Antigenic Variation of Varicella Zoster Virus Fc Receptor gE:Loss of a Major B Cell Epitope in the Ectodomain

Richard A. Santos, Jorge A. Padilla, Christopher Hatfield, and Charles Grose1

Department of Microbiology and the Immunology Program, University of Iowa College of Medicine, Iowa City, Iowa 52242

Received April 7, 1998; returned to author for revision May 15, 1998; accepted June 26, 1998

Varicella zoster virus (VZV) is considered to possess a genetically stable genome; only one serotype is recognized aroundthe world. The 125-kbp genome contains ;70 open reading frames. One that has received particular attention is openreading frame 68, which codes for glycoprotein gE, the predominant 623-residue viral envelope product that harbors both Band T cell epitopes. This report describes the initial characterization of a community-acquired VZV isolate that was adistinguishable second serotype (i.e., it had lost a major B cell epitope defined on the gE ectodomain by a murine monoclonalantibody called mAb 3B3). The mAb 3B3 epitope was found not only on the prototype sequenced Dumas strain from Hollandand all previously tested North American isolates but also on the varicella vaccine Oka strain originally attenuated in Japan.Sequencing of the mutated gE ectodomain demonstrated that codon 150 exhibited a single base change that led to an aminoacid change (aspartic acid to asparagine). Observation of the monolayers infected with the mutant VZV strain also led to thesurprising discovery that the topography of egress was altered. Wild-type VZV emerges along distinctive viral highways,whereas the mutant strain virions were nearly uniformly distributed over the cell surface in a pattern more closely resemblingegress of herpes simplex virus 1. The mutant VZV strain was designated VZV-MSP because it was isolated in Minnesota.© 1998 Academic Press

INTRODUCTION

Varicella-zoster virus (VZV) is an ancient virus. Esti-mations of its origins have established that the mod-ern herpesviruses arose ;60–80 million years ago(McGeoch and Cook, 1994; McGeoch et al., 1995). VZVis a member of the alphaherpesvirus subfamily ofHerpesviridae. It is the etiological agent of chickenpoxin childhood, after which the virus enters a latent statein the dorsal root ganglia; decades later, the samevirus reactivates and causes the disease shingles(herpes zoster). The entire sequence of the 125-kbpVZV genome has been published by Davison and Scott(1986). With the subsequent publication of sequencedata from other herpesviruses, the alphaherpesvi-ruses have now been subdivided into two generacalled simplexvirus and varicellovirus (McGeoch andCook, 1994). VZV is considered to have one of the moststable genomes of all herpesviruses. The Oka strain ofvaricella vaccine that was derived from a Japanesechild with chickenpox has a few minor genomic differ-ences from North American strains, but to date noantigenic variation has been discovered among themajor surface immunogens of the virion (Arvin andGershon, 1996).

VZV gE is the predominant glycoprotein specified in in-fected cells and virions; this fact is in marked distinction tothe related herpes simplex virus type 1 (HSV-1), in which gEis a relatively minor constituent (Spear, 1985; Grose, 1990).In both VZV and HSV-1 infections, gE is a biologically activeFc receptor on the surface of infected cells. The VZV Fcreceptor is distinguished by the fact that it contains motifsin its cytoplasmic tail that direct export from the endoplas-mic reticulum, trafficking to the trans-Golgi network as wellas endocytosis from the outer cell membrane (Nishimuraand Balch, 1997; Zhu et al., 1995, 1996; Olson and Grose,1997, 1998). The precise role of the VZV Fc receptor activityremains to be elucidated, but the homolog gE is intimatelyinvolved in cell-to-cell spread of other alphaherpesviruses(Dingwell et al., 1994).

In VZV infection in humans, VZV gE is a major antigenicdeterminant to which numerous humoral and cytolytic re-sponses are observed (Ito et al., 1985; Arvin et al., 1986;Bergen et al., 1991). Recently, an immunodominant B cellepitope was demarcated in the gE ectodomain; the epitopeis defined by murine monoclonal antibody (mAb) 3B3 (Duusand Grose, 1996; Hatfield et al., 1997). Because of thewidely accepted genetic stability of this herpesvirus, wehad assumed this epitope would be present in all VZVisolates worldwide. In this report, we describe a communi-ty-acquired VZV strain with an unexpected mutation in thegE ectodomain that leads to the loss of an immunodomi-nant B cell epitope. The mutant virus strain also exhibits analteration in the topography of egress.

