JOURNAL OF VIROLOGY, Dec. 1993, p. 7360-73720022-538X/93/127360- 13$02.00/0Copyright © 1993, American Society for Microbiology

Herpesvirus Proteinase: Site-Directed Mutagenesis Used ToStudy Maturational, Release, and Inactivation Cleavage

Sites of Precursor and To Identify a PossibleCatalytic 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 HopkinsUniversity 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 threeprocessing cleavages. Two of these were predicted to be at highly conserved consensus sequences-one near thecarboxyl 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 alsoidentified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have usedsite-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, andhave shown that the proteinase precursor is active without cleavage at these sites. We have also shown that theP4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-siteresidues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site butnot at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage andnearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, allR-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 unstableand is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, theenzymatic 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 Mand I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., proteinconformation) role for this residue in determining substrate specificity.

Assembly of the herpesvirus capsid includes a maturationalproteolytic cleavage that appears to be an essential step in theformation of infectious virions (35). One target of this cleavageis the procapsid assembly protein precursor which loses a smallportion of its carboxyl end during the process (19, 28, 48). Theproteinase responsible for catalyzing this reaction is encodedby a viral gene that is conserved among the herpes groupviruses and contains the gene for its substrate, the assemblyprotein precursor, as its nested, in-frame, 3'-coterminal half(28, 36, 48).By using insertion and deletion mutants to study the cyto-

megalovirus proteinase in transient transfection assays andplasma desorption mass spectrometry to determine its cleavagesite in the mature assembly protein, we showed the following(48). (i) The enzymatic portion of the proteinase is containedwithin the amino half of the full-length molecule and includestwo highly conserved domains, referred to as CDI and CD2.This finding has been confirmed for both herpes simplex virus(HSV) and human cytomegalovirus (HCMV) (5, 7, 30, 47). (ii)Maturational cleavage at the carboxyl end of the assemblyprotein precursor is between Ala and Ser in the consensus

sequence V/L-X-A I S. This sequence is called the matura-

Corresponding author.t Present address: Syntex Discovery Research, Institute of Biochem-

istry and Cell Biology, Palo Alto, CA 94304.t Present address: Department of Microbiology, Medical College of

Wisconsin, Milwaukee, WI 53201.

tional cleavage site (M site) and is highly conserved in thehomologous proteins of other herpes group viruses (48). And,(iii) the proteinase precursor is cleaved twice-once at its Msite, present near its carboxyl end as a consequence of thenested relationship of the proteinase and assembly proteingenes (27, 46) (Fig. 1) and once at a site called the releasecleavage site (R site) near the middle of the proteinaseprecursor (48). The R site was predicted to be between Ala andSer in the consensus sequence Y-V/L-K/Q-A I S near themiddle of the proteinase and, like the M-site cleavage se-

quence, is highly conserved among the homologs of otherherpes group viruses (48). Amino acid sequence analyses ofbacterially synthesized products of the proteinase have shownthat cleavage occurred at the predicted sites and demonstratedthe fidelity and utility of the bacterial system (5, 16). Recently,a third cleavage site with the sequence VEA l AT has beenidentified near the middle of the HCMV 28-kDa assemblinhomolog, between CDI and CD2 (5, 7). It has been proposedto inactivate the enzyme and therefore is called the inactiva-tion site (I site).The studies reported here had four objectives. The first was

to substantiate that the predicted YVKA I1 S sequence is the Rcleavage site and to investigate the importance of specificresidues in both the R and M cleavage sites. The second was todetermine whether cleavage at the R site is required to activatethe proteinase. The third was to determine whether the simiancytomegalovirus (SCMV) proteinase has an inactivation cleav-age site similar to that identified for HCMV (5, 7). And, the

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HERPESVIRUS PROTEINASE 7361

R-siteCD2 CD3 CD1 tC.31

ml M281 ly

C3I l I II 7 I-

LM8 + I l. I I m IYVKA 249

M 281

rb APNG.5

fourth was to test specific amino acid residues within theM-site proteolytic portion of the molecule for their possible involve-

ment in the catalytic site of the enzyme. Our approach was toiNA

introduce specific changes by site-directed mutagenesis and557 then determine the phenotypic effect of the change by using a

transient transfection-based assay.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 differencebetween these studies are discussed.

Progress reports of this work have been presented at theGordon Conference on Proteolytic Enzymes and Their Inhib-

lZJ itors, 8 to 12 June 1992; the Annual Meeting of the AmericanKME%MO Society for Virology, 11 to 15 July 1992; the 17th International

-TAA Herpesvirus Workshop, 1 to 6 August 1992; and the 4thInternational Cytomegalovirus Workshop, 19 to 21 April 1993.

Base pairs i m

DNA 0 200 400 600 800 1000

r- inI *Il

A 14 kDa I

A,I 13dDa idcl

1200 140() 1600 1800

B.

ProposedInactivation

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III C21 NiI Ew

II 50 kDa II~ eI In 46 kDa

_ I

pNP1, II 37 kD)a1 EM

NP1,-

Assembly Protein:

