S132A-pPRHis (S132A). (B) Kinetics of substrate cleavage in refolding buffer with no Na2SO4 added. This is the same experiment as shown in panel A, but the reaction buffer had no Na2SO4. (C and D) Relative active site concentrations of protease preparations. Samples of the four dialyzed protease preparations tested in panels A and B were reacted with biotinylated suicide substrate and subjected to SDS-PAGE (4 to 12% gradient gel) followed by protein staining with SYPRO-R (C). The asterisk indicates the position of the T-site cleavage product. The gel shown in panel C was destained, reacted in-gel with [125I]strepavidin, and imaged, all as described in Materials and Methods. (D) A phosphorimage of the dried gel. The origin of the faint band just slower than assemblin in lanes 4, 5, 7, and 8 of this panel is unknown. (E) Relative active site concentrations of IC-assemblinHis, ICRM-pPRHis, L382A/ICRM-pPRHis, and S132A-pPRHis were calculated from measurements of the data in panels C and D, as described in Materials and Methods. Shown here is a histogram comparing the relative active site specific activities (125I-labeled active site probe [arbitrary units] per relative molar amount of protease). This experiment was done with a single gel; the same comparison of the T-site mutants was done in triplicate (see Fig. 9C, below).

Gel electrophoresis and Western immunoassay. Proteins were separated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE), using either 0.75-mm-thick 10% polyacrylamide gels with Tris-glycine electrode buffer (34), 4 to 12% polyacrylamide gradient mini-gels (NP0323BOX; Invitrogen) with 2-(N-morpholino)ethanesulfonic acid electrode buffer (NP0002; Invitrogen), or 12% polyacrylamide mini-gels (NP0343BOX; Invitrogen) with 3-(N-morpholino)propanesulfonic acid (MOPS) electrode buffer (NP0001; Invitrogen). Following SDS-PAGE, proteins were stained with Coomassie brilliant blue (CBB) or SYPRO-ruby (SYPRO-R; 170-3125; Bio-Rad, Hercules, CA), as specified. The amount of stained protease was determined by comparing its optical absorbance with a dilution series of known amounts of bovine serum albumin (BSA) and quantifying the band intensities by using a Kodak Gel Logic 200 system with 1D Image Analysis software v3.6 (Kodak, Rochester, NY).

Western immunoassays were done following electrotransfer of proteins to polyvinylidene difluoride membranes (4, 38), essentially as described by Towbin et al. (72), using [125I]protein A to detect bound antibodies. Rabbit polyclonal antisera were to synthetic peptides representing the carboxyl end of pUL80a (C1) (62) or the amino end of pUL80a (N2) (27) or to bacterially expressed HCMV assemblin (anti-pUL80a1-256) prepared by SDS-PAGE following selection by IMAC (12).

Peptide cleavage assays. Enzymes were prepared as 500 nM working stocks in RB (see above) and diluted to a final concentration of 100 nM in the reaction mixture. A 10 mM stock solution of fluorogenic peptide substrate [Abz-Tbg-Tbg-Asn(NMe2)-Ala-Ser-Ser-Arg-Tyr(3-NO2)-Arg-OH (Abz represents o-aminobenzoyl, Tbg represents tert-butylglycine, and Me stands for methyl); 4033221; Bachem, San Carlos, CA) that mimics the HCMV maturational cleavage site sequence (6) was prepared in dimethyl sulfoxide (Me2SO) and stored at –20°C. A working stock of 150 µM substrate was prepared in Me2SO for further dilution into reaction buffers.

Two different buffer conditions were used for the cleavage assays: (i) the activation buffer mixture, 50 mM Tris, pH 8.0, 0.5 M Na2SO4, 0.1 mM EDTA, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride, 100 nM protease, 5 µM substrate, and 3.3% Me2SO from the substrate solution (derived from reference 5) and 10 mM sodium phosphate, pH 8.0, 60 mM NaCl, 3% glycerol, and 10 mM DTT from the enzyme solution; (ii) refolding buffer mixture, 50 mM sodium phosphate buffer, 300 mM NaCl, pH 8.0, 50 mM DTT, 15% glycerol (to approximate gradient conditions), 100 nM protease, 5 µM substrate, and 3.3% Me2SO from the substrate solution.