1 To whom reprint requests should be addressed at University ofIowa Hospital/2501 JCP, 200 Hawkins Drive, Iowa City, IA 52242. Fax:(319) 356-4855. E-mail: grose@blue.weeg.uiowa.edu.

VIROLOGY 249, 21–31 (1998)ARTICLE NO. VY989313

0042-6822/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

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RESULTS

Analysis of the VZV isolate by confocal microscopy

The virus designated VZV-MSP was initially isolated inhuman fibroblast monolayers. The cytopathic effect(CPE) was compatible with VZV, but the isolate waspoorly reactive with antibodies in a commercial VZVdiagnostic kit. Because the isolate did not stain withantibodies to HSV types 1 and 2 nor did its CPE resemblethat of HSV, the virus isolate was sent to our laboratoryfor further analysis. When the isolate (VZV passage 1)was received, the infected cell monolayer was trypsin-

dispersed and inoculated onto human melanoma cells(VZV passage 2). When CPE was apparent in 5 days, theinfected cell monolayer was trypsin-dispersed one moretime and inoculated onto 35-mm monolayers for exami-nation by laser scanning confocal microscopy (VZV pas-sage 3). The low-passage VZV-32 strain was included inseparate dishes as a control virus. When CPE covered;70% of each monolayer, the infected monolayers wereprobed with mAb 3B3 against gE and with mAb 6B5against gI and examined by confocal microscopy. In priorstudies, we showed that these two mAbs do not cross-react with other viral or cellular proteins (Grose, 1990).

FIG. 1. Loss of mAb epitope 3B3 in the VZV-MSP strain. Low-passage laboratory strain VZV-32 (A and B) and community strain VZV-MSP (C andD) were inoculated onto human melanoma cells. After 3 days, the cultures were probed with mAb 3B3 against gE (A and C) and mAb 6B5 againstgI (B and D). Nuclei are stained red and the viral glycoproteins are stained green when analyzed by laser scanning confocal microscopy. (C) Notethe absence of green staining of the mutant VZV-MSP strain.

22 SANTOS ET AL.

FIG. 4. Epitope mapping with mAb 3B3 in VZV gL. Individual monolayers were transfected with pTM1 plamids carrying VZV gL alone, gL 3B3.11,and gL 3B3.13. The construction of the plasmids is described in Materials and Methods. The attachment of mAb 3B3 to VZV gL 3B3.11 (A) and VZVgL 3B3.13 (B) was evaluated by laser scanning confocal microscopy. Monolayers transfected with VZV gL alone were nonreactive with mAb 3B3 (notshown). (Magnification bars) 30 mm.

23VZV gE ANTIGENIC VARIATION

As expected from numerous published experiments,mAb 3B3 and mAb 6B5 reacted with the laboratory strainVZV-32 (Fig. 1). In marked contrast, the anti-gE mAb 3B3did not attach to cells infected with the VZV-MSP strain,even though mAb 6B5 did bind the infected cells strongly(Fig. 1). As an additional control, the anti-gH mAb 206was added to cultures individually infected with both VZVstrains; all VZV-infected cultures were positive in thisassay (data not shown).

We next immediately addressed the question ofwhether the VZV-MSP strain fails to express the entireglycoprotein or has lost an epitope on the glycopro-tein. In this quest, we were fortunate in that we hadbegun epitope mapping of gE. Through this method-ology, we established that the 3B3 epitope consistedof at least 11 amino acids (151–161) of gE (Duus andGrose, 1996). We also determined that our panel ofmAbs included reagents against other nonoverlappingepitopes on gE; one of these antibodies, mAb 711,attaches to another as-yet-undefined epitope on theectodomain of gE. Therefore, the above experimentwas repeated with mAb 711 as the immunoprobe ofVZV-32- and VZV-MSP-infected monolayers. Bothmonolayers stained positively, a result that indicatedthat gE was expressed in VZV-MSP-infected cells butappeared to have lost either a small segment of itsectodomain or just the 3B3 epitope (Fig. 2).