ADC0 OL 3Okf'a

- ~~~~c,'-IS4 kDa

FIG. 1. SCMV proteinases and assembly protein precursor genes,and their protein products. (A) The four principal genes used in thesestudies, their plasmid designations, and proteolytic activities. Mutantsof the proteinase were derived from plasmid AW4, and mutants of theassembly protein precursor were derived from plasmid AWI. Theoverlapping relationship of the genes encoding the proteinase(APNG1) and the assembly protein precursor (APNG.5) is indicatedat the bottom of the panel. The R and M cleavage sites are indicatedby arrows; three of the five conserved domains (CD1 to CD3) areindicated by shaded rectangles; a 15-amino-acid epitope (C3) insertedinto the amino half of LM3 is indicated by a solid rectangle; thetranslational start methionines of the proteinase (M-1) and theassembly protein precursor (M-281) and the carboxyl-terminal resi-dues of assemblin (YVKA), the mature assembly protein (VNA) andits precursor (KME) are also indicated. (B) The full-length proteinaseprecursor, pNPI, its three principal cleavage sites, and the proteinproducts expected from cleavage at these sites. Also shown are theassembly protein precursor, pAP, and the two products of its cleavage atthe maturational site. The designation of each protein is shown to theleft of the line representing it, and the computer-predicted size of eachis indicated. The carboxyl-terminal residues of the mature assemblyprotein (VNA), assemblin (YVKA), and the amino half of I-site-cleavedassemblin (INA) are indicated at the top. Antisera were made tosynthetic peptides representing the amino (NI) and carboxyl (Cl) endsof the assembly protein precursor (i.e., anti-Ni and anti-Cl, respective-ly), and the carboxyl end of assemblin (NP1I) (C2) (i.e., anti-C2).

MATERLALS AND METHODS

Construction of mutants. Standard techniques were used toconstruct, clone, and propagate the plasmids (39). The assem-bly protein nested gene 1 (APNG1, which encodes the full-length proteinase precursor, pNP1) and APNG.5 (which en-codes the assembly protein precursor, pAP) genes weresubcloned from the plasmids AW4 and AWl (48), respectively,into the plasmid M13mpl8. Mutations were introduced byusing a kit (RPN1523; Amersham, Arlington Heights, Ill.)based on the primer-directed mutagenesis technique describedby Eckstein and coworkers (42, 43). The mutations wereconfirmed by dideoxy sequencing (40) with the modified T7polymerase, Sequenase (United States Biochemical Corp.,Cleveland, Ohio). Mutant genes were subcloned fromM13mpl8 into pRSV.neo (31) for analysis in transient trans-fection assays as follows. (i) Single-stranded DNA was isolatedfrom Escherichia coli that had been infected with the mutantconstructs in Ml3mpl8 (33). (ii) Five micrograms of single-stranded DNA was annealed to 1 pmol of the M13 universalprimer (United States Biochemical Corp.). (iii) Double-stranded DNA was synthesized in vitro by incubating theannealed single-stranded DNA with Sequenase buffer (40 mMTris [pH 7.5], 20 mM MgCl2, 50 mM NaCl), 100 ,uM eachdeoxynucleoside triphosphate, 10 mM dithiothreitol, and 1.7 Uof the modified T7 polymerase, Sequenase (United StatesBiochemical Corp.), at 37°C for 15 min. (iv) The resultingdouble-stranded DNA was then phenol-chloroform extracted,ethanol precipitated, and dried. (v) The resulting double-stranded DNA was suspended in buffer and cleaved withappropriate restriction enzymes (i.e., SalI-BamHI for APNGIand APNG.5) to release the desired fragment for subcloning.And, (vi) the excised fragments were then ligated into pRSV-.neo that had been cleaved with Sall and BamHI, and theresulting construct was transformed into DH5cx E. coli (lackingF' and nonpermissive for M13) and selected by replication inthe presence of ampicillin. To insure that only the intendedsite-directed change was present in mutants of putative active-site amino acids, a small DNA restriction fragment containingthe mutation was excised (i.e., mutations of D-15, E-20, E-22,and H-47 in a SalI-EcoR5 fragment; of D-104, S-118, S-120,and S-121 in an EcoR5-Kspl fragment; and of H-142, C-146,and S-147 in a KspI-Eco47III fragment), substituted for thecorresponding fragment of the wild-type gene in AW4, andsequenced (including the flanking insertion site region).LM3 was derived from the plasmid AW4 and encodes a

minimally active proteinase that contains a 15-amino-acidinsert (C3, poliovirus epitope) between conserved domains 1and 2 (CD1 and CD2) (see Fig. IA and reference 48).

A.ProteoIytib

PEsd A

AW4 +

LM3

AW1

SCMV Genes r*APNG1

Proteinase:

- 60 kDa

27 kDa I Assentln -

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7362 WELCH ET AL.

Transfection assay. Approximately 1 x 106 human embry-onal kidney (HEK) cells (line 293; American Type CultureCollection) were seeded onto Lab-Tek 2-well slides (Nunc,Inc., Naperville, Ill.) or into 2-cm2 wells (3047; Becton Dick-inson Labware, Oxnard, Calif.) on day 1 and then transfectedwith the appropriate plasmids (2 ,ug of DNA total per well) onday 2 by a modified calcium phosphate technique (11). Aplasmid (RSV.TAg) encoding simian virus 40 T antigen wasadded to each transfection (0.2 ,ug per transfection) to increasethe copy number of the RSV.neo-derived constructs (37). Cellswere harvested 48 to 72 h after transfection (as indicated in thefigure legends) by aspirating the medium, adding 60 to 70 [L of2 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) sample buffer to the cell layer, and removing theviscous lysate by pipette and heating it for 3 min in a boilingwater bath. Samples were stored at - 80°C until analyzed bySDS-PAGE.SDS-PAGE and Western immunoassay. SDS-PAGE was

done essentially as described by Laemmli (25); the ratio ofN,N'-methylenebisacrylamide (bis) or diallyltartardiamide toacrylamide was 0.735:28 or 1.09:28, respectively; SDS was fromBio-Rad. SDS-PAGE sample buffer contained 2% SDS, 5%beta-mercaptoethanol, 10% glycerol, 25 mM Tris (pH 7), and0.01% bromophenol blue.

Electrotransfer was done essentially as described by Towbinet al. (45). A semidry transfer unit was used, the membranewas Immobilon P (Millipore, Bedford, Mass.), the buffer was50 mM Tris-20% methanol, and the time of transfer wascalculated by the formula gel width x height x 2.5 =milliamperes per 30 min. The membrane was blocked in 5%bovine serum albumin, reacted sequentially with antiserumand then with "2'I-protein A, and exposed to X-ray film,usually with a calcium tungstate intensifying screen (26).The antisera used were prepared by injecting rabbits with

keyhole limpet hemocyanin-conjugated synthetic peptides (41)representing the carboxyl 13 residues of the assembly proteinprecursor (anti-Cl), the amino 21 residues of the assemblyprotein (anti-Ni), or the carboxyl 14 residues of assemblinNPln (anti-C2) (Fig. 1).

Pulse-chase radiolabeling and immunoprecipitation. Twodays after transfection, the culture medium was replaced withmethionine-free medium (7732-18-5; GIBCO, Grand Island,N.Y.) containing 50 pCi of [35S]methionine per ml. Fifteenminutes later, the radiolabeling medium was removed from allcultures; one was harvested immediately (pulse) by aspiratingthe medium, adding 150 RI of lysis buffer (1% Nonidet P-40,0.5% deoxycholate, 0.5 M KCl, and 1 mM phenylmethylsulfo-nyl fluoride in phosphate buffer, pH 7.4), recovering the lysatefrom the well, vortexing it, and freezing it at - 80°C. Labelingmedium on the remaining cultures was removed and replacedfour times (5 min each time) with normal growth medium todilute the intracellular pool of unincorporated radiolabel.Additional cultures were harvested in the same way at 30 minand at 1, 2, 4, 8, 12, and 22 h after the pulse radiolabelinginterval. Cells transfected with only the plasmid encoding Tantigen were processed in parallel and harvested after thepulse and 22-h time points. Prior to immunoprecipitation, thelysates were thawed, vortexed, and clarified by centrifugation(12,000 x g, 10 min, 4°C). A similar experiment was also donewith a 5-min pulse or radiolabel and chase periods of 5, 10, 15,and 30 min.

