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. coli (lacking into the amino half of LM3 is indicated by a solid rectangle; the translational start methionines of the proteinase (M-1) and the F' and nonpermissive for M13) and selected by replication in assembly protein precursor (M-281) and the carboxyl-terminal resi- the presence of ampicillin. To insure that only the intended dues of assemblin (YVKA), the mature assembly protein (VNA) and site-directed change was present in mutants of putative active- its precursor (KME) are also indicated. (B) The full-length proteinase site amino acids, a small DNA restriction fragment containing precursor, pNPI, its three principal cleavage sites, and the protein the mutation was excised (i.e., mutations of D-15, E-20, E-22, products expected from cleavage at these sites. Also shown are the and H-47 in a SalI-EcoR5 fragment; of D-104, S-118, S-120, assembly protein precursor, pAP, and the two products of its cleavage at and S-121 in an EcoR5-Kspl fragment; and of H-142, C-146, the maturational site. The designation of each protein is shown to the and S-147 in a KspI-Eco47III fragment), substituted for the left of the line representing it, and the computer-predicted size of each corresponding fragment of the gene in and is indicated. The carboxyl-terminal residues of the mature assembly wild-type AW4, protein (VNA), assemblin (YVKA), and the amino half of I-site-cleaved sequenced (including the flanking insertion site region). assemblin (INA) are indicated at the top. Antisera were made to LM3 was derived from the plasmid AW4 and encodes a synthetic peptides representing the amino (NI) and carboxyl (Cl) ends minimally active proteinase that contains a 15-amino-acid of the assembly protein precursor (i.e., anti-Ni and anti-Cl, respective- insert (C3, poliovirus epitope) between conserved domains 1 ly), and the carboxyl end of assemblin (NP1I) (C2) (i.e., anti-C2). and 2 (CD1 and CD2) (see Fig. IA and reference 48). 7362 WELCH ET AL. J. VIROL.

Transfection assay. Approximately 1 x 106 human embry- (P3391; Sigma) (100 mg/ml of phosphate buffer, pH 7.4) was onal kidney (HEK) cells (line 293; American Type Culture added to each tube, and incubation was continued for 60 min Collection) were seeded onto Lab-Tek 2-well slides (Nunc, at room temperature. The reacted beads were collected by Inc., Naperville, Ill.) or into 2-cm2 wells (3047; Becton Dick- centrifugation (12,000 x g, 4°C, 1 min), washed four times in inson Labware, Oxnard, Calif.) on day 1 and then transfected immunoprecipitation buffer (lysis buffer with no KCl), com- with the appropriate plasmids (2 ,ug of DNA total per well) on bined with an equal volume of 2 x SDS-PAGE sample buffer, day 2 by a modified calcium phosphate technique (11). A heated in a boiling water bath for 3 min, and stored at - 80°C plasmid (RSV.TAg) encoding simian virus 40 T antigen was until analyzed. Following SDS-PAGE, gels were stained with added to each transfection (0.2 ,ug per transfection) to increase Coomassie brilliant blue (18) and destained, and proteins were the copy number of the RSV.neo-derived constructs (37). Cells then detected by fluorography following sodium salicylate were harvested 48 to 72 h after transfection (as indicated in the enhancement (9). figure legends) by aspirating the medium, adding 60 to 70 [L of 2 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis RESULTS (SDS-PAGE) sample buffer to the cell layer, and removing the viscous lysate by pipette and heating it for 3 min in a boiling Four sets of mutations were made by site-directed mutagen- water bath. Samples were stored at - 80°C until analyzed by esis: one within the release recognition-cleavage sequence (R SDS-PAGE. site, YVKA249 l S) near the middle of the proteinase precur- SDS-PAGE and Western immunoassay. SDS-PAGE was sor, a second within the maturational recognition-cleavage done essentially as described by Laemmli (25); the ratio of sequence (M site, VNA5.6 5)S near the carboxyl end of the N,N'-methylenebisacrylamide (bis) or diallyltartardiamide to proteinase, a third in the homologous M site at the carboxyl acrylamide was 0.735:28 or 1.09:28, respectively; SDS was from end of the assembly protein precursor, and a fourth within the Bio-Rad. SDS-PAGE sample buffer contained 2% SDS, 5% proteolytic domain of assemblin (i.e., residues 1 to 249). A beta-mercaptoethanol, 10% glycerol, 25 mM Tris (pH 7), and deletion mutation was also made in the putative inactivation 0.01% bromophenol blue. sequence (I site, INA127 1 AD) near the middle of the assem- Electrotransfer was done essentially as described by Towbin blin. et al. (45). A semidry transfer unit was used, the membrane Proteinase mutants were transfected (i) alone, to test their was Immobilon P (Millipore, Bedford, Mass.), the buffer was metabolic stability and ability to self cleave, (ii) together with 50 mM Tris-20% methanol, and the time of transfer was AW1 (which encodes the assembly protein precursor), to test calculated by the formula gel width x height x 2.5 = their ability to cleave the M site in trans, (iii) together with milliamperes per 30 min. The membrane was blocked in 5% LM3 (which encodes enzymatically inactive proteinase), to test bovine serum albumin, reacted sequentially with antiserum their ability to cleave the R site in trans, or (iv) together with and then with "2'I-protein A, and exposed to X-ray film, LM8 (which encodes assemblin, NP1n), to test their own usually with a calcium tungstate intensifying screen (26). susceptibility to cleavage in trans. Mutants of the assembly The antisera used were prepared by injecting rabbits with protein precursor were transfected (i) alone, to test their keyhole limpet hemocyanin-conjugated synthetic peptides (41) metabolic stability, or (ii) together with LM8 or AW4 (which representing the carboxyl 13 residues of the assembly protein encodes assemblin precursor pNP1) to test the effect of the precursor (anti-Cl), the amino 21 residues of the assembly mutations on M-site recognition and cleavage. Transfected cell protein (anti-Ni), or the carboxyl 14 residues of assemblin lysates were subjected to SDS-PAGE, and proteins of interest NPln (anti-C2) (Fig. 1). were identified by Western immunoassays with antisera to the Pulse-chase radiolabeling and immunoprecipitation. Two amino end or carboxyl end of the assembly protein precursor, days after transfection, the culture medium was replaced with pAP (i.e., anti-Ni and anti-Cl, respectively) or to the carboxyl methionine-free medium (7732-18-5; GIBCO, Grand Island, end of assemblin, NP1, (i.e., anti-C2) (Fig. 1). N.Y.) containing 50 pCi of [35S]methionine per ml. Fifteen Landmarks of the proteinase and its assembly protein minutes later, the radiolabeling medium was removed from all substrate are shown in Fig. 1. The amino acids of the assembly cultures; one was harvested immediately (pulse) by aspirating protein have been numbered according to their positions in the the medium, adding 150 RI of lysis buffer (1% Nonidet P-40, longest open reading frame (i.e., APNG1) with which it is in 0.5% deoxycholate, 0.5 M KCl, and 1 mM phenylmethylsulfo- frame, overlapping, and 3' coterminal; thus, the translational nyl fluoride in phosphate buffer, pH 7.4), recovering the lysate start methionine for the assembly protein is methionine 281. from the well, vortexing it, and freezing it at - 80°C. Labeling Cleavage at the R site is not required for proteolytic activity. medium on the remaining cultures was removed and replaced One deletion and five substitution mutations were made in the four times (5 min each time) with normal growth medium to predicted R-site consensus sequence, YVKA 4 S, of the cyto- dilute the intracellular pool of unincorporated radiolabel. megalovirus (CMV) Colburn assemblin precursor, pNP1 (48). Additional cultures were harvested in the same way at 30 min As described here, four of these eliminated or greatly de- and at 1, 2, 4, 8, 12, and 22 h after the pulse radiolabeling creased R-site cleavage as measured by (i) increased amounts interval. Cells transfected with only the plasmid encoding T of the noncleaved precursors, pNP1 and NP1, and (ii) reduced antigen were processed in parallel and harvested after the amounts of the expected cleavage products, assemblin (NP1.) pulse and 22-h time points. Prior to immunoprecipitation, the and NP1c. lysates were thawed, vortexed, and clarified by centrifugation Deletion of the entire R-site consensus sequence (Y246- (12,000 x g, 10 min, 4°C). A similar experiment was also done S250- ) eliminated R-site cleavage and resulted in the accumu- with a 5-min pulse or radiolabel and chase periods of 5, 10, 15, lation of the assemblin precursor, NP1, and the absence of its and 30 min. cleavage products, NP1c (Fig. 2; Fig. 3A, lane 12; and Fig. 4, Immunoprecipitations were done by combining 50 ,ul of lane 30) and assemblin (NPln, Fig. 3B, lane 28). Substitution of clarified lysate with 20 [lI of antiserum anti-Ni, anti-Cl, or alanine for the P4 tyrosine (Y246A) strongly inhibited cleavage anti-C2 (described above and in the legend to Fig. 1). Incuba- at the R site, but small amounts of assemblin (NPln) and NP1c tion of lysates with antisera was done at room temperature were detected (Fig. 2; and Fig. 3, lanes 6 and 23). A less (-24°C) for 90 min. Fifty microliters of protein A beads complete inhibition of R-site cleavage was observed when VOL. 67, 1993 HERPESVIRUS PROTEINASE 7363