Sequence analysis of VZV-MSP gE

To further investigate the nature of the gE mutation, weused polymerase chain reaction (PCR) amplificationtechniques to first determine whether a full-length gEgene (VZV ORF 68) was present in the mutant strain. Wewere able to amplify a full-length gene of an appropriatesize. Thereafter, we substituted primers that amplifiedoverlapping portions of the gE gene, each ;300 bases insize, beginning at the upstream region of ORF 68. Eachfragment was subjected to DNA sequencing, and eachsequence was compared with the published (Davisonand Scott, 1986) sequence of the Dumas strain (Fig. 3A;accession No. X04370). After analysis of the first 337codons of VZV-MSP gE ORF, we found the first and mostimportant base change in codon 150 (Fig. 3B, arrow); thesubstitution involved a replacement of a guanine by anadenine. Of great interest, this point mutation led to achange in amino acid from aspartic acid to asparagine(Fig. 3B). Because this alteration in gE occurred oneamino acid away from the deduced 3B3 epitope, which isunderlined in Fig. 3A, the sequence data strongly sug-gested that amino acid 150 was a previously unrecog-nized contributor to the 3B3 epitope. Further sequencingof VZV-MSP gE revealed one silent mutation in codon341 of VZV-MSP gE. All other codons were identical tothose in the gE sequence of the Dumas strain.

Epitope mapping of VZV-MSP gE

In an earlier experiment, we had evaluated the 3B3epitope by inserting the 11-amino-acid sequence into theunrelated VZV ORF 60, namely, the gL glycoprotein (Duusand Grose, 1996). The epitope tag within gL was recog-nized by mAb 3B3 when observed by laser scanningconfocal microscopy. To evaluate the contribution of theaspartic acid residue to formation of the epitope, the gLepitope mapping and tagging experiment was repeatedto insert the aspartic acid residue in its correct locationat the N terminus of the 3B3 epitope. To obtain the properparameters for the mutagenesis primers, one additionalcodon was inserted along with an aspartic acid (Fig. 3B).The pTM-1 expression plasmids, including gL-3B3.11 andgL-3B3.13, were transfected into HeLa cells and ob-served by confocal microscopy after labeling with mAb3B3 (Fig. 4). Cells transfected with the gL-3B3.11 plasmidwere positive in a restricted cytoplasmic pattern, aspreviously described by Duus and Grose (1996). Not onlywere cells transfected with the gL-3B3.13 more intenselystained, but also the pattern was more widely distributed

FIG. 2. Expression of VZV gE in wild-type and mutant VZV strains.Individual monolayers were infected with the VZV-32 strain (A) and theVZV-MSP strain (B). The monolayers were incubated with mAb 711 andexamined by laser scanning confocal microscopy on day 3 p.i. Anuninfected monolayer had a negative background (not shown).

24 SANTOS ET AL.

throughout the cytoplasm. Cells transfected with thepTM-1 gL plasmid alone were negative.

Subcloning the VZV-MSP gE ORF

After completion of the above experiment, wesought to confirm the epitope experiments by ampli-fying the entire gE gene from VZV-MSP DNA andinserting it into a pTM-1 expression vector. We hadpreviously cloned wild-type gE into the same expres-sion vector; the same primers were used for the sec-ond cloning experiment (Yao et al., 1993). After tran-sient transfection with these two forms of VZV gE aswell as the pTM-1 vector as a control, the cell lysateswere solubilized and subjected to electrophoresis fol-lowed by transfer to membranes. Additional controlsamples for the transfection immunoblotting experi-ments included MeWo cell monolayers infected withthree VZV strains: VZV-32, VZV-Oka, and VZV-MSP.Uninfected MeWo cells served as a negative control.All samples were blotted with mAb 3B3 followed bychemiluminescence (Fig. 5). The mAb attached to VZV-32, VZV-Oka, and VZV gE wild-type but not to VZV-MSP,VZV-MSP gE, or the vector and uninfected cell con-trols. When VZV-MSP gE was subsequently immuno-blotted with a polyclonal monospecific antibody to gE,the result was positive. Thus, the results in Figure 5

confirmed that VZV-MSP gE by itself was expressedbut lacked the 3B3 epitope.