Immunoprecipitations were done by combining 50 ,ul ofclarified lysate with 20 [lI of antiserum anti-Ni, anti-Cl, oranti-C2 (described above and in the legend to Fig. 1). Incuba-tion of lysates with antisera was done at room temperature(-24°C) for 90 min. Fifty microliters of protein A beads

(P3391; Sigma) (100 mg/ml of phosphate buffer, pH 7.4) wasadded to each tube, and incubation was continued for 60 minat room temperature. The reacted beads were collected bycentrifugation (12,000 x g, 4°C, 1 min), washed four times inimmunoprecipitation buffer (lysis buffer with no KCl), com-bined with an equal volume of 2 x SDS-PAGE sample buffer,heated in a boiling water bath for 3 min, and stored at - 80°Cuntil analyzed. Following SDS-PAGE, gels were stained withCoomassie brilliant blue (18) and destained, and proteins werethen detected by fluorography following sodium salicylateenhancement (9).

RESULTS

Four sets of mutations were made by site-directed mutagen-esis: one within the release recognition-cleavage sequence (Rsite, YVKA249 l S) near the middle of the proteinase precur-sor, a second within the maturational recognition-cleavagesequence (M site, VNA5.6 S5) near the carboxyl end of theproteinase, a third in the homologous M site at the carboxylend of the assembly protein precursor, and a fourth within theproteolytic domain of assemblin (i.e., residues 1 to 249). Adeletion mutation was also made in the putative inactivationsequence (I site, INA127 1 AD) near the middle of the assem-blin.

Proteinase mutants were transfected (i) alone, to test theirmetabolic stability and ability to self cleave, (ii) together withAW1 (which encodes the assembly protein precursor), to testtheir ability to cleave the M site in trans, (iii) together withLM3 (which encodes enzymatically inactive proteinase), to testtheir ability to cleave the R site in trans, or (iv) together withLM8 (which encodes assemblin, NP1n), to test their ownsusceptibility to cleavage in trans. Mutants of the assemblyprotein precursor were transfected (i) alone, to test theirmetabolic stability, or (ii) together with LM8 or AW4 (whichencodes assemblin precursor pNP1) to test the effect of themutations on M-site recognition and cleavage. Transfected celllysates were subjected to SDS-PAGE, and proteins of interestwere identified by Western immunoassays with antisera to theamino end or carboxyl end of the assembly protein precursor,pAP (i.e., anti-Ni and anti-Cl, respectively) or to the carboxylend of assemblin, NP1, (i.e., anti-C2) (Fig. 1).Landmarks of the proteinase and its assembly protein

substrate are shown in Fig. 1. The amino acids of the assemblyprotein have been numbered according to their positions in thelongest open reading frame (i.e., APNG1) with which it is inframe, overlapping, and 3' coterminal; thus, the translationalstart methionine for the assembly protein is methionine 281.

Cleavage at the R site is not required for proteolytic activity.One deletion and five substitution mutations were made in thepredicted R-site consensus sequence, YVKA 4 S, of the cyto-megalovirus (CMV) Colburn assemblin precursor, pNP1 (48).As described here, four of these eliminated or greatly de-creased R-site cleavage as measured by (i) increased amountsof the noncleaved precursors, pNP1 and NP1, and (ii) reducedamounts of the expected cleavage products, assemblin (NP1.)and NP1c.

Deletion of the entire R-site consensus sequence (Y246-S250- ) eliminated R-site cleavage and resulted in the accumu-lation of the assemblin precursor, NP1, and the absence of itscleavage products, NP1c (Fig. 2; Fig. 3A, lane 12; and Fig. 4,lane 30) and assemblin (NPln, Fig. 3B, lane 28). Substitution ofalanine for the P4 tyrosine (Y246A) strongly inhibited cleavageat the R site, but small amounts of assemblin (NPln) and NP1cwere detected (Fig. 2; and Fig. 3, lanes 6 and 23). A lesscomplete inhibition of R-site cleavage was observed when

J. VIROL.

HERPESVIRUS PROTEINASE 7363

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pNP1 oNP1

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R-site mutations were made: a deletion mutant (Y246-S250-) thateliminated all 5 amino acids in the R-site consensus sequence and asubstitution mutant (Y246A) in which Tyr-246 was changed to an Ala.HEK cells were transfected (-48 h) with the mutant genes, eitheralone ( - ) to test the enzyme's self-cleavage properties, with anassembly protein precursor (+Sub., i.e., with the AWl construct) totest the mutant enzyme's ability to cleave substrate in trans, or withassemblin (+Enz., i.e., with the LM8 construct) to test its ability to becleaved in tracns at its release site. Transfected cells were processed andsubjected to SDS-PAGE in a 10Cc bis-cross-linked gel followed byWestern immunoassay using anti-NI. Shown here is an autoradio-graphic exposure of the resulting Immobilon membrane. Protein desig-nations are indicated in the left margin. Nuclear and cytoplasmicfractions of Colburn-infected cells were included to provide referenceproteins. LM3 was included as a positive control substrate to verify thatLM8 was active in the cotransfections, and to provide a marker for NP1c.Open circles to the left of Y246A lanes indicate the positions of pNP1,NP,, and NP1,.

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glycine was substituted for the P1 alanine (A249G) (Fig. 3,lanes 9 and 25; Fig. 4, lanes 27 and 37). When both of thesesubstitutions were combined in the same mutant (Y246A/A249G), R-site cleavage was inhibited almost as completely asin the Y246-S25o- deletion mutant (Fig. 3, lanes 11 and 27; andFig. 4, lanes 29 and 39), but prolonged fluorographic exposurerevealed trace amounts of NP1C not seen in the deletionmutant (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 approxi-mated those in cells transfected with the wild-type proteinasegene (Fig. 3, lanes 10 and 26; and Fig. 4, lane 28). The findingthat each of these five mutant proteinases was active incleaving at its own M site (i.e., conversion of pNPl to NP1) andat the M site of the assembly protein precursor (i.e., conversionof pAP to AP) (Fig. 2; and Fig. 4, lanes 45 and 47 through 50)demonstrates that R-site cleavage is not absolutely requiredfor proteolytic activity. These data also show that some of theR-site mutants, A249G in particular, gave rise to a 68-kDa band(discussed below) not typically seen (for examples, see Fig. 3,lanes 9 and 25 [asterisks]). It is likely that this band corre-sponds to the computer-predicted 46-kDa protein indicated inFig. lB and represents pNP1 cleaved at both its M and I sitesbut not at its R site (i.e., observed sizes of -86 kDa [pNP1] -4 kDa [C'] - 14 kDa [A,j] = -68 kDa).To test the susceptibility of the R-site mutations to cleavage

in trans, they were used as substrates in cotransfections withwild-type assemblin (i.e., LM8 plasmid). Although the pres-ence of wild-type assemblin did not appear to significantlyincrease the relative extent of cleavage at the mutant R sites, itdid reduce the relative amount of pNP1,. in mutant K248N (Fig.4, lane 36) and increase the relative amount of the 68-kDaband in at least four (Fig. 4, lanes 35 and 38 through 40) of thesix R-site mutants.

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FIG. 3. Self-cleavage properties of R- and M-site proteinase mutants. R- and M-site proteinase mutants were tested for self-cleavage bytransfection and Western immunoassay. 72-h-transfected cell proteins were separated by SDS-PAGE in a 10% bis-cross-linked gel, and reactionswere 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 andcytoplasmic fractions of Colburn-infected cells (Col. Nuc. and Col. Cyto.) are shown for reference. Circles indicate bands corresponding to theprotein designations; asterisks denote the 68-kDa band referred to in text.

VOL. 67, 1993

"O

7364 WELCH ET AL.

E