Assays were done by equilibrating 30 µl of enzyme stock with 70 µl of reaction buffer cocktail, rapidly adding 50 µl of substrate (15 µM in corresponding reaction buffer), and quickly transferring the reaction mixture into a quartz cuvette. Substrate cleavage was measured at 25°C in a Spex FluoroMax-3 fluorometer (Jobin Yvon, Inc., Edison, NJ) using 312-nm excitation and 415-nm emission wavelengths and 1.0- and 4.0-nm excitation and emission slit widths, respectively. Initial reaction rates were calculated by using a linear best fit algorithm. Fluorescence change was related to the molar amount of substrate cleaved determined using o-aminobenzoic acid (anthranilic acid; A89855; Sigma) solutions prepared in each reaction buffer.

pAP cleavage assay. Samples of each gradient fraction to be assayed were combined with enough 2x cleavage buffer (2x is 1.0 M Na2SO4, 20% glycerol, 20 mM DTT, 200 mM MOPS, pH 7.2) to give a final concentration of 1x. A one-sixth volume of IMAC-purified pAP substrate in 2 M urea (~9 µM/reaction mixture) was added, and the reaction mixture was incubated at 30°C for 15 min and arrested by adding 4x Laemmli protein sample buffer. The fraction of pAP cleaved to AP was determined by SDS-PAGE, followed by protein staining and image analysis.

Active site titration. Enzymes at a final concentration of 100 nM were combined with 50 µM biotinylated, diphenylphosphonate peptide inhibitor [biotinyl-NH-(CH2)5-CO-Tyr-Tbg-Glu-AlaP-(OPh)2; gift of C. Craik and A. Marnett] in 1x activation buffer and incubated at room temperature for 3 to 4 h. Although this inhibitor was designed and optimized for the Kaposi's sarcoma-associated herpesvirus protease (43), it also showed strong reactivity and selectivity for the CMV protease over other proteins in the bacterial lysates (data not shown). Samples were solubilized in NuPAGE sample buffer (three parts NP0007 [Invitrogen] plus two parts 1 M DTT). The amount of protease protein in each sample was determined by SDS-PAGE followed by staining with SYPRO-R, imaging, and quantification, as described above.

After imaging, the amount of bound biotinylated inhibitor was measured by an in-gel assay (988-08329; LI-COR Biosciences, Lincoln, NE): SYPRO-R-stained gels were soaked 15 min in 50% isopropanol, 5% acetic acid and an additional 15 min in distilled deionized water and then incubated overnight at 4°C with [125I]streptavidin (IM236; GE Healthcare) diluted 1:1,000 in TN buffer (10 mM Tris, pH 7.4, 9% [wt/vol] NaCl) containing 5% BSA and 0.1% Tween 20 (P5927; Sigma, St. Louis, MO). Nonbound [125I]streptavidin was removed by rinsing the gel three times in TN containing 0.1% Tween 20 and three times in TN. The gel was then dried onto Whatman 3M filter paper, and radioactivity was detected and quantified by using a BAS-2500 PhosphorImager with ImageGauge v4.22 (Fuji Medical Systems, Stamford, CT).


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RESULTS
 
Point mutations stabilize HCMV pPR against autoproteolysis. Assemblin and pPR were stabilized against autoproteolysis by using conservative, single amino acid substitutions intended to prevent recognition of the cleavage sites with minimal effect on protein structure. On the basis of earlier site-directed mutagenesis studies (20, 31, 51, 54, 66, 79), we substituted valine for the absolutely conserved P1-alanine residues (nomenclature of Schechter and Berger [61]) at the I, C, R, and M sites (Ala143, Ala209, Ala256, and Ala643, respectively) (Fig. 1A). Only partial inhibition of R-site cleavage was achieved with this mutation (data not shown), and so we used a point mutation at the P1' site (S257V) that had been characterized and shown to block cleavage without noted effect on proteolytic activity (31). The HCMV pPR mutant with all four substitutions is called ICRM-pPR.

We expressed the protease in bacteria to simplify comparing our data with previous studies of purified assemblin and pPR, most of which were done with material recovered from bacteria. Our initial objective was to prepare and characterize soluble nondenatured enzyme. We found that ~75% of ICRM-pPR was soluble (supernatant after 100,000 x g for 30 min) following expression at 16°C overnight (Fig. 1B, lanes 10 to 12), whereas most was insoluble (pellet after 100,000 x g for 30 min) following expression at 37°C for 5 h (Fig. 1B, lanes 4 to 6).

When tested by Western immunoassays, none of the self-cleavage products (e.g., PR, PRc, Asbln, An, and Ac) (Fig. 1A) produced by wild-type pPR (Fig. 1C, lanes 2 and 5) was detected in lysates of bacteria expressing ICRM-pPR, whose patterns were indistinguishable from those of S132A-pPR, an enzymatically inactive mutant (14, 68, 79) (Fig. 1C). The smaller immunoreactive fragments in both ICRM- and S132A-pPR are attributed to cleavage by bacterial proteases at sites other than the I, C, R, and M sites or to premature termination of translation (i.e., lack of a corresponding set of fragments reactive with antibodies to the carboxyl end of pPR). We conclude from these results and data shown below that the four point mutations made to create ICRM-pPR effectively stabilize it against autoproteolysis at these sites.

