precursor (pAP), are members of the CMV assembly protein nested gene family ..... Baum, E. Z., G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and.
JOURNAL OF VIROLOGY, July 1995, p. 4524–4528 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 69, No. 7
Human Cytomegalovirus Proteinase: Candidate Glutamic Acid Identified as Third Member of Putative Active-Site Triad GREGORY A. COX,1 MARK WAKULCHIK,1 LORETTA M. SASSMANNSHAUSEN,1 WADE GIBSON,2 AND ELCIRA C. VILLARREAL1* Infectious Diseases Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285,1 and Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 212052 Received 22 December 1994/Accepted 4 April 1995
The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.
absolutely conserved histidine and serine, respectively, that have been implicated by site-directed mutagenesis as catalyticsite residues in herpes simplex virus type 1 (HSV-1) (22) and simian CMV (SCMV) (39). Confirmation that CD3 Ser (e.g., HSV-1 Ser-129 in Fig. 2) is the active-site nucleophile has recently been provided by affinity labeling with the serine proteinase inactivator diisopropyl fluorophosphate (DFP) (11, 17). Classical serine hydrolases (i.e., serine proteinases, lipases, acetylcholinesterase, and thioesterase) typically contain an aspartic or glutamic acid at the catalytic site, in addition to the histidine and the serine nucleophile (10, 19, 26). Identification of a specific conserved aspartate or glutamate as the anticipated third member of the assemblin catalytic site has not been confirmed. A double mutation of two adjacent glutamates in HSV-1 assemblin (i.e., HSV-1 CD3 Glu-114 and Glu-115 in Fig. 2) inactivated the proteinase (23). However, these mutations were not tested separately, and changing a computeraligned counterpart residue in SCMV (i.e., Asp-104) did not abolish its activity (39). The objective of this study was to verify the predicted CD3 Ser and CD2 His members of the HCMV catalytic site and to identify a candidate Asp or Glu member of the triad. We have used site-directed mutagenesis to change these histidine and serine residues, as well as nine other amino acids of interest, within absolutely and partially conserved domains of HCMV assemblin. Mutant proteinases were tested in transient transfection assays for their ability to cleave substrate pAP to AP, and the results were determined by Western immunoassays. We show here that CD3 Ser and CD2 His, as well as an
The human cytomegalovirus (HCMV) UL80a open reading frame encodes the proteinase assemblin as an active precursor that undergoes at least three autoproteolytic cleavages (4, 39, 41). One of these is at the maturational (M) site near the carboxyl end of the 85-kDa precursor (Fig. 1A). A second occurs near the midpoint of the precursor at the release (R) site (Fig. 1A). Although neither M-site nor R-site cleavage is absolutely required for proteolytic activity (18, 23, 39), R-site cleavage releases the 28-kDa assemblin and may potentiate its activity (23, 39) (Fig. 1B). The final autoproteolytic cleavage occurs near the midpoint of assemblin at the internal (I) site (Fig. 1A). This cleavage converts assemblin from an active single-chain enzyme to an active two-chain form (16, 17). Both the proteinase and its substrate, the assembly protein precursor (pAP), are members of the CMV assembly protein nested gene family (38). The substrate, pAP, is synthesized from a separate in-frame transcript as a 38-kDa protein and is cleaved by the proteinase at its carboxyl end to form the 32kDa mature assembly protein (AP) (14, 38) (Fig. 1C). The AP is an abundant phosphoprotein found in immature intranuclear capsids but not in mature virions (14). The AP is thought to be functionally analogous to the bacteriophage scaffolding protein in facilitating capsid assembly (6, 21, 27), and its cleavage is essential for the production of infectious virus (12, 28). Alignment of the available amino acid sequences of herpesvirus assemblin homologs reveals five highly conserved domains (CD1 through CD5), as well as areas of partial conservation (39). Two of these domains, CD2 and CD3, contain an * Corresponding author. 4524
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FIG. 1. Schematic representation of the HCMV proteinase and the assembly protein precursor. (A) The UL80a gene product is the precursor proteinase that is autoprocessed at the sites indicated by the arrows, which are described in the text. The five conserved domains (CD1 through CD5) are indicated by shaded rectangles. (B) LM12 plasmid contains the assemblin gene that was cloned into the RSV.5neo vector at the XbaI site (40). The mutant proteinase genes were made from the assemblin gene that was cloned into M13mp19 at the XbaI and BamHI sites and mutated by site-directed mutagenesis as described in the text. (C) LM11 plasmid contains the assembly protein precursor (pAP) gene that was cloned into the RSV.5neo vector at the XbaI site (40). Active proteinases will cleave this gene product (pAP) into the mature assembly protein (AP) at the M site.