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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 theproteins 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). Specificprotein bands are indicated in the left and right margins; transfecting plasmids are indicated above each lane; mutations are designated by theamino 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 indicatecorrespondence to protein designations in the right margin. Asterisks identify two cross-reacting host cell proteins; the -68-kDa band denoted withan 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 ina 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 asparaginefor P2 lysine (K248N), had a comparatively unusual phenotype.It cleaved well at the R site (i.e., it had a strong NPlc band anda comparatively weak NP1 band), but it left relatively morepNP1 and pNPlc than the other R-site mutants, indicating areduced cleavage efficiency at its own M site (Fig. 3, lanes 8and 24; and Fig. 4, lanes 26, 36, and 46). Compared with thewild-type enzyme, this mutant also showed reduced cleavage intrans of the assembly protein precursor M site (Fig. 5, lane 12).Additionally, the amount of NP1n was generally higher withthis mutant than with the wild type (i.e., AW4 and LM8) or theother R-site mutants (Fig. 3B). It is unlikely that this pheno-type is due to a second site mutation, since the same resultswere obtained with three additional K248N mutants that werederived from an independent mutagenesis experiment andfrom a recombinant (K248Nb in Fig. 5, lanes 13 and 14)constructed by replacing a 703-bp nucleotide sequence of thewild-type proteinase gene with the corresponding but alteredsequence from the original K248N mutant gene.The relative inhibitory effect (strongest to weakest) of these

mutations on R-site cleavage was estimated to be Y246-S250-> Y246A/A249G > Y246A > A249G > S25oG > K248N.Cleavage at the M site of proteinase precursor, pNP1, is not

required for proteolytic activity. Two deletion and threesubstitution mutations were made in the M-site consensus

sequence, VNA k S, of pNPI. Deletion of the entire M site(V554-S557-, Fig. 5, lane 4) or of alanine at the M-site sisslebond (Fig. 3, lane 16; and Fig. 4, lane 34) eliminated M-site butnot R-site cleavage of pNPI, as indicated by the accumulationof pNPIc rather than NPlc. Substitution of alanine for P3valine inhibited cleavage of the pNP1 M site significantly (i.e.,increased the proportion of pNPIc relative to NP1J, butsubstitution of glycine for either the P1 alanine (A556G) or P1'serine (S557G) had little inhibitory effect on cleavage of thepNPI M site (i.e., produced relatively more NPlc than pNPI0)(Fig. 3, lanes 13, 15, and 17, respectively; and Fig. 4, lanes 31through 33, respectively). None of these mutations eliminatedcleavage of pNPI at its R site (i.e., produced little NP1 orpNP1; comparatively more NPlc or pNP1c) or cleavage of theassembly protein precursor at its M site (Fig. 4, lanes 51through 53; and Fig. 5, lanes 4 and 5), indicating that M-sitecleavage is not essential for proteolytic activity.The relative inhibitory effect (strongest to weakest) of these

mutations on the proteinase M-site cleavage was estimated tobe V554-S557 - -A556 - > V554A > A556G > S557G.

Deletion of the I site does not prevent either R- or M-sitecleavage. A third cleavage site, with the sequence VEA c AT,has been identified between CD1 and CD3 in the proteolyticdomain of HCMV assemblin (5, 7). Cleavage at this putative Isite gives rise to an amino half (An; 16 kDa) and a carboxyl

J. VIROL.

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cm*. -NP1

-Ac1 2

FIG. 6. Deletion of INAAD-129 sequence eliminates I-site cleav-age. The wild-type (Wt) proteinase (AW4) and a deletion mutantlacking the sequence INAAD (1125-DI29-) were analyzed by transfec-tion and Western immunoassay for I-site cleavage. A 10 to 20%gradient gel (Novagen, Madison, Wis.) was used for SDS-PAGE, andproteins were detected with anti-C2. Shown here is a direct autoradio-graphic exposure of the resulting immunoblot. Protein bands areidentified in the right margin.

w _ -NP1 s

1 7 3 4 5 6 7 8 9 10 11 12 13 14

FIG. 5. Phenotypes of M- and I-site deletion mutants and of theR-site P2 lysine mutant. Proteinase mutants lacking the M-site con-

sensus sequence (V554-S557-) or the putative I-site sequence (1125-D129-) or having an asparagine in place of P2 lysine at the R site(K248N), were tested in transfection assays for their ability to selfcleave (lanes 4, 6, and 11) and to cleave the assembly protein precursor

(lanes 5, 7, and 12). K248Nb was constructed as described underMaterials and Methods and was tested in the same way (lanes 13 and14) to verify that this mutant had no unexpected second site changes.Proteins from 72-h-transfected cells were separated in a 12% bis-cross-linked gel. Shown here is a fluorogram prepared following Westernimmunoassay of the SDS-PAGE-separated proteins. The Immobilonreplica was probed with anti-NI, exposed, and then probed again withanti-C2. Cytoplasmic and nuclear fractions of Colburn-infected cells(Col. Cyto. and Col. Nuc.) were included for reference (lanes 1 and 2).Mock, preparation from cells transfected with T-antigen plasmid only.Protein designations are shown in the right margin.

fragment (Ac; 13 kDa) (5). We have detected an apparentlycorresponding 13-kDa protein in CMV Colburn that is reactivewith an antiserum to the carboxyl end of assemblin (anti-C2[Fig. 1]). By sequence alignment, we determined that thisfragment could be produced by cleavage at a site, INA J AD,which has similarity to the HCMV I site (Fig. 9, CMV I-sitearrow). To verify this potential counterpart I site in strainColburn and to determine the effect of altering it on proteo-lytic activity, an I-site deletion mutant was made (1125-D129 -).Consistent with the 5-amino-acid deletion, this mutant gave a

comparatively smaller assemblin protein (NP1n in Fig. 5, lane6; and Fig. 6, lane 1) and no 13-kDa Ac fragment (Fig. 6). Thismutant cleaved both its own M site and that of the assemblyprotein precursor with approximately the same efficiency as thewild-type enzyme but appeared to be less efficient than thewild-type proteinase in cleaving at its R site (i.e., it hadcomparatively more NP1 [Fig. 5, lanes 5 and 6]).

Pulse-chase radiolabeling shows cleavage products of pro-

teinase precursor have differential stabilities. A pulse-chaseradiolabeling experiment was done in an attempt to determinewhether a temporal order of cleavage could be seen betweenthe M, R, and I sites. Cells transfected with the gene for theproteinase precursor (i.e., AW4) were pulse labeled with

[35S]methionine for 15 min, chased in the absence of radiolabelfor periods of up to 22 h, and then harvested and analyzed byimmunoprecipitation with one of three antisera (i.e., anti-NI,anti-Cl, or anti-C2 [Fig. 1]) followed by SDS-PAGE. Experi-mental details are presented under Materials and Methods.

Results of the experiment (Fig. 7) can be summarized as

follows. (i) Products of M-site cleavage (e.g., NPI and NP1I)were present in the pulse-labeled sample (lanes 1, 11, and 21),indicating that M-site cleavage had taken place within the15-min labeling period. (ii) Both pNP1 and NP1 decreased inintensity following the 15-min labeling period, consistent withtheir proteolytic conversion to product forms by R-site cleav-age. (iii) Products of R-site cleavage (e.g., NP1 , NP1c, andpNPlc) were present in the pulse-labeled sample (lanes 1, 11,

and 21), indicating the R-site cleavage had also taken placewithin the 15-min labeling period. The presence of pNP1c(lanes 11 and 21) shows that R-site cleavage can occur prior toM-site cleavage. (iv) Assemblin (NPIn) decreased in intensityfollowing the 15-min labeling period (anti-C2 panel), demon-strating that it is metabolically unstable and consistent with itsconversion to the 14-kDa An and 13-kDa AC fragments follow-ing I-site cleavage (Fig. 1). (v) Ac, the 13-kDa carboxyl half ofassemblin, increased dramatically in intensity (-18-fold) dur-ing the first 30 min of the chase period and then decreasedthroughout the rest of the chase (Fig. 7, anti-C2 panel),consistent with its being derived from a precursor but more