Rate-velocity sedimentation of nondenatured ICRM-pPR and IC-assemblin. Attempts to purify soluble ICRM-pPR under nondenaturing conditions were complicated by its binding to membranes and chromatography media. This property was mapped to the region of pPR containing nuclear localization signals 1 and 2 (Fig. 1A) but was not further delineated (8).

As an alternate way to separate full-length pPR from the smaller fragments, we tested rate-velocity sedimentation in glycerol gradients. Four different forms of the protease were prepared and analyzed in parallel for comparison: ICRM-pPR, S132A-pPR (inactive), IC-assemblin, and L382A/ICRM-pPR, having a point mutation in its ACD that disrupts pPR oligomerization (39, 83). The resulting gradients were fractionated and analyzed by SDS-PAGE and Western immunoassays. Both ICRM-pPR and S132A-pPR were asymmetrically distributed, trailing from relatively high amounts in the range of 200 to 220 kDa to lesser amounts in the 66-kDa range of BSA, whereas the highest amounts of L382A/ICRM-pPR sedimented as a symmetrical band near the position of BSA (Fig. 2A) (39). All three preparations contained fragments from the amino end of pPR (i.e., reactive with anti-N2), most of which were large enough to include the assemblin domain and potentially have proteolytic activity. IC-assemblin sedimented as a symmetrical band at ~57 kDa, consistent with the expected homo-dimer of its 28-kDa subunits (16, 42, 76).


Figure 2
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  		FIG. 2. Separation and assay of nondenatured protease by sedimentation in glycerol gradients. (A) Distribution of nondenatured proteases following sedimentation. Nondenatured ICRM-pPR, S132A-pPR, L382A/ICRM-pPR, and IC-assemblin were prepared and subjected to centrifugation and gradient fractionation as described in Results and in Materials and Methods. Samples of each fraction from all gradients were solubilized and subjected to SDS-PAGE, followed by Western immunoassay using anti-N2. Samples of the starting preparations (Load) and pellets from each tube (Pellet) were included with each gradient set. The positions of full-length protease (pPR) and assemblin (Asbln) are indicated. The asterisk denotes the gradient fraction containing the highest concentration of protease. (B) Survey of proteolytic activity in gradient-separated, nondenatured preparations. Samples of each gradient fraction, except those from S132A-pPR (inactive), were combined with substrate (pAP) and monitored for cleavage of pAP->AP following incubation, SDS-PAGE, and protein staining with SYPRO-R, as described in Results and Materials and Methods. Shown here are images of the stained gels. Fraction numbers and asterisk positions correspond with respective gradients in panel A. A sample of each starting lysate was reacted and tested in parallel and is shown at the left of each gradient set (Lysate). (C) Quantification of substrate cleavage. The percentage of pAP cleaved to AP was calculated for each fraction in Panel B and plotted as the ratio [AP]/[pAP+AP]. Symbols on the ordinate of the graph show activities in starting lysates.

Testing these fractions for proteolytic activity against pAP, the biological substrate, showed those containing the highest amounts of ICRM-pPR, L382A/ICRM-pPR, and IC-assemblin (Fig. 2A) also cleaved the most pAP to AP (Fig. 2B), demonstrating that all three enzymes partially purified from bacterial lysates are enzymatically active. The relative specific activity of ICRM-pPR approximated that of IC-assemblin (i.e., ±16%), when estimated from these nondenatured preparations (Fig. 2C, fraction 5 for IC-assemblin and fraction 10 for ICRM-pPR). Activity in the upper portion of the ICRM-pPR gradient, where the amount of full-length pPR is lower, suggests that fragments of pPR in those fractions have enzymatic activity (Fig. 2C). The relative activity of L382A/ICRM-pPR versus IC-assemblin and ICRM-pPR could not be determined because the full-length monomer and smaller fragments cosedimented (Fig. 2A). Although this method separated full-length ICRM-pPR from most of the smaller fragments, a different approach was required to eliminate the larger fragments.

Purification of urea-denatured protease by IMAC and recovery of enzymatic activity. Denaturing ICRM-pPR in 8 M urea eliminated its tendency to adhere to membranes and other surfaces (data not shown). Therefore, as an alternative purification method, we added a six-His purification tag to the carboxyl end of ICRM-pPR (ICRM-pPRHis) and used IMAC to selectively recover the full-length protein from bacteria lysed in a buffer containing 8 M urea. Denatured ICRM-pPRHis was highly enriched by this procedure, whether expressed overnight at 16°C as soluble protein (Fig. 3) or at 37°C as inclusions (Fig. 4, lane 8; see also Fig. 7, lanes 1, 5, and 8, below).