apparently conserved Glu within CD3, are essential for the proteolytic activity of HCMV assemblin and suggest that these three amino acids constitute the catalytic triad of the active site. Construction and analysis of mutants. A DNA fragment of UL80a encoding the first 256 amino acids of the precursor, which expresses wild-type HCMV assemblin, was subcloned from the LM12 plasmid (Fig. 1B) (40) into the M13mp19 plasmid at the XbaI and BamHI sites. Mutations were introduced by site-directed mutagenesis (35) by using the Sculptor in vitro mutagenesis system (Amersham, Arlington Heights, Ill.). The mutations were confirmed by dideoxy sequencing (31) with the Sequenase 2.0 kit (United States Biochemical, Cleveland, Ohio). The mutant proteinase genes were then subcloned from M13mp19 back into the vector RSV.5neo (24), and the mutant assemblin genes were sequenced to ensure that only the intended change was present. The plasmid LM11 contains the gene that encodes the pAP (Fig. 1C) (40). The plasmid RSV.TAg contains a gene that encodes the simian virus 40 T antigen and was used in all transfections to increase expression of RSV.5neo-derived constructs (29). The transfection assays were performed as described before (41). Briefly, on day 1, human embryonal kidney (HEK) cells (line 293; American Type Culture Collection, Rockville, Md.) were seeded into 24-well plates. On day 2, cells were transfected with 2 mg of the plasmid LM11, 0.5 mg of plasmid encoding the mutant or wild-type mature proteinase (e.g., LM12), and 0.25 mg of the plasmid RSV.TAg. On day 3, exhausted medium was removed and replaced with fresh medium, and on day 4, the cells were harvested. Samples were stored at 2808C until they were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoassays. SDS-PAGE was performed essentially as described by Laemmli (20) by using a Novex electrophoresis system with 12% Tris-glycine gels (Novex, San Diego, Calif.). Electrotransfer to nitrocellulose membrane (Novex) was performed essentially as described by Towbin et al. (37). Western immunoassays by
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FIG. 2. Amino acid sequence alignment of conserved domains 2 and 3 among assemblin homologs as shown by Welch et al. (39). Sequences for the following are from GenBank: HCMV strain AD169 (7), SCMV strain Colburn (38), HSV-1 (25), varicella-zoster virus (VZV) (9), equine herpesvirus (EHV) (36), infectious laryngotracheitis virus (ILTV) (15), herpesvirus saimiri (HVS) (1), and Epstein-Barr virus (EBV) (2). Classification as alpha-, beta-, or gammaherpesviruses is based on the nomenclature of Roizman et al. (30). Amino acids shown in boldface type are described in the text as being essential for proteolytic activity; underlined amino acids are described in the text as not being essential for proteolytic activity. Boxes are drawn around the conserved serine nucleophile, the conserved histidine, and the candidate glutamic acid of the assemblin putative catalytic triad.
enhanced chemiluminescence (42) were performed according to instructions for use of the ECL detection system (Amersham). The primary antibody B1N1A was a monoclonal antibody against a synthetic peptide of the first 20 amino acids of the assembly protein precursor (5). The primary antibody B22-27 was prepared by Deena Hepburn at Eli Lilly and Company and is a monoclonal antibody against HCMV assemblin. The secondary antibody was goat anti-mouse immunoglobulin G coupled to horseradish peroxidase (Amersham). Mutational analysis of highly and partially conserved serine, histidine, and cysteine residues of the HCMV proteinase. Among the three most highly conserved domains of assemblin, (i) CD1 contains an absolutely conserved cysteine which is not essential for activity in either HSV-1 or SCMV (23, 39) and an absolutely conserved histidine which is not essential for activity in SCMV (39) but may be in HSV-1 (22), (ii) CD2 also contains an absolutely conserved histidine that is thought to be part of the catalytic triad (22, 39), and (iii) CD3 contains an absolutely conserved serine that is evidenced to be the catalytic nucleophile (11, 17, 39). These and other amino acid residues with a potential to contribute to the catalytic site of the HCMV proteinase were mutated and tested for activity. Amino acids were changed to alanines to minimize the possibility of structural distortions (3, 8). We mutated the CD3 serines that were either absolutely or highly conserved (Fig. 2) and found that only one of these mutations, the S-to-A mutation at position 132 (S132A), abolished proteolytic activity (Fig. 3A, lane 6). This same residue was selectively labeled by DFP (17), consistent with it being the active-site nucleophile. The other two serine mutations, S134A
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FIG. 3. Histidine 63 and serine 132 are absolutely essential for proteolytic activity. Site-directed mutagenesis was used to make specific amino acid substitutions in highly conserved histidine or serine residues, as described in the text. The mutant constructs were transfected with the pAP plasmid LM11 and the plasmid RSV.TAg. The resultant proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with B1N1A (A) and B22-27 (B) antibodies as described in the text. The mutant plasmids used for the transfections are indicated above the respective lanes. Lanes 1 contain pAP alone, lanes 2 contain wild-type assemblin alone, and lanes 3 show the transfection of pAP and wild-type assemblin. Lanes 4 to 8 show transfections of pAP and the mutant proteinases. Lanes 9 contain the nuclear fraction of CMV strain AD169-infected human foreskin fibroblasts (HFF). Numbers on the left indicate molecular masses (in kilodaltons) of the protein markers (lanes M). Protein designations are shown on the right.