glycine was substituted for the P1 alanine (A249G) (Fig. 3, UO>fiY) C/ coi< lanes 9 and 25; Fig. 4, lanes 27 and 37). When both of these substitutions were combined in the same mutant (Y246A/ A249G), R-site cleavage was inhibited almost as completely as in the Y246-S25o- deletion mutant (Fig. 3, lanes 11 and 27; and pNP1 o Fig. 4, lanes 29 and 39), but prolonged fluorographic exposure NP1 revealed trace amounts of NP1C not seen in the deletion mutant (data not shown). Substitution of glycine for serine 250 (S25oG) had little effect on R-site cleavage; both product (e.g., NPIC and NP1) and precursor (e.g., NP1) bands approximated those in cells transfected with the wild-type proteinase gene (Fig. 3, lanes 10 and 26; and Fig. 4, lane 28). The finding NP1c- _o o that each of these five mutant proteinases was active in pAP - cleaving at its own M site (i.e., conversion of pNPl to NP1) and at the M site of the assembly protein precursor (i.e., conversion AP - of pAP to AP) (Fig. 2; and Fig. 4, lanes 45 and 47 through 50) demonstrates that R-site cleavage is not absolutely required for proteolytic activity. These data also show that some of the - - Sub. +Enz. R-site mutants, A249G in particular, gave rise to a 68-kDa band FIG. 2. R-site cleavage is not required to activate proteinase. Two R-site mutations were made: a deletion mutant (Y246-S250-) that (discussed below) not typically seen (for examples, see Fig. 3, eliminated all 5 amino acids in the R-site consensus sequence and a lanes 9 and 25 [asterisks]). It is likely that this band corre- substitution mutant (Y246A) in which Tyr-246 was changed to an Ala. sponds to the computer-predicted 46-kDa protein indicated in HEK cells were transfected (-48 h) with the mutant genes, either Fig. lB and represents pNP1 cleaved at both its M and I sites alone ( - ) to test the enzyme's self-cleavage properties, with an but not at its R site (i.e., observed sizes of -86 kDa [pNP1] - assembly protein precursor (+Sub., i.e., with the AWl construct) to 4 kDa [C'] - 14 kDa [A,j] = -68 kDa). test the mutant enzyme's ability to cleave substrate in trans, or with To test the susceptibility of the R-site mutations to cleavage assemblin (+Enz., i.e., with the LM8 construct) to test its ability to be in trans, they were used as substrates in cotransfections with cleaved in tracns at its release site. Transfected cells were processed and wild-type assemblin (i.e., LM8 plasmid). Although the pres- subjected to SDS-PAGE in a 10Cc bis-cross-linked gel followed by ence did Western immunoassay using anti-NI. Shown here is an autoradio- of wild-type assemblin not appear to significantly graphic exposure of the resulting Immobilon membrane. Protein desig- increase the relative extent of cleavage at the mutant R sites, it nations are indicated in the left margin. Nuclear and cytoplasmic did reduce the relative amount of pNP1,. in mutant K248N (Fig. fractions of Colburn-infected cells were included to provide reference 4, lane 36) and increase the relative amount of the 68-kDa proteins. LM3 was included as a positive control substrate to verify that band in at least four (Fig. 4, lanes 35 and 38 through 40) of the LM8 was active in the cotransfections, and to provide a marker for NP1c. six R-site mutants. Open circles to the left of Y246A lanes indicate the positions of pNP1, NP,, and NP1,.

'3.,CDm CI, A. B. r ,. N4 Ln) <, r'j c3zz ~~i >-< ~~~~~~Z (D~~~CD >l-e: UI- C: CD C '- z CD (D < LU < ,D O ,KrU)oU)LO r- z '3r U) U - >~, c=, m U LI) L) LsrI LrI o o 2 i> N\ " rJ " r-l 1N, In U) Ln Ln -j - - nJ CXJ n N UJ U) Li) LO Li) , , Ji < >-, Y <:tJ} >- > > < < Lo >- Y, < Un >- >- > < < UZ

pNP1 - NP1- oW 4h*4S

* 40

pNPlc- 6b "O NP1 c- pAP- AP-

NP1N.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 FIG. 3. Self-cleavage properties of R- and M-site proteinase mutants. R- and M-site proteinase mutants were tested for self-cleavage by transfection and Western immunoassay. 72-h-transfected cell proteins were separated by SDS-PAGE in a 10% bis-cross-linked gel, and reactions were performed with either anti-NI (A) or anti-C2 (B) following electrotransfer to Immobilon. Shown here are the resulting fluorographic images. Specific protein bands are indicated in the left margin; transfecting plasmids are indicated at the top. Lanes marked "-" were blank. Nuclear and cytoplasmic fractions of Colburn-infected cells (Col. Nuc. and Col. Cyto.) are shown for reference. Circles indicate bands corresponding to the protein designations; asterisks denote the 68-kDa band referred to in text. 7364 WELCH ET AL. J. VIROL.

co00 C(Co _j °° B H:B E Bj2B < S n S SX LIn --i vD0C;0QtHD-J. VCDvsi; C3a "j-~~~~~~*n ~ . 00 OL OC...... L)SS>DtL:4Si0...... +r0JSt4)Stco cc6n4t,,)S r: r + ++ z Oc) z Y><

a3pNPl- NP1- -pNP1

-pNP1, 0 '... o _odo-...... NP1c A; 0 11111Ii-Ill'o 0 * *w . "INAk -00.00* doll. -Z .q_m.A pAP- _ _ .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

AP- *_ .04w_ 0_

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 39 40 41 42 4344 45 46 47 4849 -0 51 352- -Alone- - +Enz. Alone -wt Eniz +Sb tratt- Controls Substrate Mutants Enzyme Mutants - FIG. 4. Survey assay of cleavage properties of R- and M-site mutants. Shown here is a Western immunoassay (probed with anti-NI) of the proteins produced in cells transfected (-48 h) with (i) M- and R-site mutants of the proteinase, alone (lanes 25 through 34) or with assemblin, LM8 (lanes 35 through 44), or assembly protein precursor AW,, (lanes 45 through 54), or (ii) M-site mutants of the assembly protein precursor, AWI, alone (lanes 13 through 16) or with assemblin, LM8 (lanes 17 through 20), or proteinase precursor, AW4 (lanes 21 through 24). Specific protein bands are indicated in the left and right margins; transfecting plasmids are indicated above each lane; mutations are designated by the amino acid changed (e.g., V554) followed by its substitution. Nuclear and cytoplasmic fractions of Colburn-infected cells (Col. Nuc. and Col. Cyto.) are shown for reference. A dot to the left of a band indicates correspondence to the protein designation in left margin; open circles indicate correspondence to protein designations in the right margin. Asterisks identify two cross-reacting host cell proteins; the -68-kDa band denoted with an asterisk in Fig. 3 is approximately the same size as the smaller of these host proteins (see lane 39). Samples were subjected to SDS-PAGE in a single 10%, bis-cross-linked gel that was cut in half (between lanes 24 and 25) for electrotransfer.

