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Herpesvirus Proteinase JOURNAL OF VIROLOGY, Dec. 1993, p. 7360-7372 Vol. 67, No. 12 0022-538X/93/127360- 13$02.00/0 Copyright © 1993, American Society for Microbiology Herpesvirus Proteinase: Site-Directed Mutagenesis Used To Study Maturational, Release, and Inactivation Cleavage Sites of Precursor and To Identify a Possible Catalytic Site Serine and Histidine ANTHONY R. WELCH,t LISA M. McNALLY,t MATTHEW R. T. HALL, AND WADE GIBSON* Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205 Received 27 July 1993/Accepted 18 September 1993 The cytomegalovirus maturational proteinase is synthesized as a precursor that undergoes at least three processing cleavages. Two of these were predicted to be at highly conserved consensus sequences-one near the carboxyl end of the precursor, called the maturational (M) site, and the other near the middle of the precursor, called the release (R) site. A third less-well-conserved cleavage site, called the inactivation (I) site, was also identified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have used site-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, and have shown that the proteinase precursor is active without cleavage at these sites. We have also shown that the P4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site but not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Among those tested, only 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity. Assembly of the herpesvirus capsid includes a maturational tional cleavage site (M site) and is highly conserved in the proteolytic cleavage that appears to be an essential step in the homologous proteins of other herpes group viruses (48). And, formation of infectious virions (35). One target of this cleavage (iii) the proteinase precursor is cleaved twice-once at its M is the procapsid assembly protein precursor which loses a small site, present near its carboxyl end as a consequence of the portion of its carboxyl end during the process (19, 28, 48). The nested relationship of the proteinase and assembly protein proteinase responsible for catalyzing this reaction is encoded genes (27, 46) (Fig. 1) and once at a site called the release by a viral gene that is conserved among the herpes group cleavage site (R site) near the middle of the proteinase viruses and contains the gene for its substrate, the assembly precursor (48). The R site was predicted to be between Ala and precursor, as 3'-coterminal half protein its nested, in-frame, Ser in the consensus sequence Y-V/L-K/Q-A I S near the (28, 36, 48). middle of the proteinase and, like the M-site cleavage se- mutants to the cyto- By using insertion and deletion study quence, is highly conserved among the homologs of other in transient transfection assays and megalovirus proteinase herpes group viruses (48). Amino acid sequence analyses of plasma desorption mass spectrometry to determine its cleavage bacterially synthesized products of the proteinase have shown in mature we the site the assembly protein, showed following that occurred at the sites and demonstrated the is contained cleavage predicted (48). (i) The enzymatic portion of proteinase the and of the bacterial system 16). Recently, within the amino half of the full-length molecule and includes fidelity utility (5, a third cleavage site with the sequence VEA l AT has been two highly conserved domains, referred to as CDI and CD2. This finding has been confirmed for both herpes simplex virus identified near the middle of the HCMV 28-kDa assemblin (HSV) and human cytomegalovirus (HCMV) (5, 7, 30, 47). (ii) homolog, between CDI and CD2 (5, 7). It has been proposed the inactiva- Maturational cleavage at the carboxyl end of the assembly to inactivate the enzyme and therefore is called tion site protein precursor is between Ala and Ser in the consensus (I site). here had four The first was sequence V/L-X-A I S. This sequence is called the matura- The studies reported objectives. to substantiate that the predicted YVKA I1 S sequence is the R cleavage site and to investigate the importance of specific residues in both the R and M cleavage sites. The second was to Corresponding author. determine whether cleavage at the R site is required to activate t Present address: Syntex Discovery Research, Institute of Biochem- istry and Cell Biology, Palo Alto, CA 94304. the