Figure 3
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  		FIG. 3. Recovery of proteolytic activity by denatured ICRM-pPR following centrifugal buffer exchange. ICRM-pPRHis and pAPHis were selected by IMAC as described in Materials and Methods, with the exceptions that in this one experiment (i) cultures were grown overnight at 16°C, (ii) the bacterial pellet was lysed (French press) in NLB containing 8 M urea, and (iii) elution from the column was in buffer containing 8 M urea. ICRM-pPRHis and pAPHis in 8 M urea were made 1x in cleavage buffer by adding an equal volume of 2x buffer and subjected to twofold step-wise dilutions in 1x cleavage buffer. Each step reduced the volume by centrifugal filtration and then reduced the urea concentration by dilution. Samples of the mixture were removed at each step and subjected to SDS-PAGE (12% mini-gel). Proteins bound to the concentrator "membrane" (Memb.) were recovered in a volume of 0.5% SDS, 5 mM Tris (pH 8.0), and 0.5 mM EDTA equal to solute volume at the time of sampling and compared in lane 10. Shown here is an image of the CBB-stained proteins.


Figure 4
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  		FIG. 4. Comparison of protease preparation methods. S132A-pPRHis (S132AHis), ICRM-pPRHis (ICRMHis), L382A/ICRM-pPRHis (L382AHis), and IC-assemblinHis (IC-AsblnHis) were recovered from bacterial lysates (Lysate) as inclusions (Inclusion), denatured in 8 M urea, and affinity selected by IMAC (Purified). Proteases were renatured by sedimentation in glycerol gradients (Gradient) or by dialysis (Dialyzed), and samples of the renatured preparations were subjected to SDS-PAGE and SYPRO-R protein staining. Shown here is an image of the stained gel. Molecular masses (in kDa) of marker proteins (Mark 12) are shown at the left. The asterisk indicates the position of the putative T-site cleavage product; the empty circle indicates the position of an unexplained protein seen in this S132A-pPRHis preparation but no others (e.g., Fig. 5B, 7, and 8B).


Figure 7
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  		FIG. 7. Stabilizing pPR against T-site cleavage. ICRM-pPRHis and L382A/ICRM-pPRHis were additionally mutated to change A676 to Gln (A676Q), expressed in bacteria, purified from denatured inclusions by IMAC, and renatured by gradient sedimentation or dialysis as described in Results and in Materials and Methods. Samples of the resulting preparations [ICRMT, L382A/(T-)] were compared with ICRM-pPRHis, S132A-pPRHis, L382A/ICRM-pPRHis, and IC-assemblinHis by SDS-PAGE and protein staining with SYPRO-R. Shown here is an image of the stained gel. Molecular masses (in kDa) of marker proteins (Mark 12) are shown at left. The asterisk indicates the position of the T-site cleavage product.

To test renaturation of the IMAC-selected denatured ICRM-pPRHis, we combined it with purified pAP substrate in a centrifugal concentrator and incrementally lowered the urea concentration from 8 M to ~0.03 M, as described in the legend to Fig. 3. Small samples were removed after each concentration step. Analysis of the samples by protein staining following SDS-PAGE showed that cleavage of pAP to AP was first detected when the urea concentration was reduced below 2 M. However, as the concentration of urea was further decreased, pPR, pAP, and AP began adsorbing to the membrane, where the majority of all three proteins was recovered at the end of the experiment (Fig. 3, lane 10).

Since ICRM-pPR remained soluble in 2 M urea, we modified the IMAC procedure to include an on-column buffer exchange from 8 M to 2 M urea before elution, as described in Materials and Methods. pPR prepared by this procedure was a single homogeneous band that constituted >75% of the SYPRO-R-stained material in the sample lane following SDS-PAGE (Fig. 4, lane 8).

Renaturing ICRM-pPR by rate-velocity sedimentation or by dialysis. Since eliminating urea by exchanging the IMAC elution buffer directly into cleavage buffer caused pPR to adhere to the membrane (Fig. 3), we tested renaturation by rate-velocity sedimentation as an alternative. IMAC-selected ICRM-pPRHis in 2 M urea was layered onto glycerol gradients lacking urea and subjected to ultracentrifugation. S132A-pPRHis, L382A/ICRM-pPRHis, and IC-assemblinHis were prepared and analyzed in parallel (Fig. 4, lanes 3, 8, 13, and 18). Following centrifugation, the gradients were fractionated and samples of each fraction were analyzed by SDS-PAGE and protein staining with SYPRO-R (Fig. 5). The size distributions of the proteins, relative to marker proteins in a companion gradient, were consistent with those of the nondenatured proteins (Fig. 2A), indicating that the urea-denatured proteins renatured to the same oligomer compositions as the soluble, nondenatured proteins. The 70-kDa fragment cosedimenting with ICRM-pPR and L382A/ICRM-pPR, but not with S132A-pPR, results from cleavage at a new site described below.