slowly than the M- and R-site cleavage products. (vi) Theintensity of NPIc, the carboxyl half of NP1 cleaved at its R site,remained essentially the same throughout the chase interval(anti-Ni panel), demonstrating that it is much more stablethan the other precursor and product forms of the proteinase(e.g., pNP1, NP1, NPln, pNP1I, and AC). It can also be seen inthe anti-NI panel that NP1c became progressively more het-erogeneous in size during the chase period, with one or twohigher-molecular-weight bands appearing immediately abovethe pulse-labeled NPIc band and increasing in intensity withtime. And (vii) the intensities of the pNP1, NP1, pNP1c, andNPln bands were approximately the same in a 15-min (lanes 31through 33) and 22-h (lanes 34 through 36) labeling period,indicating that a steady state with respect to their interconver-sion was approximated within the 15-min interval. Consistentwith the pulse-chase data (lanes 1 through 30), the NP1c and

4

VOL. 67, 1993

7366 WELCH ETAL.J.Vro.

--,chl~,S(, ~rn Chase Chase051 2 4 8 1222MrnMo,,, a 0Q5 1 2 4 8 1222 MPM22 a_O.51 2 48 12 22MP Md2-~,

0 g0

2;L;- ':<

pNPi1 ~

QNPI(

-pNPl-cI~

-NP1..,- qm

4',

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 periodthan in the pulse (compare lanes 31 and 34), indicating their

greater metabolic stability.A similar experiment was done, but a shorter pulse-radiola-

beling period was used. Results of this second experiment (Fig.8) showed the following significant differences from the first

(Fig. 7). (i) The pNPI band was most intense in the 5-mmn pulseand 5-mmn chase samples and then became essentially unde-

tectable in the 15-mmn chase sample, consistent with its cleav-

age to lower-molecular-weight bands. (ii) The NP1 band

increased in intensity through the 10-mmn chase period (maxi-mal intensity after 5 to 10 min of chase [Fig. 8]) and then

decreased in the 15-min chase period and further in the 30-mmnchase period, indicating slower rates of appearance and disap-pearance than those for pNP1, and consistent with its beingderived from pNPI. (iii) The NP1'nband was less intense than

the NPI band in the 5-mmn pulse sample, in contrast to its

relatively greater intensity in the 15-mmn pulse sample (Fig. 7),and increased in intensity through the 10- and 15-mmn chase

periods (maximal intensity after 10 to 15 min of chase) and

then decreased in intensity in the 30-mmn chase period. (iv) Acwas barely perceptible in the 5-mmn pulse sample, in contrast to

its greater intensity than that of the pNP1 band in the 15-mmn

pulse (Fig. 7), and increased steadily through the 30-mmn chase

period, consistent with its being derived from NP1 n. The

results of the 5- and 15-min pulse-chase experiments are

consistent in showing loss of pNPI before NP1, NPI before

NP1~, and NPln before A,; and maximal labeling of pNPI at 5

to 10 mmn, of NP1 at 10 to 15 min, of NPln at 15 to 20 min, and

of the AC at 20 to 35 min.Mutations at the maturational cleavage site of the assembly

protein precursor affect cleavage. One deletion and three

substitution mutations were made at the M site of the assembly

protein precursor. The effect of these mutations on the sus-

ceptibility of the site to cleavage was determined by cotrans-

fecting each of the mutants with the gene for either assemblin

(i.e., LM18) or its full-length precursor (i.e., AW4). Deletion of

alanine at the sissle bond (A.556 ) completely eliminated

cleavage of pAP to AP by either form of the proteinase (Fig. 4,

lanes 20 and 24). Substitution of alanine for the P3 valine

(V554A) substantially decreased the extent of pAP cleavage to

AP compared with cleavage of wild-type pAP (Fig. 4, lanes 17

and 21). Some reduction in susceptibility to cleavage was also

noticeable when glycine was substituted for the P1 alanine

(A556G), but the extent to which this substitution interfered

with M-site cleavage appeared significantly less than in the

context of the P1 position in the R site (compare lane 18 with

lanes 27 and 37 of Fig. 4). Little change in susceptibility to

cleavage was noticed when glycine was substituted for P1'

serine (S557G) (Fig. 4, lane 19).The relative inhibitory effect (strongest to weakest) of these

mutations on assembly protein precursor M-site cleavage was

estimated to be A556 > A556G = V554A > S557G.

Alignment of the herpesvirus assemblin homologs reveals

1 1.

J. VIROL.

HERPESVIRUS PROTEINASE 7367

IV

X- Chase -- -

In 5S I' 1 5' 30'

-pNP1-NP1

Rub -NP1 N

_-AC

1 2 3 4 5

Anti-C(?

FIG. 8. Pulse-chase labeling (5-min) of the Colburn proteinase,pNP1, expressed in HEK cells following transfection. Shown here is afluorogram of an 18%, diallyltartardiamide-cross-linked gel containingsamples from the 5-min pulse-chase experiment described in the text.Transfected cells were pulse radiolabeled (Pulse), or pulse labeled andchased (Chase) in nonradioactive medium for the indicated times (inminutes). Proteins were immunoprecipitated with anti-C2, proteindesignations shown in the right margin are explained in the Fig. 1Blegend, and the circles between lanes 1 and 2 indicate the positions ofthe proteins.

three domains which contain absolutely conserved residueswith the potential to contribute to known proteolytic activesites. A previous alignment of the assemblin homologs of sevenherpes group viruses identified two highly conserved domains,CD1 and CD2, and several absolutely conserved potentialactive site residues, including two histidines, a glutamic-aspar-tic acid, and a cysteine (48). More recently, we have used theprogram PileUp (University of Wisconsin) to obtain a more

complete alignment and we have included the sequence of theherpesvirus saimari (1) assemblin homolog in the comparison.Results of the analysis revealed three additional highly con-

served domains, referred to as CD3, CD4, and CD5, one ofwhich (CD3) contains the only absolutely conserved serineamong the assemblin homologs (Fig. 9, Ser-118 in the SCMVsequence). This alignment also shows the highly conservedR-site and M-site cleavage sequences, as well as the I-sitecleavage sequence that has been demonstrated only for CMVs(HCMV [5] and SCMV [Fig. 5, lanes 6 and 7]) and may not beconserved among all assemblin homologs, as suggested by theabsence of obvious sequence similarity in that region.

Absolutely conserved CD2 histidine 47 and CD3 serine 118are essential for SCMV proteolytic activity; absolutely con-served CD1 histidine 142 and CD1 cysteine 146 are not

essential. Site-directed mutagenesis was used as a means ofevaluating the importance of specific amino acids to catalyticactivity. Mutations were introduced into the APNG1 gene asdescribed in Materials and Methods (AW4 in Fig. IA). Themutant proteinase constructs were then transfected alone orcotransfected with AW1 (APNG.5), the plasmid that codes forthe assembly protein precursor, and analyzed for (i) ability ofthe proteinase mutants to cleave themselves at both the R andM sites, as monitored by the appearance of NP1, pNP1c, andNP1c, and (ii) ability of the proteinase mutants to cleave theassembly protein precursor (pAP) in trans at its M site, asmonitored by the appearance of the mature assembly protein,AP. The immunoblot shown in Fig. 10 was probed with anti-NIand reveals both cleaved (e.g., NP1, NP1c, pNP1c, and AP) andnoncleaved (e.g., pNP1 and pAP) forms of the proteinase andassembly protein precursor (Fig. 1B).