The sixth mutation in this set, a substitution of asparagine sequence, VNA k S, of pNPI. Deletion of the entire M site for P2 lysine (K248N), had a comparatively unusual phenotype. (V554-S557-, Fig. 5, lane 4) or of alanine at the M-site sissle It cleaved well at the R site (i.e., it had a strong NPlc band and bond (Fig. 3, lane 16; and Fig. 4, lane 34) eliminated M-site but a comparatively weak NP1 band), but it left relatively more not R-site cleavage of pNPI, as indicated by the accumulation pNP1 and pNPlc than the other R-site mutants, indicating a of pNPIc rather than NPlc. Substitution of alanine for P3 reduced cleavage efficiency at its own M site (Fig. 3, lanes 8 valine inhibited cleavage of the pNP1 M site significantly (i.e., and 24; and Fig. 4, lanes 26, 36, and 46). Compared with the increased the proportion of pNPIc relative to NP1J, but wild-type enzyme, this mutant also showed reduced cleavage in substitution of glycine for either the P1 alanine (A556G) or P1' trans of the assembly protein precursor M site (Fig. 5, lane 12). serine (S557G) had little inhibitory effect on cleavage of the Additionally, the amount of NP1n was generally higher with pNPI M site (i.e., produced relatively more NPlc than pNPI0) this mutant than with the wild type (i.e., AW4 and LM8) or the (Fig. 3, lanes 13, 15, and 17, respectively; and Fig. 4, lanes 31 other R-site mutants (Fig. 3B). It is unlikely that this pheno- through 33, respectively). None of these mutations eliminated type is due to a second site mutation, since the same results cleavage of pNPI at its R site (i.e., produced little NP1 or were obtained with three additional K248N mutants that were pNP1; comparatively more NPlc or pNP1c) or cleavage of the derived from an independent mutagenesis experiment and assembly protein precursor at its M site (Fig. 4, lanes 51 from a recombinant (K248Nb in Fig. 5, lanes 13 and 14) through 53; and Fig. 5, lanes 4 and 5), indicating that M-site constructed by replacing a 703-bp nucleotide sequence of the cleavage is not essential for proteolytic activity. wild-type proteinase gene with the corresponding but altered The relative inhibitory effect (strongest to weakest) of these sequence from the original K248N mutant gene. mutations on the proteinase M-site cleavage was estimated to The relative inhibitory effect (strongest to weakest) of these be V554-S557 - -A556 - > V554A > A556G > S557G. mutations on R-site cleavage was estimated to be Y246-S250- Deletion of the I site does not prevent either R- or M-site > Y246A/A249G > Y246A > A249G > S25oG > K248N. cleavage. A third cleavage site, with the sequence VEA c AT, Cleavage at the M site of proteinase precursor, pNP1, is not has been identified between CD1 and CD3 in the proteolytic required for proteolytic activity. Two deletion and three domain of HCMV assemblin (5, 7). Cleavage at this putative I substitution mutations were made in the M-site consensus site gives rise to an amino half (An; 16 kDa) and a carboxyl VOL. 67, 1993 HERPESVIRUS PROTEINASE 7365

0V)

coo c ;c O ,LO1 N 4- L Ln L17n 1- -') >, : ~CA /) ' z z z V Z v L.~ / CD oD * L Lin LA LA LA - Nr o o o ur Lnu, N B N N rqi ) u M > > = < -, *. -NP1 cm -pNPI -NP1

-Ac R. -pNPl c 1 2 _wam _m -NP1 c FIG. 6. Deletion of INAAD-129 sequence eliminates I-site cleav- _ :, _ Om age. The wild-type (Wt) proteinase (AW4) and a deletion mutant .-l - am - -pAP lacking the sequence INAAD (1125-DI29-) were analyzed by transfection and Western immunoassay for I-site cleavage. A 10 to 20% -AP 4 am gradient gel (Novagen, Madison, Wis.) was used for SDS-PAGE, and proteins were detected with anti-C2. Shown here is a direct autoradiographic exposure of the resulting immunoblot. Protein bands are identified in the right margin. w _ -NP1 s