Figure 5
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  		FIG. 5. Gradient renaturation of proteases. Preparations of IMAC-selected, denatured proteases were subjected to sedimentation in glycerol gradients, followed by gradient fractionation, as described in Results and in Materials and Methods. Samples of the resulting fractions were subjected to SDS-PAGE, followed by protein staining with SYPRO-R. A separate gradient containing marker proteins (standards) (E) was analyzed in parallel. Shown here are images of the stained gels. Braces indicate fractions with highest protein concentrations (combined fractions shown in Fig. 4, lanes 4, 9, 14, and 19); asterisks indicate the position of the putative T-site cleavage product. Molecular masses (in kDa) of marker proteins are indicated beneath panel E.

A two-step dialysis procedure (see Materials and Methods), first against lysis buffer supplemented with 1 M arginine and then against refolding buffer, was also effective for renaturing pPR from the 2 M urea-containing IMAC elution buffer, without appreciable loss to the membrane. As a comparison of the two methods, the inclusion preparations that had been used for the gradient renaturation experiment shown in Fig. 5 were used as starting material to purify the four urea-denatured proteins by IMAC and then renature them by the two-step dialysis method. Samples of the preparations refolded by dialysis or by sedimentation were compared by SDS-PAGE and protein staining (Fig. 4). With the exception of an unexplained minor band in the dialyzed S132A-pPR preparation (Fig. 4, lane 5), no difference between pairs was evident (Fig. 4, lanes 4, 9, 14, and 19 versus 5, 10, 15, and 20). The additional band appearing in both the ICRM-pPRHis and L382A/ICRM-pPRHis preparations after renaturation (i.e., Fig. 4, compare lane 8 with lanes 9 and 10 and compare lane 13 with lanes 14 and 15) is due to cleavage at the T site (see below). The dialysis-refolded preparations had the same oligomer distributions following gradient sedimentation (data not shown) that were observed for the nondenatured (Fig. 2A) and gradient-renatured (Fig. 5) preparations, indicating that dialysis did not alter the extent to which the proteins interact.

pPR and assemblin have similar enzyme kinetics. The enzymatic activities of the gradient-refolded and dialyzed preparations were compared using an in vitro assay and two different reaction buffers: activation buffer, containing Na2SO4 to promote protein interaction, and refolding buffer, containing glycerol and DTT to mimic gradient conditions but no Na2SO4. When compared pair-wise for the different preparation and assay conditions, the activities of ICRM-pPR and assemblin were about the same (Table 1): (i) ~0.7 µM substrate cleaved/min/µM of enzyme when refolded by dialysis and assayed in activation buffer; (ii) ~1 µM substrate cleaved/min/µM of enzyme when refolded in gradients and assayed in activation buffer; (iii) ~0.3 µM substrate cleaved/min/µM of enzyme when refolded by dialysis and assayed in refolding buffer; and (iv) ~0.5 µM substrate cleaved/min/µM of enzyme when refolded in gradients and assayed in refolding buffer. One notable difference in these comparisons was that, although the activity of mutant L382A/ICRM-pPR was similar to those of ICRM-pPRHis and IC-assemblinHis in activation buffer containing 0.5 M Na2SO4, it was significantly lower in refolding buffer without Na2SO4, whether prepared by dialysis (~8-fold lower) or sedimentation (~4-fold lower). Thus, maximal activity of pPR appears to require its oligomerization, which is normally accomplished by self-interaction through the ACD of its scaffolding domain but can also be promoted by 0.5 M Na2SO4 in the absence of the ACD interaction (i.e., L382A mutant) (Fig. 2A and 5; see also Fig. 8, below). As expected, the S132A-mutant prepared and tested in parallel was inactive. These data are presented graphically in Fig. 6A and B.


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  		TABLE 1. Catalytic activities of ICRM-pPRs and IC-assemblin


Figure 8
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  		FIG. 8. Gradient renaturation of mutants with blocked T site. Denatured ICRMT-pPRHis and L382A/ICRMT-pPRHis were IMAC selected and subjected to renaturation by sedimentation in glycerol gradients as described in Results and in Materials and Methods. S132A-pPRHis and IC-assemblinHis were included for comparison. SDS-PAGE of gradient fractions was in 10% Tris-glycine gels; shown here are images of the CBB-stained gels. Samples of starting lysates are shown in the left-most lane of each set. Braces indicate the most concentrated fractions (combined fractions shown in Fig. 7, lanes 3 to 6). Positions of molecular mass markers (in kDa) are indicated beneath panel D.

pPR and assemblin active sites renature to a comparable extent. To compare active site renaturation for the three enzymes assayed in Table 1, we used the dialyzed preparations and reacted each with a biotinylated suicide substrate (gift of C. S. Craik) that selectively and covalently binds to functional active sites (43) and then calculated the amount bound (Fig. 6D) per molar amount of protein (Fig. 6C), as described in Materials and Methods. The relative percentage of active sites differed by less than 15% between the three enzyme preparations, supporting the conclusion from kinetic assay data (Table 1) that the enzymatic activities of IC-assemblin and ICRM-pPR are equivalent. The specificity of this assay for active enzyme was demonstrated by the comparatively weak to undetected reactivity of enzymatically inactive S132A-pPRHis (Fig. 6C and D, lanes 1 and 2, and E).