Because of their importance in known proteolytic active sitesand their absolute conservation among the assemblin ho-mologs (Fig. 9), histidines 47 and 142, serine 118, and cysteine146 were mutated first. Substitution of an alanine (SI18A) forthe only absolutely conserved serine residue, Ser-1 18, abol-ished the proteolytic activity, as did mutation of histidine 47 toeither an alanine or a glutamine (Si 18A and H47A or H47Q,respectively [Fig. 10]). Mutants in which the conserved cysteineresidue was changed to an alanine or threonine (C146A andC146T [Fig. 10]) retained enzymatic activity, indicating that thisresidue is not an essential active-site nucleophile. To insurethat the neighboring serine 147 was not somehow replacing thecysteine as an active-site nucleophile, the double mutationC146A/S147A was also tested and was found to have little effecton activity (C146A,S147A [Fig. 10]). Mutation of histidine 142to either alanine or glutamine severely reduced, but did notabolish, proteolytic activity (H142A or H142Q [Fig. 10]).

In addition to the above-mentioned absolutely conservedresidues, a number of highly conserved and several noncon-served residues were also mutated. Serine 195 was suggestedinitially as a potential catalytic residue because of its positionalsimilarity to the chymotrypsin nucleophile (48), but its changeto 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 conservedin CD3, serines 120 and 121, were mutated together(SI2oA,S121A) and then individually (SI2oA and S121A). Thesemutations reduced proteolytic activity but did not abolish it(S12oA,S121A,S12oA and S121A [Fig. 10]). Two nonconservedserines were also changed to alanine (Ser-198 and Ser-199),resulting in an electrophoretically faster-migrating NP1 butwith little, if any, loss of proteolytic activity (Fig. 10A). Becauseof their role in metalloprotease activity and as members ofactive-site triads of chymotrypsin-like serine and cysteine pro-teinases, highly conserved glutamic acids and aspartic acidswere also mutated. A double mutation at glutamic acids 20 and22 and a single mutation at glutamic acid 22 severely reducedbut did not eliminate proteolytic activity. The E22A mutant wasinteresting in that it appeared to be relatively unaffected in itsability to cleave at the M site but had impaired ability to cleaveat the R site (E20A,E22A and E22A in Fig. lOB; also seeDiscussion). Mutations at the only residue showing a high levelof conservation as aspartic acid (Asp-15) and at the conservedacidic residue, aspartic acid 104 (a glutamic acid in most otherassemblin homologs [Fig. 9]), also reduced the proteolyticactivity (as in D15A and D15N and in DIo4A and D104N [Fig.10]). LM3 was included in this experiment as an example of anessentially inactive proteinase (48).

VOL. 67, 1993

4.11W -

't ff.

7368 WELCH ET AL.

CD5 CD2MAAEADEENC E AL .YVYA.GYLALY SK.DEG EL NITPEI,V.RSA L.PP .TSKIPI -RKDC.V'E,V'EIAMDAYTVDGNA V S L.P::. IYVA.GYIA.LY DMGDGG. TLTRET AAA .............P: RASRLPINID.''"''RNGCVE V-.:" LSMAADAPGDRM EEPLPDRAV.P IYYAGFL.LY' SGDSG EL ALDPD:TVRAA L .DNPLPINVDEDNG LLA

DIKY .'I,VA':Y'L,'.VVY D,HOESAGRE'.Y E.L,TREOSKSA ',HESSCV,VGTV LT

'LHAQGQGPS'SVA"EH't'N'DDTAQGQG'A..................................RGP'LDP'DLE,NV:DESATVGYV

HMLDPDPLT:.::VKSOLY:LKK:PELTVELDASVAGF.-,.VDAYPKVDP.V:LY.LNL'...R.::':..D... L.VSKCL'ATK1'GLE'LPESTIG:HT IG

* * * *

RESIDUE 5 15 20 22 24 34 38 47 57

CD4I I ED I'.RG-.'IPRFLGIVPCPAV OLLHAVLFEAAHI V DDARGP F,.FL.G.. II..:'.NCPRO LGAVLAT AAGVVDDP1RGP F F V G.L:I: AC V LERV L.ET'AASI LD LP.:RG::.L' F:C,'LG:V VMST A L AP I FLS Y V ODMOSVRD...... F.... ..SC.V..T.'P..R.g LEIVRMFSVPDC~C--L-G-`CV,,::TSP:R :F::L:E IVRPASE

LNVRAGL FCtt.GR:VTSRK F'LD I VOKAS EL Y OS R AGL FS AAS.I'.'TS:GDDFL.S LLDS I Y HLYAVTHGV FCVGVIHS'EK F..L'H LT EN LFS

59 67 7 7

CD3S N F .FGN RD S VPDFFGE LS EGAA I F.'ERRGPPDA LFANAEEGKS E LVSRGPVKS E LVSRGPPDCDI AOSORLNSCV AOATSK

8 7

CMV I-Site4

LS. P LERA LYLVTNYLPS VSL'SK LSP NEIPD'G .

:LS EOE'RL tLYLVSNY:LPS& ASLS:S::R-RLt.GP D:E.E;PD'E........ .. ......... . ...................LS. REER L L.Y LI TNYL: P.S VSSLATKIR.LGG EAHP'DR....

MV'L-T. ET.E.KF L:Y L:L.S.N I L.PS LS'S:,REE.....S P.LO P D K V V EF L'S::GT Y AG LS L.S:S CDD V EA ATTS LS G.SE SS$::0L'R P DG V -LEt'F L5'G S Y S G L.SI'.LSS`R.R-.. D I N AAD:GAAGD

.PL'PRE.PK V.E-A'L'.H AW L PS LSSlAS.LHP. . D I POT T ADG... FLP Y OP L LJE'.M'L.H TW L P A LSL5,l SS. L C P.. ..... TAONA

* * **

97 104 107 1 18 125

CD1vzvE H VHS V

I LTVHCMVSCMV

EBVH V S

VzV

E H VHSV

I LTVHCMVSCMV

EBVHVS

FFTNVAM'tX '-LC'VVG~RRVG.T VVN'YDCTPES S I,EPF'RVL"S.M ESKARL... L SLVKDYA... GLNKVWKVSE DKL... AKVL LST.AVNL RA CVSLCVIGRRVGT IVTYDAT EN AVAPFKRLSP SS E:. . L ITAREAQSRL GDAATWHLSE DTL- .TRVL STAVNLFA- LCAIGHL..SEG.

RLAAEAELAL S.GRTWAPGV EAL THTL LSTAVRFFAI.-V::A '.LC.ELGR:REG:T' VA1Y.G:ATASE AI.GAFD S A PIKEE'QO.L. Y EIATREK... CAEVPRELSR PEI ..TRVL MKKFIHPFKIALAC.SVGRR.RG RYRPEWVTORFPDLTA ADDRDGL RAOW QRCGSTAVDA SG DPFRSDSYGL tLGNS:'CFKR`VA ,LC:SVG R'.GT LA G.RODW VMER P,P )LTE ADR'EAL'RNOL SGSGEVAAKE SAESSAAAAV D'PFOSDSYGL GNSS VD-FF'DV.S ICALGRRRGT' TAVYTDLAWVLKH.SD.LEP...A.SIAAOI ENDANAAKRE .....CPED HPLP LTK. .L IAKA ID:MFQF4g.,V,L.SCL:C.ALG'RRRGT V VYSMN LE D AI-:SOFCSIO A.AEVEN I YODSKNVDIN SL,

* **

142 146 155 165 175 185 195 205 215

R-Site M-Site

NMLL RDRWDVVAKR RREAG',REI.;MG.H VY:LOASTGYG 333... .NAVEA4I.S'SK VaricellazostervirusL.'L NR:NR'WNLVARR R:R:EAGIT::'EG. H T.YLOASASFG. .375.. .OT, DA''AS. Equine herpes virus

NMM 'L R.D.R,:WS LV AE'R' $':R,.OQAGI:'AG. H TYLAN.A4SSA... Herpes simplex virus

GAF.'L M::D.RGTC:L;KT.R. REMAWAVVYN.P KY:LQ:AJNEV .310 .ETVDAISM.M Infectious laryngotracheitis virusALY I RERLPKLR YD K L V V T E R E SVKSVSPE. .377.. .GV CR. Humancytomegalovirus (AD169)ALYI OERLPKLRYI3 KLV.GVTAE'AVGV SPA .GV''N"CR. Simiancytomegalovirus(Colburn)AGF;L RNRVET; R OD R`GVAN I .PA. E S Y.LKA SD A P D .324.. K L,VO:A4S'AS Epstein-Barr virus'AG F I KDJRLQLLKTD.'.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 werecompared by using the University of Wisconsin program PileUp. Each sequence began at the amino-terminal methionine and stopped at the sisslealanine of the R-site consensus sequence (e.g., Ala-249 for SCMV) (48). The sequence data used were from the following sources: varicella-zostervirus open reading frame (ORF) 33 (VZV) (14); equine herpesvirus ORF (EHV) (44), HSV type 1 UL26 ORF (HSV) (32), infectiouslaryngotracheitis 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 thenomenclature of Roizman et al. (38). Shaded residues indicate identity in at least four of the eight sequences shown. Five highly conserveddomains, 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 Msites indicate the number of amino acids (not shown) between the two flanking residues. Numbering below the alignment corresponds to the SCMVsequence; the right-hand digit is directly below the indicated SCMV residue. Asterisks are directly below residues in the SCMV proteinase thatwere mutated.

DISCUSSION

Herpesviruses encode a maturational proteinase that re-

moves the carboxyl end from an abundant, phosphorylatedcapsid protein during assembly (28, 36, 48). Without thiscleavage, infectious virus is not produced (35). The proteinaseresponsible for this cleavage is encoded by the viral genome,and its gene overlaps that of its substrate, the assembly proteinprecursor (27, 28, 36, 46, 48). On the basis of evidenceobtained from experiments done with the SCMV maturationalproteinase, assemblin, a working model of its synthesis andprocessing was proposed and generalized to other herpesgroup viruses (48). Two key predictions of the model were that(i) the proteinase precursor is cleaved at a highly conserved5-amino-acid consensus sequence near its midpoint to yield themature proteinase, assemblin, and (ii) enzymatically importantamino acids are conserved among the herpesvirus assemblin

homologs and are located in comparatively well-conserveddomains of the enzyme. The work reported here was done totest these predictions and related questions.