Alterations in topography of egress of viral particles

In previously published studies, we documented thatthe egress of wild-type VZV particles onto the surface ofinfected cells occurs in a distinctive pattern that wastermed “viral highways” (Harson and Grose, 1995). Theviral highways are composed of thousands of viral par-ticles that emerge in long rows across the surface of thesyncytia. When the distributions of VZV-32 and VZV-MSPparticles were compared at a low-magnification level byscanning electron microscopy (SEM), wild-type virionswere again arranged in a pattern consistent with viralhighways (Figs. 6A–6C). Cells infected with the VZV-Okastrain show a similar pattern of viral highways (Grose,1996). In contrast, no such topographical pattern wasobserved on samples infected with VZV-MSP; instead,viral particles were distributed more uniformly over thecell surface (Figs. 6D–6F). After the observation of nu-merous monolayers by SEM, the viewer was left with theimpression that the number of VZV wild-type virionspresent on the cell surface was less than those of VZV-MSP (compare Figs. 6C and 6F). The topographical ar-rangement of VZV-MSP particles also exhibited a high

FIG. 3. Sequence of the ectodomain of VZV gE. (A) The deduced amino acid sequence of the N-terminal 400 of 623 codons of wild-type VZV gE.(Underlined) Previously defined mAb 3B3 epitope. (Arrowhead at codon 150) The aspartic acid residue altered in VZV-MSP. (Arrowhead at codon 341)The silent mutation. (B) DNA and deduced amino acid sequence of the mAb 3B3 epitope in the wild-type VZV-32 strain and the mutant VZV-MSP strain.(Underlined and arrows) The altered nucleotides (G–A) and amino acids (D–N). The two additional codons inserted into the expression plasmiddescribed in Figure 4 are designated 3B3.2, and the original 3B3 epitope is designated 3B3.11. (See Materials and Methods.)

25VZV gE ANTIGENIC VARIATION

degree of similarity with that of HSV-1, in which thou-sands of particles covered the cell surface (Fig. 7).

The observation that CPE in a VZV-infected monolayerfollows the longitudinal axis of the cells in the monolayerwas first made by Weller (1953). In VZV-infected humanmelanoma cell cultures, individual syncytial foci enlargeand eventually merge until the entire monolayer hasbecome a single syncytium. Virions emerge only aftersyncytia are formed, but the virions are never releasedinto the culture medium. If syncytial formation is blockedby the addition of anti-gH antibody into the culture me-dium, virions do not egress until the antibody is removed(Rodriguez et al., 1993). Based on the imaging studies inthis as well as previous reports, we postulate that virionsexit at the leading edge of syncytial foci that are merging.We further postulate that VZV egress (gE/gI mediated)and VZV-induced cell-to-cell fusion (gH/gL mediated) areseparate but interdependent events. The mutation onVZV-MSP gE appears to lessen that interdependence.

DISCUSSION

Among the human herpesviruses, VZV has alwaysbeen considered to have a very stable genome (Rentier,1995). Generally, one serotype is recognized around theworld. There are some minor differences noticed byrestriction-fragment-length-polymorphism analyses be-tween Japanese VZV strains and VZV strains from NorthAmerica, but these genetic changes have never been

correlated with changes in specific epitopes (LaRussa etal., 1992). A geographic-specific sequence variation inthe ORF 10 late gene product between Japanese andNorth American strains has been described by Kinching-ton and Turse (1995). They prepared an antibody directedagainst a synthetic peptide consisting of the N-terminal14 amino acids of the Dumas strain of ORF 10. Theantibody bound to 10 VZV isolates collected in the UnitedStates but failed to bind to 10 VZV isolates obtained inJapan. Whether this 14-mer represents a B cell epitope towhich the VZV-infected human host usually manifests animmune response was not determined in the abovestudy, but it seems unlikely because ORF 10 is notpresent on the surface of the virion. More likely, the ORF10 sequence variant represents a VZV equivalent of the“founder effect,” in which a human genetic diseaseemerges within one ethnic group or a geographicallyisolated population.