Identification and mutation of the "tail" cleavage site creates fully stable ICRMT-pPR. Refolding of ICRM-pPRHis and L382A/ICRM-pPRHis by either dialysis or sedimentation resulted in a prominent cleavage product, accounting for as much as 25% of the total protein, that was undetected in the denatured preparations (Fig. 4, lanes 8 to 10 and 13 to 15, and 5A and C). Inactive S132A-pPRHis did not give rise to this band (Fig. 4, lanes 3 to 5, and 5B), indicating that it is a previously unrecognized autoproteolytic cleavage product of pPR. Because this site could compete with the intended peptide substrate in activity assays, we identified and mutated it.

The estimated size of the fragment (70 kDa) and its reactivity with an antipeptide antiserum to the amino end of pPR (Fig. 7, lanes 1 and 2), but not with one to the carboxyl end (data not shown), indicated that the putative cleavage site is within the "tail" domain of pPR. A candidate site (VVLA675{downarrow}AAAQ) in this region would yield a 70.5-kDa product consistent with the size of the observed fragment. Substituting Val for either Ala675 or Ala676 as used to block the I, C, and M sites would generate new, potentially recognizable cleavage sites. For that reason, and to avoid using a charged or bulky hydrophobic substitution that could disrupt structure, we replaced Ala676 with Gln as a strategy to prevent cleavage in ICRM-pPRHis and L382A/ICRM-pPRHis. When these mutants, called ICRMT-pPRHis and L382A/ICRMTHis, were purified by IMAC under denaturing conditions and renatured by dialysis or sedimentation, no T-site cleavage was evident (Fig. 7, lanes 5 and 6 and lanes 8 and 9). This result supports identification of the sequence VLA675{downarrow}AAA as the likely pPR T site, pending confirmation by direct sequence analysis.

ICRMT-pPR and IC-assemblin have comparable catalytic activities. ICRMT-pPRHis and L382A/ICRMT-pPRHis mutant proteases were expressed, purified, and renatured by sedimentation (Fig. 8) and by dialysis as described above. The resulting preparations (Fig. 7, lanes 5 and 6 and lanes 8 and 9) were indistinguishable from those of ICRM-pPRHis and L382A/ICRM-pPRHis (Fig. 7, lanes 1 and 2) with the notable absence of the 70-kDa T-site cleavage product. IC-assemblinHis was prepared in parallel for comparison of catalytic activities.

The proteolytic activities of these ICRMT-pPRHis, L382A/ICRMT-pPRHis, and IC-assemblinHis preparations were then measured using the fluorogenic peptide substrate in activation buffer and in refolding buffer, as described above for the ICRM-pPR mutants. Results of this comparison (Table 2; Fig. 9) were consistent with those summarized in Table 1 and Fig. 6 for the ICRM-pPR mutants and assemblin. First, the enzymatic activities of ICRMT-pPRHis, L382A/ICRMT-pPRHis, and IC-assemblinHis were indistinguishable within error (Fig. 9A) in activation buffer, whether renatured by dialysis or by gradient sedimentation, (~0.7 µM/s/µM enzyme) (Table 2). Second, the activities of ICRMTHis and IC-assemblinHis were also similar in refolding buffer but were approximately twofold lower than in activation buffer (~0.3 µM/s/µM enzyme) (Table 2). And third, the activity of L382A/ICRMT-PRHis in refolding buffer was ~4-fold lower than the other two in the same buffer.


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  		TABLE 2. Catalytic activities of ICRMT-pPRs and IC-assemblin


Figure 9
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  		FIG. 9. Proteolytic activity of ICRMT mutants. (A) Kinetics of substrate cleavage in the activation buffer containing 0.5 M Na2SO4. All was as described for Fig. 6A but done with ICRMT-pPRHis and L382A/ICRMT-pPRHis. (B) Kinetics of substrate cleavage in refolding buffer with no 0.5 M Na2SO4 added. All was as described for Fig. 6B but done with ICRMT-pPRHis and L382A/ICRMT-pPRHis. Abbreviations in all panels are as for Fig. 6. (C) Relative active site concentrations of ICRMT-pPR mutants. ICRMT-pPRHis, L382A/ICRMT-pPRHis, and IC-assemblinHis were analyzed as described in Materials and Methods and in the legend to Fig. 6C to E. Shown here are the relative active site concentrations, calculated from reactions done in triplicate, with standard errors indicated (bars).

The relative percentage of active enzyme in each dialysis-refolded preparation was determined as described above, after a 4-h reaction with biotinylated suicide substrate. The relative number of active sites (i.e., amount of [125I]streptavidin bound) per molar amount of protein (SYPRO-R staining) for IC-assemblinHis, ICRMT-pPRHis, and L382A/ICRMT-pPRHis was the same within error, indicating all three denatured enzymes regained activity to a comparable extent. Thus, T-site cleavage in pPR was without detected effect on its activity, relative to IC-assemblin.