1 7 3 4 5 6 7 8 9 10 11 12 13 14 [35S]methionine for 15 min, chased in the absence of radiolabel FIG. 5. Phenotypes of M- and I-site deletion mutants and of the for periods of up to 22 h, and then harvested and analyzed by R-site P2 lysine mutant. Proteinase mutants lacking the M-site con- immunoprecipitation with one of three antisera (i.e., anti-NI, sensus sequence (V554-S557-) or the putative I-site sequence (1125- anti-Cl, or anti-C2 [Fig. 1]) followed by SDS-PAGE. Experi- or having an asparagine in place of P2 lysine at the R site D129-) mental details are presented under Materials and Methods. (K248N), were tested in transfection assays for their ability to self Results of the experiment (Fig. 7) can be summarized as cleave (lanes 4, 6, and 11) and to cleave the assembly protein precursor (lanes 5, 7, and 12). K248Nb was constructed as described under follows. (i) Products of M-site cleavage (e.g., NPI and NP1I) Materials and Methods and was tested in the same way (lanes 13 and were present in the pulse-labeled sample (lanes 1, 11, and 21), 14) to verify that this mutant had no unexpected second site changes. indicating that M-site cleavage had taken place within the Proteins from 72-h-transfected cells were separated in a 12% bis-cross- 15-min labeling period. (ii) Both pNP1 and NP1 decreased in linked gel. Shown here is a fluorogram prepared following Western intensity following the 15-min labeling period, consistent with immunoassay of the SDS-PAGE-separated proteins. The Immobilon their proteolytic conversion to product forms by R-site cleav- replica was probed with anti-NI, exposed, and then probed again with age. (iii) Products of R-site cleavage (e.g., NP1 , NP1c, and anti-C2. Cytoplasmic and nuclear fractions of Colburn-infected cells were present in the pulse-labeled sample (lanes 1, 11, (Col. Cyto. and Col. Nuc.) were included for reference (lanes 1 and 2). pNPlc) Mock, preparation from cells transfected with T-antigen plasmid only. and 21), indicating the R-site cleavage had also taken place Protein designations are shown in the right margin. within the 15-min labeling period. The presence of pNP1c (lanes 11 and 21) shows that R-site cleavage can occur prior to M-site cleavage. (iv) Assemblin (NPIn) decreased in intensity following the 15-min labeling period (anti-C2 panel), demon- fragment (Ac; 13 kDa) (5). We have detected an apparently strating that it is metabolically unstable and consistent with its corresponding 13-kDa protein in CMV Colburn that is reactive conversion to the 14-kDa An and 13-kDa AC fragments follow- with an antiserum to the carboxyl end of assemblin (anti-C2 ing I-site cleavage (Fig. 1). (v) Ac, the 13-kDa carboxyl half of [Fig. 1]). By sequence alignment, we determined that this assemblin, increased dramatically in intensity (-18-fold) dur- fragment could be produced by cleavage at a site, INA J AD, ing the first 30 min of the chase period and then decreased which has similarity to the HCMV I site (Fig. 9, CMV I-site throughout the rest of the chase (Fig. 7, anti-C2 panel), arrow). To verify this potential counterpart I site in strain consistent with its being derived from a precursor but more Colburn and to determine the effect of altering it on proteo- slowly than the M- and R-site cleavage products. (vi) The lytic activity, an I-site deletion mutant was made (1125-D129 -). intensity of NPIc, the carboxyl half of NP1 cleaved at its R site, Consistent with the 5-amino-acid deletion, this mutant gave a remained essentially the same throughout the chase interval comparatively smaller assemblin protein (NP1n in Fig. 5, lane (anti-Ni panel), demonstrating that it is much more stable 6; and Fig. 6, lane 1) and no 13-kDa Ac fragment (Fig. 6). This than the other precursor and product forms of the proteinase mutant cleaved both its own M site and that of the assembly (e.g., pNP1, NP1, NPln, pNP1I, and AC). It can also be seen in protein precursor with approximately the same efficiency as the the anti-NI panel that NP1c became progressively more het- wild-type enzyme but appeared to be less efficient than the erogeneous in size during the chase period, with one or two wild-type proteinase in cleaving at its R site (i.e., it had higher-molecular-weight bands appearing immediately above comparatively more NP1 [Fig. 5, lanes 5 and 6]). the pulse-labeled NPIc band and increasing in intensity with Pulse-chase radiolabeling shows cleavage products of pro- time. And (vii) the intensities of the pNP1, NP1, pNP1c, and teinase precursor have differential stabilities. A pulse-chase NPln bands were approximately the same in a 15-min (lanes 31 radiolabeling experiment was done in an attempt to determine through 33) and 22-h (lanes 34 through 36) labeling period, whether a temporal order of cleavage could be seen between indicating that a steady state with respect to their interconver- the M, R, and I sites. Cells transfected with the gene for the sion was approximated within the 15-min interval. Consistent proteinase precursor (i.e., AW4) were pulse labeled with with the pulse-chase data (lanes 1 through 30), the NP1c and 7366 WELCH ET AL.J.Vro.J. VIROL.

2; --, chl~,S(, ~rn Chase Chase L;- 051 2 4 8 1222 MrnMo,,, a 0Q5 1 2 4 8 1222 MPM22 a_O.51 2 48 12 22MP Md2-~, ':<

pNPi1 ~

QNPI(

0 g0 -pNPl-cI~

-NP1..,- qm

4',

1 1. 14 1 .15 B 9 20. 2 24A 2_5, 2 2 .7 2 2 c: 31 32 33' 3: .,

Aniti C2 ___--- -- Anti-Nil -_-- Anti Cl PLulse Long

FIG. 7. Pulse-chase labeling (15 min) of the Colburn proteinase, pNPI, expressed in HEK cells following transfection. Shown here is a fluorogram of a single 18%, diallyltartardiamide-cross-linked gel containing samples from the pulse-chase experiment described in the text. Transfected cells were pulse radiolabeled (Pulse), or pulse labeled and chased (Chase) in nonradioactive medium for the indicated times (in hours). Mock-transfected controls for the pulse (MP') and the longest chase period (M22) are indicated. Portions of each sample were subjected to immunoprecipitation with the three antisera (anti-C2, anti-NI, and anti-Cl). The three pulse-radiolabeled samples were analyzed side by side (lanes 31 through 33) to aid in identifying the bands, and samples radiolabeled continuously for 22 h (lanes 34 through 36) were also examined. Circles to the left of bands indicate their correspondence to protein designations in the space between lanes 30 and 31.

AC bands were much more intense in the long labeling period NP1~, and NPln before A,; and maximal labeling of pNPI at 5 than in the pulse (compare lanes 31 and 34), indicating their to 10 mmn, of NP1 at 10 to 15 min, of NPln at 15 to 20 min, and greater metabolic stability. of the AC at 20 to 35 min. A similar experiment was done, but a shorter pulse-radiola- Mutations at the maturational cleavage site of the assembly beling period was used. Results of this second experiment (Fig. protein precursor affect cleavage. One deletion and three 8) showed the following significant differences from the first substitution mutations were made at the M site of the assembly (Fig. 7). (i) The pNPI band was most intense in the 5-mmn pulse protein precursor. The effect of these mutations on the sus- and 5-mmn chase samples and then became essentially unde- ceptibility of the site to cleavage was determined by cotrans- tectable in the 15-mmn chase sample, consistent with its cleav- fecting each of the mutants with the gene for either assemblin age to lower-molecular-weight bands. (ii) The NP1 band (i.e., LM18) or its full-length precursor (i.e., AW4). Deletion of increased in intensity through the 10-mmn chase period (maxi- alanine at the sissle bond (A.556 ) completely eliminated mal intensity after 5 to 10 min of chase [Fig. 8]) and then cleavage of pAP to AP by either form of the proteinase (Fig. 4, decreased in the 15-min chase period and further in the 30-mmn lanes 20 and 24). Substitution of alanine for the P3 valine chase period, indicating slower rates of appearance and disap- (V554A) substantially decreased the extent of pAP cleavage to pearance than those for pNP1, and consistent with its being AP compared with cleavage of wild-type pAP (Fig. 4, lanes 17 derived from pNPI. (iii) The NP1'nband was less intense than and 21). Some reduction in susceptibility to cleavage was also the NPI band in the 5-mmn pulse sample, in contrast to its noticeable when glycine was substituted for the P1 alanine relatively greater intensity in the 15-mmn pulse sample (Fig. 7), (A556G), but the extent to which this substitution interfered and increased in intensity through the 10- and 15-mmn chase with M-site cleavage appeared significantly less than in the periods (maximal intensity after 10 to 15 min of chase) and context of the P1 position in the R site (compare lane 18 with then decreased in intensity in the 30-mmn chase period. (iv) Ac lanes 27 and 37 of Fig. 4). Little change in susceptibility to was barely perceptible in the 5-mmn pulse sample, in contrast to cleavage was noticed when glycine was substituted for P1' its greater intensity than that of the pNP1 band in the 15-mmn serine (S557G) (Fig. 4, lane 19). pulse (Fig. 7), and increased steadily through the 30-mmn chase The relative inhibitory effect (strongest to weakest) of these consistent with its derived from NP1 period, being n. The mutations on assembly protein precursor M-site cleavage was = results of the 5- and 15-min pulse-chase experiments are estimated to be A556 > A556G V554A > S557G. consistent in showing loss of pNPI before NP1, NPI before Alignment of the herpesvirus assemblin homologs reveals VOL. 67, 1993 HERPESVIRUS PROTEINASE 7367