The 3B3 epitope within the gE ectodomain is easilydetectable by immunoelectron microscopy on the sur-face of the enveloped virion (Jones and Grose, 1988).Based on both published and unpublished data accumu-lated after the initial characterization of mAb 3B3, the 3B3epitope has been detected on all wild-type North Amer-ican isolates examined in this laboratory since 1983(Grose et al., 1983; Weigle and Grose, 1983). The epitopeas defined in this report is present in the sequence of theprototype VZV strain analyzed by Davison and Scott

FIG. 5. Immunoblotting of VZV strains and gene products. An immunoblot was prepared with each of the following lysates as antigens: melanomacells alone (lane 1), wild-type gE alone (lanes 2, 7, and 10), VZV-32 strain-infected cells (lane 3), VZV-Oka-infected cells (lane 4), VZV-MSP-infected cells(lane 5), vaccinia virus alone (lanes 6 and 9), and mutant VZV-MSP gE alone (lanes 8 and 11). Lanes 1–8 were probed with mAb 3B3, and lanes 9–11were incubated with a rabbit polyclonal monospecific anti-gE antibody (Rab 2).

26 SANTOS ET AL.

(1986); based on both results in Figure 5 and gE se-quence data provided by the Merck Company (A. Shaw,unpublished data), the same epitope is present in theOka varicella vaccine strain originating in and represen-tative of VZV strains from Japan. The fact that our initialepitope tagging experiments were successful without

the N-terminal aspartic acid suggests that by chance, theamino acids in the gL ORF immediately N terminal to theinserted 3B3 epitope partially compensated for the ab-sent aspartic acid in the epitope. When an aspartic acidwas inserted into the epitope tag on gL and the imagingexperiment was repeated, the intensity of staining by

FIG. 6. Topography of egress of VZV particles at three magnifications. (A–C) Surface of VZV-32 wild-type syncytium with emergent virions distributedin viral highways. (D–F) Surface of VZV-MSP syncytium with emergent virions distributed in a more uniform pattern. (Magnification bars) A and D, 20mm; B and E, 2 mm; and C and F, 1 mm.

27VZV gE ANTIGENIC VARIATION

mAb 3B3 under confocal microscopic visualization wasincreased severalfold.

The alteration in the VZV gE epitope from a communityisolate resembles antigenic drift in influenza A virus; thatis, a single base change within an epitope leads to asingle amino acid change, which in turn leads to the loss

of an epitope on the major cell surface hemagglutinin(HA) molecule (Wharton et al., 1989; Smith and Palese,1989). Generally, these genetic changes in influenza vi-rus are restricted to sites on the HA1 domain of the HAmolecule, usually on a loop. The HA1 nucleotide muta-tion rate has been estimated at 0.0067 substitution per

FIG. 7. Topography of egress of HSV-1 particles. A low-magnification SEM of HSV-1 virions over the cell surface resembles the pattern seen withVZV-MSP strain (Figs. 6D–6F). (Magnification bar) 1 mm.

28 SANTOS ET AL.

site per year. Clustering of the substitutions within a fewsites suggests the influence of immune pressure. Onesuch HA1 change is identical to that seen in VZV gE,namely, a switch from an aspartic acid to an asparagineresidue. The silent nucleotide substitution rate for otherinfluenza virus genes is lower and ranges from 0.0018 to0.0031 substitution per site per year. Even though anti-genic drift is a less drastic change than antigenic shift, itleads to sufficient antigenic change to allow escape fromimmunological surveillance by existing antibodies in thehost. We note that the asparagine mutation in VZV gEdoes not specify a new potential site of N-linked glyco-sylation.