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DISCUSSION
 
We conclude from this study that assemblin and its precursor, pPR, have comparable activities and that self-interaction of pPR through its scaffolding domain plays an important role in activating the enzyme. A major consideration in implementing this work was that the methods themselves did not change the catalytic properties of either enzyme. For this reason both were stabilized against autoproteolysis by using characterized point mutations, and both were purified, refolded, and assayed in the same way. Substituting Val for the respective P1 Ala's to block the I, C, and M sites was based on its previous use in preparing assemblin for structural and enzymatic studies. However, this substitution at the R site failed to completely block that cleavage, and a P1' substitution (S257V) that had been demonstrated to block the site with no apparent effect on activity (31) was used instead.

Denaturing conditions were used to purify pPR from inclusions by IMAC when attempts to purify the native protein were complicated by its tendency to adhere to chromatography media and membranes and when rate-velocity sedimentation failed to resolve it from several nearly full-length fragments. The one-step IMAC selection procedure eliminated all but trace amounts of other proteins; however, it necessitated adding the purification tag to opposite ends of assemblin and pPR. The six-His tag was added to the amino end of assemblin, since modifications and short additions, including purification tags, at its carboxyl end reduce activity (10, 15, 65, 79). The tag was added to the carboxyl end of pPR to select full-length pPR and avoid coselecting amino-terminal fragments, some of which have proteolytic activity that would interfere with assays of pPR. This sole difference in the purification methods is not expected to affect either enzyme, since (i) structure and activity determinations are similar for assemblin purified with (28, 63) or without (54, 59, 71) an amino six-His tag, and (ii) the putative elongated structure of pPR, inferred from its relatively slow sedimentation (39) and from its deduced radial density (48, 73), is presumed to distance its carboxy-terminal tag from its amino catalytic domain.

Refolding purified, denatured pPR was also complicated by the "sticky" nature of the scaffolding domain. This problem was overcome by sedimenting the denatured proteins gradually from 2 M urea into nondenaturing glycerol gradients. To our knowledge this approach has not been reported before, but it worked well with pPR and may be useful with other adherent proteins. A two-step dialysis procedure, including 1 M arginine in the first buffer and avoiding the use of high salt concentrations, was also effective and used for comparison. Each method provided specific advantages. Gradient renaturation incorporated an additional fractionation step, increasing purity and quaternary homogeneity, but yielded less concentrated preparations. Importantly, this method appears to faithfully reconstitute the mass of the native pPR oligomers (i.e., compare pPR distributions in Fig. 2A with those in Fig. 5 and 8). Dialysis was comparatively fast, easy, and maintained concentration but afforded no further resolution of the proteins beyond that achieved by IMAC.

Supporting our conclusion that the activities of pPR and assemblin are similar, we obtained consistent results with four different forms of pPR (i.e., ICRM- and ICRMT-pPRHis and L382A/ICRM- and L382A/ICRMT-pPRHis) that were renatured by two different methods (gradients and dialysis), assayed using two different buffer conditions (i.e., activation buffer with 0.5 M Na2SO4 or refolding buffer with no Na2SO4), and compared twice for extent of active site reconstitution. The reaction rates determined for both assemblin and pPR were approximately 1 µM substrate cleaved per minute per µM of protease, which is within the range reported by others for assemblin (6, 54). It also has been concluded from live cell transfection assays using stabilized pPR coexpressed with its biological substrate, pAP (pUL80.5), that pPR and assemblin are equally active (31).

In the only other comparison of the purified enzymes, pPR was determined to have a ~6-fold-lower activity than assemblin against pAP substrate and an 11-fold-lower activity against a peptide substrate (82). We suggest that this contrast with our conclusion, that the activities are similar, may be due to one or a combination of differences in preparing pPR in the earlier study that decreased its activity. In that study, for example, it was not determined whether the mutations made to block self-cleavage affected pPR activity. Since changes at the R site can reduce or alter enzymatic activity (10, 15, 65, 79), the double, nonconservative substitution made at that site (i.e., A256T and S257P at the P1 and P1' positions) may have been deleterious. It is noteworthy, however, that herpes simplex virus pPR with a similarly nonconservative substitution of the R-site sissile pair (i.e., Ala256Ser257->ProArg) retained at least partial M-site cleavage activity (35). Absence of a substitution to block C-site cleavage, and the leaky nature of the M-site substitution, may also have reduced pPR activity by providing endogenous cleavage sites able to compete the intended exogenous substrate in vitro and in cell-based assays. Another factor possibly contributing to the lower activity of pPR in the first study was the use of different methods to purify pPR (denaturing/renaturing conditions) and assemblin (nondenaturing conditions). Related to this, placing the six-His purification tag at the amino end of pPR, as in the previous study (82), would neither select for full-length pPR nor against amino-end fragments with proteolytic activity that could interfere with quantification in enzyme assays. It is unresolved, however, which if any of these differences accounts for the discrepancy in conclusions between the two comparisons.