IV essential. Site-directed mutagenesis was used as a means of X- Chase -- - evaluating the importance of specific amino acids to catalytic In 5S I' 1 5' 30' activity. Mutations were introduced into the APNG1 gene as described in Materials and Methods (AW4 in Fig. IA). The mutant proteinase constructs were then transfected alone or cotransfected with AW1 (APNG.5), the plasmid that codes for -pNP1 the assembly protein precursor, and analyzed for (i) ability of -NP1 the proteinase mutants to cleave themselves at both the R and M sites, as monitored by the appearance of NP1, pNP1c, and NP1c, and (ii) ability of the proteinase mutants to cleave the assembly protein precursor (pAP) in trans at its M site, as monitored by the appearance of the mature assembly protein, AP. The immunoblot shown in Fig. 10 was probed with anti-NI and reveals both cleaved (e.g., NP1, NP1c, pNP1c, and AP) and 4.11W - noncleaved (e.g., pNP1 and pAP) forms of the proteinase and 't ff. assembly protein precursor (Fig. 1B). Because of their importance in known proteolytic active sites Rub -NP1 N and their absolute conservation among the assemblin homologs (Fig. 9), histidines 47 and 142, serine 118, and cysteine 146 were mutated first. Substitution of an alanine (SI18A) for the only absolutely conserved serine residue, Ser-1 18, abol- ished the proteolytic activity, as did mutation of histidine 47 to either an alanine or a glutamine (Si 18A and H47A or H47Q, respectively [Fig. 10]). Mutants in which the conserved cysteine residue was changed to an alanine or threonine (C146A and C146T [Fig. 10]) retained enzymatic activity, indicating that this _-AC residue is not an essential active-site nucleophile. To insure that the neighboring serine 147 was not somehow replacing the 1 2 3 4 5 cysteine as an active-site nucleophile, the double mutation Anti-C(? C146A/S147A was also tested and was found to have little effect FIG. 8. Pulse-chase labeling (5-min) of the Colburn proteinase, on activity (C146A,S147A [Fig. 10]). Mutation of histidine 142 pNP1, expressed in HEK cells following transfection. Shown here is a to either alanine or glutamine severely reduced, but did not fluorogram of an 18%, diallyltartardiamide-cross-linked gel containing abolish, proteolytic activity (H142A or H142Q [Fig. 10]). samples from the 5-min pulse-chase experiment described in the text. In addition to the above-mentioned absolutely conserved Transfected cells were pulse radiolabeled (Pulse), or pulse labeled and noncon- chased (Chase) in nonradioactive medium for the indicated times (in residues, a number of highly conserved and several minutes). Proteins were immunoprecipitated with anti-C2, protein served residues were also mutated. Serine 195 was suggested designations shown in the right margin are explained in the Fig. 1B initially as a potential catalytic residue because of its positional legend, and the circles between lanes 1 and 2 indicate the positions of similarity to the chymotrypsin nucleophile (48), but its change the proteins. to alanine (S195A) had little effect on proteolytic activity, indicating that this serine is not an active-site residue (S195A [Fig. 10]). Two other serine residues that are highly conserved in three domains which contain absolutely conserved residues CD3, serines 120 and 121, were mutated together with the potential to contribute to known (SI2oA,S121A) and then individually (SI2oA and S121A). These proteolytic active mutations reduced proteolytic activity but did not abolish it sites. A previous alignment of the assemblin homologs of seven (S12oA,S121A,S12oA and S121A [Fig. 10]). Two nonconserved herpes group viruses identified two highly conserved domains, CD1 and CD2, and several absolutely conserved potential serines were also changed to alanine (Ser-198 and Ser-199), in active site residues, including two histidines, a glutamic-aspar- resulting an electrophoretically faster-migrating NP1 but tic acid, and a cysteine (48). More recently, we have used the with little, if any, loss of proteolytic activity (Fig. 10A). Because program PileUp (University of Wisconsin) to obtain a more of their role in metalloprotease activity and as members of complete alignment and we have included the sequence of the active-site triads of chymotrypsin-like serine and cysteine pro- herpesvirus saimari (1) assemblin homolog in the comparison. teinases, highly conserved glutamic acids and aspartic acids Results of the analysis revealed three additional highly con- were also mutated. A double mutation at glutamic acids 20 and served domains, referred to as CD3, CD4, and CD5, one of 22 and a single mutation at glutamic acid 22 severely reduced which (CD3) contains the only absolutely conserved serine but did not eliminate proteolytic activity. The E22A mutant was among the assemblin homologs (Fig. 9, Ser-118 in the SCMV interesting in that it appeared to be relatively unaffected in its sequence). This alignment also shows the highly conserved ability to cleave at the M site but had impaired ability to cleave R-site and M-site cleavage sequences, as well as the I-site at the R site (E20A,E22A and E22A in Fig. lOB; also see cleavage sequence that has been demonstrated only for CMVs Discussion). Mutations at the only residue showing a high level (HCMV [5] and SCMV [Fig. 5, lanes 6 and 7]) and may not be of conservation as aspartic acid (Asp-15) and at the conserved conserved among all assemblin homologs, as suggested by the acidic residue, aspartic acid 104 (a glutamic acid in most other absence of obvious sequence similarity in that region. assemblin homologs [Fig. 9]), also reduced the proteolytic Absolutely conserved CD2 histidine 47 and CD3 serine 118 activity (as in D15A and D15N and in DIo4A and D104N [Fig. are essential for SCMV proteolytic activity; absolutely con- 10]). LM3 was included in this experiment as an example of an served CD1 histidine 142 and CD1 cysteine 146 are not essentially inactive proteinase (48). 7368 WELCH ET AL. J. VIROL.

Virus CD5 CD2

MAAEADEENC E AL .YVYA.GYLALY SK.DEG EL NITPEI,V.RSA L.PP .TSKIPI -RKDC.V'E,V'EIA ELVZ MDAYTVDGNA V S L.P::. IYVA.GYIA.LY DMGDGG. TLTRET AAA ...... P: RASRLPINID.''"''RNGCVE V-.:" LS HSV MAADAPGDRM EEPLPDRAV.P IYYAGFL.LY' SGDSG EL ALDPD:T VRAA L .DNPLPINVDEDNG LLA LI LTV DIKY .'I,VA':Y'L,'.VVY D,HOESAGRE'.Y E.L,TREOSKSA ',HESSCV,VGTV LT R[ HCMV 'LHAQGQGPS'SVA"EH't'N'DDTAQGQG'A ...... RGP'LDP'DLE,NV:DESATVGYV Sy[EBV HMLDPDPLT:.::VKSOLY:LKK:PELTVELDASV LHVS AYPKVDP.V:LAGF.-,.VDY.LNL'...R.::':..DSKCL'...V ATKL.1'GLE'LPESTIG:HT IG * * * *

RESIDUE 5 15 20 22 24 34 38 47 57

CMV I-Site CD4 CD3 4 vzv I I E D I'.R G-.'IPRFLGIVPCPAV OLLHAVLFEAAH S N F .FGN RD S V LS. P LERA LYLVTNYLPS V SL'SK LSP NEIPD'G . .. N EH V I V DDARGP F,.FL.G.. II..:'.NCPRO LGAVLAT AAG PDFFGE LS EG :LS EOE'RL tLYLVSNY:LPS& ASLS:S::R-RLt.GP D:E.E;PD'E.... T HS V VVDDP1RGP F F VG.L:I: AC V LERV L.ET'AAS AA I F.'ERRGPP LS. REER L L.Y LI TNYL: P.S VSSLATKIR.LGG EAHP'DR.... T I LTV I LD LP.:RG::.L' F:C,'LG:V VMS T A L AP I FLS Y V OD DA LFANAEEG MV'L-T. ET.E.KF L:Y L:L.S.N I L.PS LS'S:,REE... D HCMV MFSVPDCMOSVRD...... F....~C--L..S-G-`CV,,::TSP:C.V..T.'P..R.gR :F::L:ELEIVRIV RPAS E KS E LVSRGPV ..S P.LO P D K V V EF L'S::G T Y AG LS L.S:S CDD V E A ATTS LS G. S ETT SCMV LNVRAGL FCtt.GR:VTSRK F'LD I VOKAS E KS E LVSRGPP S E SS$::0L'R P D G V -LEt'F L5'G S Y S G L.SI'.LSS`R.R-.. D I N AAD:GAAGD AETA EBV L Y OS R AGL FS AAS.I'.'TS:GDDFL.S LLDS I Y H DCDI AOSORL .PL'PRE.PK V.E-A'L'.H AW L PS LSSlAS.LHP. . D I POT T ADG GK LS HVS LYAVTHGV FCVGVIHS'EK F..L'H LT EN LFS NSCV AOATSK ... FLP Y OP L LJE'.M'L.H T W L P A LSL5,l SS. L C P...... TAONA ANTN * ...... * ** 59 67 7 7 8 7 97 104.... 107 1 18 125