FIG. 4. Conserved cysteine residues 84 and 161 are not essential for proteolytic activity. Site-directed mutagenesis was used to make specific amino acid substitutions in highly conserved cysteine residues as described in the text. The mutant constructs were transfected with the pAP plasmid LM11 and the plasmid RSV.TAg. The resultant proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with B1N1A (A) and B22-27 (B) antibodies as described in the text. The mutant plasmids used for the transfections are indicated above the respective lanes. Lanes 1 contain pAP alone, lanes 2 contain wild-type assemblin alone, and lanes 3 show the transfection of pAP and wildtype assemblin. Lanes 4 and 5 show the transfections of pAP and the mutant assemblins. Lanes 6 contain the nuclear fraction of CMV strain AD169-infected HFF cells. Numbers on the left indicate the molecular masses (in kilodaltons) of the protein markers (lanes M). Protein designations are shown on the right.
and S135A, had no effect on substrate cleavage (Fig. 3A, lanes 7 and 8). We also tested the two histidine residues that are absolutely conserved among assemblin homologs. Only CD2 H63A abolished proteolytic activity (Fig. 3A, lane 4). By sequence alignment, HCMV His-63 is homologous to SCMV His-47 and to HSV-1 His-61 (Fig. 2), both of which were essential for proteolytic activity in similar assays (22, 39). The other histidine mutation, HCMV CD1 H157A, had no effect on substrate cleavage (Fig. 3A, lane 5). HCMV CD1 His-157 is homologous to SCMV CD1 His-142, which was not essential for proteolytic activity (39), and to HSV-1 CD1 His-148, which was essential (22). Our finding that only the CD2 His of HCMV is essential favor it being the second catalytic-site residue, as suggested before (39). All serine and histidine mutant proteinase plas-
mids expressed assemblin (Fig. 3B, lanes 4 to 8), indicating that the inability of H63A and S132A mutants to cleave substrate was not due to their lack of expression. Because of their importance in known proteolytic active sites and their presence in the conserved domains, we mutated cysteine residues 84 (partially conserved residue in CD4) and 161 (absolutely conserved residue in CD1) to alanines (C84A and C161A). Both mutants retained enzymatic activity, indicating that these residues are not essential for proteolytic activity (Fig. 4A, lanes 4 and 5). This is consistent with previous reports that the conserved CD1 cysteine in HSV-1 and SCMV is not essential for proteolytic activity (23, 39), and with the finding that HCMV assemblin retains activity when its cysteines are oxidized by sulfitolysis (33). Both HCMV cysteine mutant proteinase clones expressed assemblin (Fig. 4B, lanes 4 and 5).
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FIG. 5. Glutamic acid 122 is also absolutely essential for proteolytic activity. Site-directed mutagenesis was used to make specific amino acid substitutions in partially conserved aspartic acid or glutamic acid residues as described in the text. The mutant constructs were transfected with the pAP plasmid LM11 and the plasmid RSV.TAg. The resultant proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with B1N1A (A and B) and B22-27 (C) antibodies as described in the text. The mutant plasmids used for the transfections are indicated above the respective lanes. Lanes 1 contain pAP alone, lanes 2 contain wild-type assemblin alone, and lanes 3 show the transfection of pAP and wild-type assemblin. In panels A and C, lanes 4 to 7 show the transfections of pAP and the mutant assemblins and lanes 8 contain the nuclear fraction of CMV strain AD169-infected HFF cells. In panel B, lane 4 shows the transfection of pAP and the E122A mutant proteinase and lane 5 contains the nuclear fraction of CMV strain AD169-infected HFF cells. Numbers on the left indicate the molecular masses (in kilodaltons) of the protein markers (lanes M). Protein designations are shown on the right.