During the course of this work a fifth cleavage site in pPR became evident when a ~70-kDa protein appeared following renaturation of the homogeneous, 74-kDa pPR purified by IMAC (Fig. 4, lanes 8 to 10 and 13 to 15). The size and immunological reactivity of the fragment indicated it lacks the carboxyl end of pPR and led to recognition of a candidate cleavage sequence (VVLA675{downarrow}AAAA) called the tail site. The substitution A676Q in that sequence blocked T-site cleavage, consistent with its correct identification. Amino acid sequence alignments indicate that the proposed T site is located in an insertion that is not present in the homologs of other closely related herpesviruses. Because this fragment is negligible in soluble (active) and inclusion body (inactive) preparations of pPR (Fig. 1B, lanes 10 and 11, and Fig. 4, lanes 7 and 12) and absent from purified denatured pPR recovered by IMAC (Fig. 4, lanes 8 and 13), we favor the interpretation that its production is caused or enhanced by refolding from a denatured state. This could occur, for example, if the catalytic domain of denatured pPR regained activity faster than the carboxyl end regained its fold, leaving the T site exposed to attack.

Finally, we conclude that self-interaction of pPR plays an important role in potentiating enzymatic activity. We show here that the L382A mutation not only disrupts the ability of pPR to interact through its scaffolding domain, as reported before (39, 83), but also prevents or reduces interaction through its assemblin domain under conditions where assemblin itself is dimeric (Fig. 5 and 8). Consistent with the requirement that monomeric assemblin dimerizes to activate, we found that under conditions where pPR is predominantly monomeric (i.e., the L382A mutant), its activity was only 20 to 30% that of dimeric IC-assemblin and oligomeric ICRMT-pPR.

Display of any activity by the mutant under these conditions suggests either that its assemblin domain is capable of transient interaction, producing dimers not obvious in sedimentation assays (Fig. 5 and 8), or that pPR monomers have some intrinsic activity. Arguing against intrinsic activity, when the possibility of transient interaction by the L382A/ICRMT-pPR monomers was eliminated by additionally mutating either Leu221 or Leu222 in their assemblin dimer interface (based on reference 57), neither the resulting assemblin nor pPR mutants showed activity (data not shown from assays using crude preparations of the dimer interface mutants). This observation favors interaction of the assemblin dimer interface being required for pPR activity and reduction but not elimination of L382A/ICRMT-pPR activity being due to undetected transient interaction of its monomers through that interface. We interpret these results to indicate that activation of pPR requires interaction of its subunits through the ACD of its scaffolding domain to align, stabilize, or otherwise increase interaction of the assemblin dimer interface domain, which is weaker in the context of pPR than in assemblin.

Since activation of assemblin involves conformational changes communicated by interactions at the dimer interface (1, 10, 49), differences in the organization of interface elements between assemblin and pPR may be reflected in differences in their active sites. Such differences have been proposed before to account for results of chemical rescue experiments showing restored I-site cleavage with an H63G active-site mutant of assemblin, but not with the same mutant of pPR (46). Structural comparisons of assemblin with pPR are expected to help determine how their activation mechanisms differ and provide insight into what triggers the process during capsid formation. Since controlling the time and site of pPR activation is likely to be critical for efficient production of infectious virus, and considering that pPR is a theoretically more accessible drug target in the cytoplasm during early steps of capsid assembly than is assemblin within capsids located in the nucleus, learning how the structure and interactions of pPR relate to its function is important to understanding the biology of the virus and ways to manipulate or block specific steps in its replication.


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ACKNOWLEDGMENTS
 
We thank Christopher Cherry and LaShon Ussin for doing the initial experiments to evaluate preparing pPR from bacterial inclusions, Charles Craik and Alan Marnett for generously providing the biotinyl-peptide inhibitor, Jim Stivers for use of the fluorometer, and Amy Loveland for technical assistance and help preparing the manuscript.

E.J.B. was in the Biochemistry, Cellular, and Molecular Biology graduate program; a portion of this work was submitted in partial fulfillment of the requirements for that program.

This work was aided by Public Health research grants AI32957 and AI13718 from the National Institute for Allergy and Infectious Diseases.


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FOOTNOTES
 
* Corresponding author. Mailing address: Virology Laboratories, The Department of Pharmacology & Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Phone: (410) 955-8680. Fax: (410) 955-3023. E-mail: wgibson{at}jhmi.edu Back

{triangledown} Published ahead of print on 7 February 2007. Back


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Journal of Virology, April 2007, p. 4091-4103, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02821-06
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