CD1 vzv FFTNVAM'tX '-LC'VVG~RRVG.T VVN'YDCTPES S I,EPF'RVL"S.M ESKARL... L SLVKDYA... GLNKVWKVSE D KL... AKVL LST.AVN E H V L RA CVSLCVIGRRVGT IVTY DAT EN AVAPFKRLSP SS E:. . L ITAREAQSRL GDAATWHLSE DTL- .TRVL STAVN LCAIG E LS H S V LFA- HL..SEG.TAVRRLAAEAELAL S.GRTWAPGV AL THTL I LTV FFAI.-V::A '.LC.ELGR:REG:T' VA1Y.G:ATASE AI.GAFD S A PIKEE'QO.L. Y EIATREK... CAEVPRELSR PEI ..TRVL MKKFIH HCMV PFKIALAC.SVGRR.RG RYRPEWVTORFPDLTA ADDRDGL RAOW QRCGSTAVDA SG DPFRSDSYGL tLGNS:' SCMV CFKR`VA ,LC:SV G R'.GT LA G.RODW VMER P,P )LTE ADR'EAL'RNOL SGSGEVAAKE SAESSAAAAV D'PFOSDSYGL GNSS VD- EBV FF'DV.S ICALGRRRGT'VLKH.SD.LEPTAVYTDLAW...A.SIAAOI ENDANAAKRE ... ..CPED HPLP LTK. .L IAKA ID: H V S MFQF4g.,V,L.SCL:C.ALG'RRRGT V VYSMN LE D AI-:SOFCS IO A.AEVEN I YODSKNVDIN SL, * ** 142 146 155 165 175 185 195 205 215

R-Site M-Site

VzV NMLL RDRW DVVAKR RREAG',REI.;MG.H VY:LOASTGYG 333... .NAVEA4I.S'SK Varicellazostervirus E H V L.'LR:NR'WNN LVARR R:R:EAGIT::'EG. H T.YLOASASFG. .375.. .OT, DA''AS. Equine herpes virus HSV N M M 'L R.D.R,:WS LV AE'R' $':R,.OQAGI:'AG. H TYLAN.A4SS A... Herpes simplex virus I LTV GAF.'L M::D.RGTC:L;KT.R. REMAWAVVY N.P KY:LQ:AJNEV .310 .ETVDAISM.M Infectious laryngotracheitis virus HCMV ALY I RERLPKLR YD K L V V T E R E SVKSVSPE. .377.. .GV CR. Humancytomegalovirus (AD169) SCMV ALYI OERLPKLRYI3 KLV.GVTAE'AVGV SPA .GV''N"CR. Simiancytomegalovirus(Colburn) EBV AG F;L RN RVET; R OD R`G V AN I P.A. E S Y.LKA SD A P D .3 24.. K L,VO:A4S'AS Epstein-Barr virus HVS 'AG F I KDJRLQLLK TD.'.KGE.V..217...VHIDA'SFA... Herpesvirus simari

* ** * * * * 225 235 245 249

FIG. 9. Amino acid sequence alignment of assemblin homologs. The amino acid sequences of eight herpesvirus assemblin homologs were compared by using the University of Wisconsin program PileUp. Each sequence began at the amino-terminal methionine and stopped at the sissle alanine of the R-site consensus sequence (e.g., Ala-249 for SCMV) (48). The sequence data used were from the following sources: varicella-zoster virus open reading frame (ORF) 33 (VZV) (14); equine herpesvirus ORF (EHV) (44), HSV type 1 UL26 ORF (HSV) (32), infectious laryngotracheitis virus p80 ORF (ILTV) (21), HCMV AD169 UL80a ORF (HCMV) (10), SCMV Colburn APNG1 ORF (SCMV) (46), Epstein-Barr virus BVRF2 ORF (EBV) (2), herpesvirus saimari ORF (HVS) (1). Classification as ox-, P-, or y-herpesviruses is based on the nomenclature of Roizman et al. (38). Shaded residues indicate identity in at least four of the eight sequences shown. Five highly conserved domains, CD] to CD5, are indicated, as are the conserved R and M cleavage sites and the CMV I cleavage site. Numbers between the R and M sites indicate the number of amino acids (not shown) between the two flanking residues. Numbering below the alignment corresponds to the SCMV sequence; the right-hand digit is directly below the indicated SCMV residue. Asterisks are directly below residues in the SCMV proteinase that were mutated.

DISCUSSION homologs and are located in comparatively well-conserved domains of the enzyme. The work here was done to Herpesviruses encode a maturational proteinase that re- reported moves the carboxyl end from an abundant, phosphorylated test these predictions and related questions. capsid protein during assembly (28, 36, 48). Without this Cleavage sites. Experiments done to verify the predicted M cleavage, infectious virus is not produced (35). The proteinase and R sites in the SCMV proteinase precursor demonstrated responsible for this cleavage is encoded by the viral genome, that when their consensus site amino acids were deleted (i.e., and its gene overlaps that of its substrate, the assembly protein VNA , S and YVKA I, S, respectively) the corresponding precursor (27, 28, 36, 46, 48). On the basis of evidence cleavages were eliminated (i.e., pNP1->NP1 and pNP1c-> obtained from experiments done with the SCMV maturational NP1I; NP1->NPI,,+NP1c, respectively). Taken together with proteinase, assemblin, a working model of its synthesis and results obtained using other M- and R-site mutants, discussed processing was proposed and generalized to other herpes below and in the Results section, these findings support the group viruses (48). Two key predictions of the model were that predicted cleavage sites. Direct amino acid sequence analyses (i) the proteinase precursor is cleaved at a highly conserved of the HSV M and R sites (16) and of the HCMV R site (5) 5-amino-acid consensus sequence near its midpoint to yield the have confirmed their predicted sequences and validated the mature proteinase, assemblin, and (ii) enzymatically important generalization of M- and R-site cleavage to other herpesvi- amino acids are conserved among the herpesvirus assemblin ruses. Both the M-site and the R-site deletion mutants were VOL. 67, 1993 HERPESVIRUS PROTEINASE 7369