In addition to influenza A virus, mutation rates havebeen tabulated for human immunodeficiency virus type 1(HIV-1) (Hahn et al., 1986). The nucleotide substitutionrate for the HIV-1 envelope gene is estimated to be atleast 0.001 per site per year, whereas that of the gaggene is less common. One site of variability in the HIVglycoprotein ORF is concentrated within or adjacent tothe immunodominant V3 loop of the HIV-1 glycoprotein(Shioda et al., 1992). The mutation rates for eukaryoticDNA genomes have been estimated to be ;1 milliontimes lower, ;1029 nucleotide substitutions per site peryear (Holland et al., 1982; Gojobori and Yokoyama, 1985).After extensive sequence analysis of five herpesviralgenes by means of both maximum parsimony and dis-tance methods, McGeoch et al. (1995) determined thatVZV and other alphaherpesviruses exhibit a nucleotidesubstitution rate between 10 and 100 times faster thanthat of eukaryotic DNA (1027 to 1028 changes per siteper year) but still much slower than that of many RNA orretroviruses.

Antigenic variation of a VZV surface protein may haveimplications for our understanding of the pathogenesisof VZV infection. The preponderance of opinion today isthat T cell-mediated immunity is crucial for the eradica-tion of primary viral infection (Arvin and Gershon, 1996;Grose, 1998). However, the loss of a B cell epitope on animmunodominant VZV glycoprotein raises the questionof whether antibody modulates an early event in VZVpathogenesis, probably cell-to-cell spread and virionegress mediated by gE. One potential mechanism forselective pressure involves a newly described T cell-independent antibody-mediated clearance of virus (Szo-molanyi-Tsuda and Welch, 1996). In the polyomavirusmouse model, there is a lethal infection in severe-combined-immunodeficiency mice, whereas in nudemice, a rapid fully protective predominantly IgG antiviralresponse is produced without any T cell assistance. Thepolyomavirus antigen is a viral capsid with a major gly-coprotein that is repeated 360 times in 72 pentameters.The authors speculate that the structural periodicity ofthe viral capsid antigen may induce a T cell-independentantibody response. This concept expands on earlier ob-servations from the Zinkernagel laboratory that B cells

are weakly responsive to the vesicular stomatitis virusglycoprotein when presented in a poorly organized pat-tern on a cell surface but are strongly responsive to thesame viral glycoprotein antigen when presented in ahighly organized repetitive pattern on the virion surface(Bachman et al., 1993). Similarly, a VZV-specific early Tcell-independent antibody response directed against gEon a capsid would explain a selective pressure favoringreplication of a mutant virus lacking an immunodominantB cell epitope. Studies are currently under way to enu-merate the precise number of gE molecules detectablein the emergent VZV envelopes.

MATERIALS AND METHODS

Viruses and cells

The mutant VZV was isolated from a 6-year-old boywith leukemia who contracted chickenpox and was hos-pitalized for intravenous acyclovir treatment in late 1995.The child’s illness responded to treatment, and no un-usual sequelae were observed. The child’s vesicle fluidwas inoculated onto MRC-5 cells in glass tubes, whichwere commercially purchased. The isolate was desig-nated VZV-MSP because the child lived in Minnesota.The VZV-32 laboratory strain was isolated in Texas in1976 from an otherwise healthy child with chickenpox(Grose, 1980). This virus has never been passaged .20times. The VZV-Oka strain was isolated from a Japanesechild with chickenpox and attenuated by M. Takahashi inJapan in the 1970s (Takahashi et al., 1975). All viruseswere subcultured in either MRC-5 cells or human mela-noma cells (MeWo strain). MeWo cells are highly permis-sive for VZV replication, but no infectious virus is re-leased into the culture medium (Grose, 1980). Therefore,transfer of infectivity is carried out by trypsin dispersionof infected cells and relayering of infected cells onto anuninfected monolayer at a ratio of 1:8 (infected-to-unin-fected cells). The HSV-1 Miyama strain was propagatedin the FL line derived from human amnion cells (Padillaet al., 1997).

Antibodies and immunodetection by confocalmicroscopy

mAb 3B3 was produced in this laboratory in 1983(Grose et al., 1983). The antigen for mouse immunizationwas VZV-32-infected cells. mAb 3B3 attaches to VZV gEeven under the stringent condition of a buffer containing1% SDS. Other mAbs to VZV gE (mAb 711), VZV gI (mAb6B5), and VZV gH (mAb 206) were also produced andcharacterized in this laboratory. Conditions for immuno-detection of VZV proteins by laser scanning confocalmicroscopy have been outlined by Duus and Grose,1996. Immunoblotting was performed with the aboveantibodies as described (Grose, 1990).