proteinase. The third was to determine whether the simian t Present address: Department of Microbiology, Medical College of cytomegalovirus (SCMV) proteinase has an inactivation cleav- Wisconsin, Milwaukee, WI 53201. age site similar to that identified for HCMV (5, 7). And, the 7360 VOL. 67, 1993 HERPESVIRUS PROTEINASE 7361 A. fourth was to test specific amino acid residues within the ProteoIytib R-site M-site proteolytic portion of the molecule for their possible involve- PEsd A CD2 CD3 CD1 t ment in the catalytic site of the enzyme. Our approach was to AW4 + C.31 introduce specific changes by site-directed mutagenesis and _ iNA ml M281 ly 557 then determine the phenotypic effect of the change by using a transient transfection-based assay. C3 LM3 I l I II 7 I- While this work was in progress, several reports describing similar studies of the HSV and HCMV proteinases appeared (5, 16, 29, 30). Important points of agreement and difference LM8 + I l. I I m I between these studies are discussed. YVKA 249 Progress reports of this work have been presented at the Gordon Conference on Proteolytic Enzymes and Their Inhib- AW1 lZJ itors, 8 to 12 June 1992; the Annual Meeting of the American M 281 KME%MO Society for Virology, 11 to 15 July 1992; the 17th International SCMV Genes r*APNG1 rb APNG.5 -TAA Herpesvirus Workshop, 1 to 6 August 1992; and the 4th International Cytomegalovirus Workshop, 19 to 21 April 1993. Base pairs i m DNA 0 200 400 600 800 1000 1200 140() 1600 1800 MATERLALS AND METHODS B. Construction of mutants. Standard techniques were used to construct, clone, and propagate the plasmids (39). The assem- Proposed bly protein nested gene 1 (APNG1, which encodes the full- Inactivation Sit Reease Sle length proteinase precursor, pNP1) and APNG.5 (which en- * 4 codes the assembly protein precursor, pAP) genes were INA127 YVKA249 subcloned from the plasmids AW4 and AWl (48), respectively, I Proteinase: I into the plasmid M13mpl8. Mutations were introduced by I C21 Ni using a kit (RPN1523; Amersham, Arlington Heights, Ill.) I Ew based on the primer-directed mutagenesis technique described - 60 kDa by Eckstein and coworkers (42, 43). The mutations were 27 kDa I Assentln - r- in confirmed by dideoxy sequencing (40) with the modified T7 I *Il A 14 kDa I polymerase, Sequenase (United States Biochemical Corp., 13dDa Cleveland, Ohio). Mutant genes were subcloned from A,Icl id II 50 kDa I M13mpl8 into pRSV.neo (31) for analysis in transient trans- e fection assays as follows. (i) Single-stranded DNA was isolated I In 46 kDa _ I from Escherichia coli that had been infected with the mutant 37 kD)a pNP1, II 1 EM constructs in Ml3mpl8 (33). (ii) Five micrograms of single- NP1,- stranded DNA was annealed to 1 pmol of the M13 universal primer (United States Biochemical Corp.). (iii) Double- Assembly Protein: stranded DNA was synthesized in vitro by incubating the annealed single-stranded DNA with Sequenase buffer (40 mM ADC0 OL 3Okf'a Tris [pH 7.5], 20 mM MgCl2, 50 mM NaCl), 100 ,uM each - ~~~~c,'-IS deoxynucleoside triphosphate, 10 mM dithiothreitol, and 1.7 U 4 kDa of the modified T7 polymerase, Sequenase (United States FIG. 1. SCMV precursor genes, proteinases and assembly protein Biochemical Corp.), at 37°C for 15 min. (iv) The resulting and their protein products. (A)I~The four principal genes used in these studies, their plasmid designations, and proteolytic activities. Mutants double-stranded DNA was then phenol-chloroform extracted, of the proteinase were derived from plasmid AW4, and mutants of the ethanol precipitated, and dried. (v) The resulting double- assembly protein precursor were derived from plasmid AWI. The stranded DNA was suspended in buffer and cleaved with overlapping relationship of the genes encoding the proteinase appropriate restriction enzymes (i.e., SalI-BamHI for APNGI (APNG1) and the assembly protein precursor (APNG.5) is indicated and APNG.5) to release the desired fragment for subcloning. at the bottom of the panel. The R and M cleavage sites are indicated And, (vi) the excised fragments were then ligated into pRSV- by arrows; three of the five conserved domains (CD1 to CD3) are .neo that had been cleaved with Sall and BamHI, and the indicated by shaded rectangles; a 15-amino-acid epitope (C3) inserted resulting construct was transformed into DH5cx E.
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