was prevented, supporting this sequence as the SCMV protein- A. < < ,-, ase I site. The I-site deletion mutant had the interesting phenotype of cleaving well at the M site but having a reduced v < Z < < C L.0 (.0 L, co % ability to cleave at the R site. One explanation of this apparent <0 00 ,,.,,(>,C:2 1o o o _ xLN N 2.selectivity is that the deletion, which is in a possible loop- b - insertion region (i.e., variable in length and amino acid se- pNPI- --- -ob,,m- .-.40wm -0-42bdft %3pNP- NP1- quence) between two highly conserved domains and near the essential CD3 serine, decreases the size of the substrate binding pocket such that the R site with its large P4 tyrosine is more difficult to accommodate than the M site, which has a P4 pNPI c- valine. NP1 c- - Other mutations in the R site and the M sites of both the AULO." AL-mis, - Aak-'a, -4Ii -pAP proteinase and the assembly protein precursor gave further *.AP insight into these recognition-cleavage sequences. The importance of the absolutely conserved P4 tyrosine and P1 alanine of 1 2 10 3Z 13 14 16 17 18 19 20 21T the R site was demonstrated by the low level of cleavage observed at this site when either residue was mutated. Inter- B. estingly, the Ala- Gly substitution appeared to be better <~~~ tolerated at the P1 position of the M site than at the P1 position of the R site (i.e., the mutant M site cleaved better LL < Z V~~~~~~~~L)<_ < < C v v 0 _ < L: than the mutant R site), suggesting that the recognition or n o 0 o tD0tD1 c00o CD rw 0 0) '- C C]X Q .,(, s t- 0E cleavage characteristics of the two sites differ. Even though the < Lx LI C) :EV)vrm `:, P1' serine is conserved in all but 1 of the 15 known R- and - M-site its mutation to had pNPI- £ \3pNPI sequences (48), glycine remarkably NPI1- _- I_ I ~ little effect on cleavage at either site, consistent with the generally greater importance of the P residues than the P' residues for serine and cysteine proteinases (34). A surprising phenotype was observed when the P3 lysine of the R site was pNPl c- changed to an asparagine, the P3 residue of the M site. Unlike -- NP1 c- 4bu_~ the P4 and P1 mutations, which reduced R site cleavability, this