Mutational analysis of highly and partially conserved aspartic and glutamic acid residues of the HCMV proteinase. Computer alignment of assemblin homologs by the Genetics Computer Group program Pileup showed no absolutely conserved aspartic acid or glutamic acid residues (16, 39). When the aspartic and glutamic acid residues of the SCMV proteinase that showed the highest degree of conservation among the different herpesvirus homologs were substituted (i.e., SCMV D15A/N, E20A/E22A, E22A, and D104A/N), none was found to abolish proteolytic activity (39). Additional Ala substitutions for SCMV D-31, D-205, D-210, and E-194 have now been tested, but again none of the mutations abolished proteolytic activity (13). Interestingly, a double mutation of two adjacent glutamic acids in the HSV-1 proteinase (E114G/E115A) did eliminate its activity (23), but experiments to evaluate the two mutations separately were not done. Amino acid sequence alignments indicated that HSV Glu-115 is homologous to SCMV D-104, which is not essential for proteolytic activity (39). Further inspection of the sequence alignment, however, revealed that of these two residues only Glu-115 is conserved among all members of the Alphaherpesvirinae subfamily (Fig. 2, varicellazoster virus, HSV-1, infectious laryngotracheitis virus, and equine herpesvirus) (39). Additionally, 4 amino acids downstream of HSV-1 Glu-115 there is a glutamic acid residue in the HCMV sequence (Glu-122) that is conserved among the assemblin homologs of all members of the Betaherpesvirinae and Gammaherpesvirinae subfamilies (Fig. 2, HCMV, SCMV, Epstein-Barr virus, and herpesvirus saimiri) (39). When HCMV Glu-122 was mutated to an alanine (E122A), proteolytic activity was abolished (Fig. 5A, lane 4). The same result was observed in a separate experiment when comparable levels of expression of the assembly protein precursor were obtained for the control and for the transfection of LM11 and the E122A mutant (Fig. 5B, lanes 1 and 4). This finding raises the possibility that HCMV Glu-122, and its positional counterparts in the other beta- and gammaherpesviruses, is functionally homologous to HSV-1 Glu-115 and its positional counterparts in the other alphaherpesviruses (Fig. 2). We also substituted
alanines for the partially conserved aspartic acid residues 186 and 212 of HCMV assemblin (D186A and D212A) and found that neither of these mutations affected proteolytic activity (Fig. 5A, lanes 5 and 6). Another partially conserved aspartic acid residue, D-217 of HCMV, demonstrated reduced proteolytic activity when substituted with an alanine (D217A) but was not completely devoid of activity (Fig. 5A, lane 7). All glutamate and aspartate mutant proteinase plasmids expressed assemblin (Fig. 5C, lanes 4 to 7), indicating that the inability of the E122A mutant to cleave substrate was not due to lack of expression. The presence of a glutamic acid that is essential for activity in HCMV assemblin, and the suggestion that it is well conserved in a subgroup-specific pattern, qualifies this residue to be an attractive candidate as the third member of the assemblin putative active-site triad. To our knowledge, this would represent the first instance of a specific glutamic acid replacing the usual aspartate in a serine proteinase. Substitution of glutamic acid for aspartic acid has been reported for acetylcholinesterase (34) and the lipase from Geotrichum candidum (32), but not for a serine proteinase. Confirmation of the residues involved in the catalytic mechanism of herpesvirus assemblin and a detailed investigation of the enzyme active site await crystallographic analyses. We thank Bruce Glover, Stanley Burgett, Deena Hepburn, and Maverick Ulmer at Lilly Research Laboratories for expert technical assistance; Anthony Welch, Matthew Hall, and Lisa McNally at Johns Hopkins for help in constructing and testing expression plasmids; Joseph Colacino and Gary Birch for helpful comments on preparing the manuscript; and Carlos Lopez for enthusiastic support of the project. REFERENCES 1. Albrecht, J.-C., J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess. 1992. Primary structure of the herpesvirus saimiri genome. J. Virol. 66:5047–5058. 2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, T. P. S. Tuffnell, and B. G. Barrell. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London) 310:207–211.
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