Cleavage sites. Experiments done to verify the predicted Mand R sites in the SCMV proteinase precursor demonstratedthat when their consensus site amino acids were deleted (i.e.,VNA , S and YVKA I, S, respectively) the correspondingcleavages were eliminated (i.e., pNP1->NP1 and pNP1c->NP1I; NP1->NPI,,+NP1c, respectively). Taken together withresults obtained using other M- and R-site mutants, discussedbelow and in the Results section, these findings support thepredicted cleavage sites. Direct amino acid sequence analysesof the HSV M and R sites (16) and of the HCMV R site (5)have confirmed their predicted sequences and validated thegeneralization of M- and R-site cleavage to other herpesvi-ruses. Both the M-site and the R-site deletion mutants were

Virus

ELVZHSV

LI LTVR[ HCMV

Sy[EBVLHVS

vzvEH VHS V

I LTVHCMVSCMVEBVHVS

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S ETTAETAGK LSANTN

J. VIROL.

HERPESVIRUS PROTEINASE 7369

A.

< Z

<0 < <

00C

v

1o o o _ N NxL

pNPI-NP1-

pNPI c-

NP1 c-

B.

1 2 10 3Z 13 14

<~~~

V~~~~~~~~L)LL < Z <_C v v 0 0 _nCD rw 0 0)o

< C C]X Lx Q .,(, s

pNPI-NPI1- _-

pNPl c-

NP1 c---

1 23 4 5 6 7 8 9 10111213141

FIG. 10. Site-directed mutagenesis impliHis-47 and Ser-1 18 as essential for prote(substitutions were made for specific residueas described in the text. The mutant constrthe assembly protein precursor gene AW]fected cell proteins were separated by SD'linked gels, and the proteins were probeelectrotransfer to Immobilon. Shown hereautoradiographic (B) exposures of the re

plasmid(s) used for transfection are indicattions are designated as explained in the leg(4 double mutations (E20A/E22E, C146A/S 47

Si99A) were analyzed; protein designations (the left and right margins; andA3pNPl in

pNP1 band of the mutant, LM3. The asterithe 68-kDa band also seen in Fig. 3 and 4.

able to cleave at their intact sites andprotein M site. Similarly, an HSV typewhich the R-site sissle pair, Ala-Ser, wa

was able to cleave at its intact M s

provide direct evidence that the prediutilized during processing of the protmature form, assemblin, and that tlrequire activational cleavage at eitherA new antiserum, anti-C2 (Fig. 1),

assemblin enabled us to visualize the mrecognize that it, like its HCMV counteThe site at which this cleavage occursi]as VEAt AT (5, 7) and has a looscounterpart in SCMV but not in the o

9). By deleting the 5-amino-acid pLColburn (INAt AD), cleavage of asamino (An: -13.7-kDa) and carboxyl

< <was prevented, supporting this sequence as the SCMV protein-

,-, ase I site. The I-site deletion mutant had the interestingphenotype of cleaving well at the M site but having a reduced

L.0 (.0 L, co % ability to cleave at the R site. One explanation of this apparent, C:2,.,,(>,2.selectivity is that the deletion, which is in a possible loop-b - %3pNP- insertion region (i.e., variable in length and amino acid se-

quence) between two highly conserved domains and near theessential CD3 serine, decreases the size of the substratebinding pocket such that the R site with its large P4 tyrosine ismore difficult to accommodate than the M site, which has a P4valine.

Other mutations in the R site and theM sites of both the- -4Ii -pAP proteinase and the assembly protein precursor gave further.*AP insight into these recognition-cleavage sequences. The impor-

tance of the absolutely conserved P4 tyrosine and P1 alanine of16 17 18 19 20 21 T the R site was demonstrated by the low level of cleavage

observed at this site when either residue was mutated. Inter-estingly, the Ala- Gly substitution appeared to be bettertolerated at the P1 position of the M site than at the P1position of the R site (i.e., the mutant M site cleaved better

<< < < than the mutant R site), suggesting that the recognition oro tD0tD1 L: c00o

:Et- '- `:,0E cleavage characteristics of the two sites differ. Even though theLI C) V)vrm P1' serine is conserved in all but 1 of the 15 known R- and

£ - \3pNPI M-site sequences (48), its mutation to glycine had remarkablyI_ I ~ little effect on cleavage at either site, consistent with thegenerally greater importance of the P residues than the P'residues for serine and cysteine proteinases (34). A surprisingphenotype was observed when the P3 lysine of the R site waschanged to an asparagine, the P3 residue of the M site. Unlike

4bu_~ the P4 and P1 mutations, which reduced R site cleavability, this5 16 17 18 19 20 21 P3 change did not noticeably affect R-site cleavage (Fig. 3 to

icates absolutely conserved 5), presumably because the mutation simply converted the Rolytic activity. Amino acid site to an M site with a P4 tyrosine. Surprisingly, however, theof the SCMV proteinase, mutation did reduce M site cleavage (e.g., producing compar-

ructs were transfected with atively less pNPI-NPl and pNP1I-.>NP1I [Fig. 3A, lane 8;1 (A) or alone (B), trans- Fig. 4, lanes 26 and 46]) and drastically lowered I-site cleavageS-PAGE in 10%° bis-cross- (e.g., producing an increased amount of NPI, and little if anyad with anti-NI following 13-kDa A, [Fig. 3B, lane 24] [data not shown for AJ]). Thisare fluorographic (A) and result suggests that the carboxyl end of assemblin can influencesuiting immunoblots. The substrate specificity or catalytic activity, or both, especially thated above each lane. Muta- pertaining to the I site. In line with this observation,extensionA,StoFA/SI1iA, andS198A/ (addition of 12 amino acids) or deletions (of 3 or 8 amino(see Fig.1) are indicated in acids) of the carboxyl end of the HCMV assemblin homologdicates the position of the that eliminate or alter the R site yield mutant enzymes able tosk indicates the position of cleave the M site but unable to cleave at the I site (5). Cleavage

activity against both the M and I sites was lost when the6-amino-acid sequence MHWHWH-C' was added to the car-boxyl end of the HCMV assemblin homolog (41a).Many proteinase precursors have internal cleavage sites.

in trans at the assembly These may be used to activate an inactive zymogen (22-24),1 proteinase mutant in alter the structure of the enzyme so that its substrate specificity

s replaced with Arg-Pro is changed (15, 49), or influence the rate of cleavage (i.e.,,ite (30). These results product fast and slow cleavage sites) as a mechanism to controlicted M and R sites are the relative time of appearance and the relative amounts of theteinase precursor to its different cleavage products (17). Data shown here for thehe precursor does not CMV M, R, and I sites and elsewhere for the HSV R site (30)site. indicate that the precursor is not a zymogen that requiresto the carboxyl end of cleavage for activation. Our data also indicate that R-siteature proteinase and to cleavage is not dependent upon preceding M-site cleavage, and-rpart, is cleaved in half. vice versa. With regard to the possibility that the differentn HCMV was identified cleavage site sequences influence the time of appearance or

,ely conserved possible the amount of product, our data are consistent with a kineti-ther herpesviruses (Fig. cally ordered cleavage of M > R > I. Products of all threeitative I site of strain cleavages appeared rapidly (within 15 min) and a 5-min pulseisemblin (NP1n) to its radiolabeling period was required to discriminate between M-(A,: -13.4-kDa) halves and R-site cleavage in this transfection system. Contrary to the

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7370 WELCH ET AL.

conclusion drawn from work done with the HCMV proteinase(5), our data indicate that assemblin (NP1n) is not metaboli-cally stable and that the 13.7-kDa (predicted amino half, An)and 13.2-kDa (predicted carboxyl half, A,) fragments arisedirectly from its cleavage (Fig. 7). Consistent with this finding,the HCMV and SCMV assemblin proteins synthesized inbacteria are also cleaved at their I sites to give the expectedfragments (reference 7 and our unpublished results, respec-tively). In addition, because little I-site cleavage (evidenced bythe 68-kDa band) was observed in the "tightest" R-site mu-tants (e.g., Y246-A249 - and Y246A/A249G [Fig. 3]), we concludethat I-site cleavage proceeds more efficiently following R-sitecleavage. If so, this may indicate that conformational changesaround the I site, rather than the sequence itself, determine itssusceptibility to cleavage. It is important to emphasize that thekinetics and order of cleavages may be very different ininfected cells than in cells transfected or transformed with theproteinase gene alone.