1 2 5 P3 did not affect R-site 3 to 3 4 5 6 7 8 9 10 1112 13141 16 17 18 19 20 21 change noticeably cleavage (Fig. FIG. 10. Site-directed mutagenesis impliicates absolutely conserved 5), presumably because the mutation simply converted the R His-47 and Ser-1 18 as essential for prote(olytic activity. Amino acid site to an M site with a P4 tyrosine. Surprisingly, however, the substitutions were made for specific residue of the SCMV proteinase, mutation did reduce M site cleavage (e.g., producing compar- as described in the text. The mutant constr ructs were transfected with atively less pNPI-NPl and pNP1I-.>NP1I [Fig. 3A, lane 8; the assembly protein precursor gene AW] 1 (A) or alone (B), trans- Fig. 4, lanes 26 and 46]) and drastically lowered I-site cleavage fected cell proteins were separated by SD' S-PAGE in 10%° bis-cross- (e.g., producing an increased amount of NPI, and little if any linked gels, and the proteins were probe ad with anti-NI following 13-kDa A, [Fig. 3B, lane 24] [data not shown for AJ]). This electrotransfer to Immobilon. Shown here are fluorographic (A) and result suggests that the carboxyl end of assemblin can influence autoradiographic (B) exposures of the re suiting immunoblots. The substrate specificity or catalytic activity, or both, especially that plasmid(s) used for transfection are indicat ed above each lane. Muta- pertaining to the I site. In line with this observation,extension tions are designated as explained in the leg( and 4 double mutations (E20A/E22E, C146A/S 47 A,StoFA/SI1iA, S198A/ (addition of 12 amino acids) or deletions (of 3 or 8 amino Si99A) were analyzed; protein designations ((see Fig.1) are indicated in acids) of the carboxyl end of the HCMV assemblin homolog the left and right margins; and A3pNPl in dicates the position of the that eliminate or alter the R site yield mutant enzymes able to pNP1 band of the mutant, LM3. The asteri sk indicates the position of cleave the M site but unable to cleave at the I site (5). Cleavage the 68-kDa band also seen in Fig. 3 and 4. activity against both the M and I sites was lost when the 6-amino-acid sequence MHWHWH-C' was added to the carboxyl end of the HCMV assemblin homolog (41a). Many proteinase precursors have internal cleavage sites. able to cleave at their intact sites and in trans at the assembly These may be used to activate an inactive zymogen (22-24), protein M site. Similarly, an HSV type 1 proteinase mutant in alter the structure of the enzyme so that its substrate specificity which the R-site sissle pair, Ala-Ser, wa s replaced with Arg-Pro is changed (15, 49), or influence the rate of cleavage (i.e., was able to cleave at its intact M ,ites (30). These results product fast and slow cleavage sites) as a mechanism to control provide direct evidence that the prediicted M and R sites are the relative time of appearance and the relative amounts of the utilized during processing of the prot teinase precursor to its different cleavage products (17). Data shown here for the mature form, assemblin, and that tl he precursor does not CMV M, R, and I sites and elsewhere for the HSV R site (30) require activational cleavage at either site. indicate that the precursor is not a zymogen that requires A new antiserum, anti-C2 (Fig. 1), to the carboxyl end of cleavage for activation. Our data also indicate that R-site assemblin enabled us to visualize the m ature proteinase and to cleavage is not dependent upon preceding M-site cleavage, and recognize that it, like its HCMV counte -rpart, is cleaved in half. vice versa. With regard to the possibility that the different The site at which this cleavage occursi] n HCMV was identified cleavage site sequences influence the time of appearance or as VEAt AT (5, 7) and has a loos,ely conserved possible the amount of product, our data are consistent with a kineti- counterpart in SCMV but not in the o ther herpesviruses (Fig. cally ordered cleavage of M > R > I. Products of all three 9). By deleting the 5-amino-acid pL itative I site of strain cleavages appeared rapidly (within 15 min) and a 5-min pulse Colburn (INAt AD), cleavage of as isemblin (NP1n) to its radiolabeling period was required to discriminate between M- amino (An: -13.7-kDa) and carboxyl (A,: -13.4-kDa) halves and R-site cleavage in this transfection system. Contrary to the 7370 WELCH ET AL. J. VIROL. conclusion drawn from work done with the HCMV proteinase tial (30) (Fig. 10, lanes 16 through 18). Moreover, initial (5), our data indicate that assemblin (NP1n) is not metaboli- substitutions of each of the other four cysteines in SCMV cally stable and that the 13.7-kDa (predicted amino half, An) assemblin indicate that none is absolutely required for activity and 13.2-kDa (predicted carboxyl half, A,) fragments arise (41b). The observation that high concentrations of Zn2+ directly from its cleavage (Fig. 7). Consistent with this finding, inhibit the HCMV (5, 7) and SCMV (our unpublished results) the HCMV and SCMV assemblin proteins synthesized in enzymes was interpreted to indicate that the HCMV enzyme bacteria are also cleaved at their I sites to give the expected has an active-site cysteine (5). An alternate explanation for this fragments (reference 7 and our unpublished results, respec- finding, considering the apparent absence of an essential tively). In addition, because little I-site cleavage (evidenced by conserved cysteine, is that the enzyme has a metal-binding the 68-kDa band) was observed in the "tightest" R-site mu- domain that when occupied inhibits activity. Such an interac- tants (e.g., Y246-A249 - and Y246A/A249G [Fig. 3]), we conclude tion could provide a regulatory mechanism to keep assemblin that I-site cleavage proceeds more efficiently following R-site or its precursor forms inactive until needed. cleavage. If so, this may indicate that conformational changes The catalytic mechanism of known serine proteinases in- around the I site, rather than the sequence itself, determine its volves an active-site triad consisting of a serine, a histidine, and susceptibility to cleavage. It is important to emphasize that the an aspartic acid. Serine 118 of CD3 has been identified here as kinetics and order of cleavages may be very different in a potential nucleophile of such a triad in assemblin. The CD2 infected cells than in cells transfected or transformed with the histidine (His-47 in SCMV) would qualify as a second residue proteinase gene alone. of the triad on the basis of its absolute conservation among the Active-site mutations. We previously identified two highly herpesvirus assemblin homologs and because it is essential for conserved domains, CD1 and CD2, and several amino acids activity in both the SCMV (Fig. 10, lanes 6 and 7) and HSV (i.e., His-47 and 142, Cys-146, Asp-104 and Ser-195 [Fig. 9]) in (29) enzymes. Although there is one other absolutely con- SCMV assemblin as potential active-site components. A more served histidine in assemblin (i.e., CD1 His, residue 142 in recent alignment of the assemblin homologs (Fig. 9) has SCMV), and it was found to be essential for activity in the HSV revealed an absolutely conserved serine within a third highly enzyme (30), it is not essential for activity in the SCMV conserved domain called CD3 (e.g., Ser-1 18 of SCMV). These enzyme (Fig. 10, lanes 14 and 15), thereby making the CD2 His and other amino acids with the potential to contribute to a stronger active-site candidate. No absolutely conserved as- known proteolytic active sites were changed by site-directed partic acid was apparent from the alignment shown in Fig. 9; mutagenesis as a means of determining their importance to the however, several positions did show a high conservation of catalytic function of the proteinase. The underlying rationale aspartic acid (e.g., CD5 Asp-15 in SCMV) or of an acidic of the experimental approach was that functionally critical charge (e.g., Asp or Glu). Four of these residues were mutated, amino acids (e.g., active site residues) will be highly conserved but none eliminated activity in the SCMV enzyme (Fig. 10). Of within a closely related group of enzymes and that mutating the four substitutions made, the double mutation such residues will inactivate the enzymes. Amino acid changes Glu20,Glu22->Ala20,Ala22 and the single mutation Glu- to alanine or to a conservative replacement (e.g., Asp-*Asn) 22->Ala-22 had the most adverse effects on activity. It is were used to minimize the possibility that the substitutions noteworthy that the E22A mutation, alone or together with would cause overall structural distortions (4, 13). E20A, completely inhibited R-site cleavage without apparent Of the 14 amino acids mutated, only changes at the abso- reduction of either M- or I-site cleavage (Fig. lOB, lanes 4 and lutely conserved histidine 47 of CD2 and serine 118 of CD3 5), suggesting a role for this highly conserved residue in eliminated activity of the proteinase in our assay. CD3 serine determining R-site specificity. Substitution of the most highly 118 (in the SCMV sequence) is the only absolutely conserved conserved aspartic acid (e.g., D15A or D15N in SCMV) had serine in assemblin. The finding that it is essential for activity little effect on proteolytic activity in either the SCMV (Fig. 10) makes it the strongest candidate active-site nucleophile so far or the HSV (29) enzyme. And although substitution of Glu-115 identified and adds to the evidence that the herpesvirus (doubly mutated with Glu-114 or E114G/E1I5A) eliminated proteinase is a member of the serine proteinase superfamily. detectable proteolytic activity of the HSV enzyme (30), substi- Other evidence comes from inhibitor studies which showed tutions of the corresponding residue of the SCMV proteinase that high concentrations of phenylmethylsulfonyl fluoride (25 (i.e., D104A or N) did not eliminate activity. Our inability to mM), a specific inhibitor of serine proteinases, inhibited the identify a strong candidate aspartic acid for the third member HSV type 1 enzyme (29) and lower concentrations inhibited of a possible serine proteinase active-site triad by this tech- the HCMV (5) and SCMV (our unpublished results) enzymes. nique is perhaps not surprising, given that this member of the Diisopropylfluorophosphate, another serine proteinase inhib- triad tends to be less well conserved than the Ser and His itor, also reduced the activity of the HSV (29) and HCMV (5, residues and also to have a weaker adverse effect on activity 7) enzymes. Although the possibility that the herpesvirus when substituted with another amino acid (8, 12). It is worth enzyme belongs to one of the three other proteinase super- noting that when substitutions similar to those used here were families (i.e., aspartic, metallo, or cysteine) cannot be ruled made in active-site triad residues of both subtilisin (8) and out, the following evidence suggests this is unlikely. First, trypsin (12), both enzymes retained measurable proteolytic unlike aspartic proteinases which have acidic pH optima, that activity, albeit reduced 3 to 6 orders of magnitude. Thus, it is of the CMV proteinase is neutral to slightly alkaline (7). In possible that apparent discrepancies in the results for the addition, an absolutely conserved aspartic acid has not been SCMV and HSV enzymes mutated at the CD2 His and at the recognized among the herpesvirus assemblin homologs. Sec- highly conserved Glu/Asp (i.e., Glu-104 in SCMV and Glu-115 ond, unlike the activity of metalloproteinases, which are typi- in HSV) may be explained by sensitivity differences in the assay cally sensitive to metal-chelating agents, activities of the HSV systems, or in the nature of the amino acid substitutions made. (29), HCMV (5, 7), and SCMV (our unpublished results) If further analyses confirm that the absolutely conserved enzymes were not reduced by the metal chelator, EDTA. And serine of CD3 is the assemblin nucleophile, the herpesvirus third, unlike cysteine proteinases, which would be expected to proteinases would appear to represent a new subclass of serine have an absolutely conserved essential cysteine, the only proteinases, since they do not contain the characteristic se- absolutely conserved cysteine (Cys-142 in SCMV) is not essen- quence motifs found near the active-site serines (or cysteines) VOL. 67? 1993 HERPESVIRUS PROTEINASE 7371 of the chymotrypsin-like or subtilisin-like proteinases (i.e., 16. Dilanni, C. L., D. A. Drier, I. C. Deckman, P. J. McCann III, F. G-X-S/C-G-G or G-T-S-M/A, respectively [3, 6, 20]). Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley. 1993. Identification of the herpes simplex virus-I protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products. J. ACKNOWLEDGMENTS Biol. Chem. 268:2048-2051. We thank Jennifer Ludford for use of the data shown in Fig. 7 and 17. Dougherty, W. G., and T. D. Parks. 1989. Molecular genetic and 8 and Rebecca Magno and Jenny Borchelt for excellent technical biochemical evidence for the involvement of the heptapeptide assistance. We also thank our colleagues at Lilly Research Laborato- cleavage sequence in determining the reaction profile at two ries for their help in making primers, sequencing mutant DNAs, and tobacco etch virus cleavage sites in cell-free assays. Virology growing plasmid stocks and for the gift of anti-C2. 172:145-155. This work was aided by research grants from the National Institutes 18. Fairbanks, G., T. L. Steck, and D. F. Wallach. 1971. Electro- of Health (Al 13718 and Al 22711) and from Eli Lilly and Co. A.R.W. phoretic analysis of the major polypeptides of the human eryth- was a student in the Pharmacology and Molecular Sciences training rocyte membrane. Biochemistry 10:2606-2617. program and was supported by PHS grant GM0726. M.R.T.H. is a 19. Gibson, W., A. Marcy, I. J. C. Comolli, and J. Lee. 1990. student in the Biochemistry, Cellular, and Molecular Biology training Identification of precursor to cytomegalovirus capsid assembly program and was supported by PHS grant GM07445. protein and evidence that processing results in loss of its carboxy- terminal end. J. Virol. 64:1241-1249. REFERENCES 20. Gorbalenya, A. E., A. P. Donchenko, V. M. Blinov, and E. V. 1. Albrecht, J.-C., J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, Koonin. 1989. Cysteine proteases of positive strand RNA viruses C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. and chymotrypsin-like serine proteases. A distinct protein super- Fleckenstein, and R. W. Honess. 1992. Primary structure of the family with a common structural fold. FEBS Lett. 243:103-114. herpesvirus saimiri genome. J. Virol. 66:5047-5058. 21. Griffin, A. M. 1990. The complete sequence of the capsid p40 gene 2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, from infectious laryngotracheitis virus. 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