Active-site mutations. We previously identified two highlyconserved domains, CD1 and CD2, and several amino acids(i.e., His-47 and 142, Cys-146, Asp-104 and Ser-195 [Fig. 9]) inSCMV assemblin as potential active-site components. A morerecent alignment of the assemblin homologs (Fig. 9) hasrevealed an absolutely conserved serine within a third highlyconserved domain called CD3 (e.g., Ser-1 18 of SCMV). Theseand other amino acids with the potential to contribute toknown proteolytic active sites were changed by site-directedmutagenesis as a means of determining their importance to thecatalytic function of the proteinase. The underlying rationaleof the experimental approach was that functionally criticalamino acids (e.g., active site residues) will be highly conservedwithin a closely related group of enzymes and that mutatingsuch residues will inactivate the enzymes. Amino acid changesto alanine or to a conservative replacement (e.g., Asp-*Asn)were used to minimize the possibility that the substitutionswould cause overall structural distortions (4, 13).Of the 14 amino acids mutated, only changes at the abso-

lutely conserved histidine 47 of CD2 and serine 118 of CD3eliminated activity of the proteinase in our assay. CD3 serine118 (in the SCMV sequence) is the only absolutely conservedserine in assemblin. The finding that it is essential for activitymakes it the strongest candidate active-site nucleophile so faridentified and adds to the evidence that the herpesvirusproteinase is a member of the serine proteinase superfamily.Other evidence comes from inhibitor studies which showedthat high concentrations of phenylmethylsulfonyl fluoride (25mM), a specific inhibitor of serine proteinases, inhibited theHSV type 1 enzyme (29) and lower concentrations inhibitedthe HCMV (5) and SCMV (our unpublished results) enzymes.Diisopropylfluorophosphate, another serine proteinase inhib-itor, also reduced the activity of the HSV (29) and HCMV (5,7) enzymes. Although the possibility that the herpesvirusenzyme belongs to one of the three other proteinase super-families (i.e., aspartic, metallo, or cysteine) cannot be ruledout, the following evidence suggests this is unlikely. First,unlike aspartic proteinases which have acidic pH optima, thatof the CMV proteinase is neutral to slightly alkaline (7). Inaddition, an absolutely conserved aspartic acid has not beenrecognized among the herpesvirus assemblin homologs. Sec-ond, unlike the activity of metalloproteinases, which are typi-cally sensitive to metal-chelating agents, activities of the HSV(29), HCMV (5, 7), and SCMV (our unpublished results)enzymes were not reduced by the metal chelator, EDTA. Andthird, unlike cysteine proteinases, which would be expected tohave an absolutely conserved essential cysteine, the onlyabsolutely conserved cysteine (Cys-142 in SCMV) is not essen-

tial (30) (Fig. 10, lanes 16 through 18). Moreover, initialsubstitutions of each of the other four cysteines in SCMVassemblin indicate that none is absolutely required for activity(41b). The observation that high concentrations of Zn2+inhibit the HCMV (5, 7) and SCMV (our unpublished results)enzymes was interpreted to indicate that the HCMV enzymehas an active-site cysteine (5). An alternate explanation for thisfinding, considering the apparent absence of an essentialconserved cysteine, is that the enzyme has a metal-bindingdomain that when occupied inhibits activity. Such an interac-tion could provide a regulatory mechanism to keep assemblinor its precursor forms inactive until needed.The catalytic mechanism of known serine proteinases in-

volves an active-site triad consisting of a serine, a histidine, andan aspartic acid. Serine 118 of CD3 has been identified here asa potential nucleophile of such a triad in assemblin. The CD2histidine (His-47 in SCMV) would qualify as a second residueof the triad on the basis of its absolute conservation among theherpesvirus assemblin homologs and because it is essential foractivity in both the SCMV (Fig. 10, lanes 6 and 7) and HSV(29) enzymes. Although there is one other absolutely con-served histidine in assemblin (i.e., CD1 His, residue 142 inSCMV), and it was found to be essential for activity in the HSVenzyme (30), it is not essential for activity in the SCMVenzyme (Fig. 10, lanes 14 and 15), thereby making the CD2 Hisa stronger active-site candidate. No absolutely conserved as-partic acid was apparent from the alignment shown in Fig. 9;however, several positions did show a high conservation ofaspartic acid (e.g., CD5 Asp-15 in SCMV) or of an acidiccharge (e.g., Asp or Glu). Four of these residues were mutated,but none eliminated activity in the SCMV enzyme (Fig. 10). Ofthe four substitutions made, the double mutationGlu20,Glu22->Ala20,Ala22 and the single mutation Glu-22->Ala-22 had the most adverse effects on activity. It isnoteworthy that the E22A mutation, alone or together withE20A, completely inhibited R-site cleavage without apparentreduction of either M- or I-site cleavage (Fig. lOB, lanes 4 and5), suggesting a role for this highly conserved residue indetermining R-site specificity. Substitution of the most highlyconserved aspartic acid (e.g., D15A or D15N in SCMV) hadlittle effect on proteolytic activity in either the SCMV (Fig. 10)or the HSV (29) enzyme. And although substitution of Glu-115(doubly mutated with Glu-114 or E114G/E1I5A) eliminateddetectable proteolytic activity of the HSV enzyme (30), substi-tutions of the corresponding residue of the SCMV proteinase(i.e., D104A or N) did not eliminate activity. Our inability toidentify a strong candidate aspartic acid for the third memberof a possible serine proteinase active-site triad by this tech-nique is perhaps not surprising, given that this member of thetriad tends to be less well conserved than the Ser and Hisresidues and also to have a weaker adverse effect on activitywhen substituted with another amino acid (8, 12). It is worthnoting that when substitutions similar to those used here weremade in active-site triad residues of both subtilisin (8) andtrypsin (12), both enzymes retained measurable proteolyticactivity, albeit reduced 3 to 6 orders of magnitude. Thus, it ispossible that apparent discrepancies in the results for theSCMV and HSV enzymes mutated at the CD2 His and at thehighly conserved Glu/Asp (i.e., Glu-104 in SCMV and Glu-115in HSV) may be explained by sensitivity differences in the assaysystems, or in the nature of the amino acid substitutions made.

If further analyses confirm that the absolutely conservedserine of CD3 is the assemblin nucleophile, the herpesvirusproteinases would appear to represent a new subclass of serineproteinases, since they do not contain the characteristic se-quence motifs found near the active-site serines (or cysteines)

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HERPESVIRUS PROTEINASE 7371

of the chymotrypsin-like or subtilisin-like proteinases (i.e.,G-X-S/C-G-G or G-T-S-M/A, respectively [3, 6, 20]).

ACKNOWLEDGMENTS

We thank Jennifer Ludford for use of the data shown in Fig. 7 and8 and Rebecca Magno and Jenny Borchelt for excellent technicalassistance. We also thank our colleagues at Lilly Research Laborato-ries for their help in making primers, sequencing mutant DNAs, andgrowing plasmid stocks and for the gift of anti-C2.

This work was aided by research grants from the National Institutesof Health (Al 13718 and Al 22711) and from Eli Lilly and Co. A.R.W.was a student in the Pharmacology and Molecular Sciences trainingprogram and was supported by PHS grant GM0726. M.R.T.H. is astudent in the Biochemistry, Cellular, and Molecular Biology trainingprogram and was supported by PHS grant GM07445.

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