29VZV gE ANTIGENIC VARIATION

Epitope mapping by recombination PCR mutagenesis

The technique of recombination PCR mutagenesishas been adapted to investigate epitope mapping andtagging (Yao et al., 1993; Hatfield et al., 1997). By thismethodology, the epitope of mAb 3B3 was initiallydefined between amino acids 151–161 in the ectodo-main of the 623-amino-acid gE glycoprotein. The meth-odology for producing plasmid pTM1-VZV gL 3B3.11 isdescribed in detail by Hatfield et al. (1997). As part ofthe investigation in Results (Fig. 8), an additional twocodons were inserted at the N terminus of the 11-amino-acid 3B3 epitope to produce a 13-amino-acidepitope tag in the VZV gL protein. The mutating prim-ers included MP23 (sense) (CAT ACT GTG TCG ACCAAA GGC AAT ACG GTG ACG TG) and MP24 (anti-sense) (TTG CCT TTG GTC GAC ACA GTA TGC GATTGT GAT AG). PCR amplification was performed underthe following parameters: 94°C denaturation for 30 s,50°C annealing for 30 s, and 72°C extension for 5 min;after 25 cycles, there was a final extension at 72°C for7 min. PCR products were combined and transformedinto cells in which the overlapping regions underwentrecombination to yield a plasmid containing the mu-tagenized insert. Plasmid purification was performedwith a Qiagen Maxi Kit. The newly designated pTM1-VZV gL 3B3.13 plasmid was partially sequenced at theUniversity of Iowa DNA Core Facility to confirm theauthenticity of the insertional mutagenesis. This PCRmutagenesis protocol is very reliable with a errorfrequency of ,0.025% (Jones and Howard, 1990).

Subcloning of VZV-MSP gE

The subcloning of wild-type VZV gE has been de-scribed previously (Yao et al., 1993). Briefly, two flankingPCR primers were used that amplified the VZV-MSP gEORF directly from VZV-MSP-infected melanoma cells.These primers also created a SacI and a SpeI restrictionenzyme site at the 59- and 39-ends, respectively. Theprimers were Nco gpI (sense) (CGA CCC GGG GAG CTCCCA TGG GGA CAG TTA ATA AAC C) and IP2 (antisense)(CGC TCT AGA ACT AGT GGA TCC CCC GGG GAA TTTGTC ACA GGC TTT T). PCR amplification was performedunder the following conditions: 94°C denaturation for40 s, 50°C annealing for 40 s, and 72°C extension for 4min; after 35 cycles, there was a final extension at 72°Cfor 7 min. After amplification, the PCR fragment wasdigested with SacI and SpeI before cloning into themultiple cloning site of the expression vector pTM1.

Imaging of viral particles

Clean coverslips were coated with carbon, hydrophi-lized, and irradiated with UV light for 12 h, followed by aglow discharge for a few seconds and then sterilizationby dry heat (160°C overnight). MeWo cells were culti-vated on the glass coverslips. When the cells becameconfluent, they were cocultivated with either VZV-32- orVZV-MSP-infected cells at a 1:8 ratio and incubated at32°C. At the designated times after infection, the cellswere prefixed with 1% glutaradehyde in phosphate-buff-ered saline (PBS) at 4°C for 1 h, rinsed in chilled PBSfollowed by postfixation with 1% osmium tetraoxide inPBS at 4°C for 1 h, and then dehydrated in a gradedethanol series. Finally, after two changes in 100% etha-nol, the specimens were subjected to a critical pointdrying method. In the case of HSV-1 samples, FL cellsprepared on coverslips were inoculated with HSV-1Miyama strain at an m.o.i. of 10. At 24 h after virusinoculation, coverslips were washed with PBS and pro-cessed as described for VZV. Subsequently, the speci-mens were mounted onto aluminum plates and observedwith either an Hitachi S-4000 or an Hitachi S-900 SEM.Imaging was performed at the University of Iowa CentralMicroscopy Research Facility and the University of Wis-consin-Madison Integrated Microscopy Facility.

ACKNOWLEDGMENTS

The authors thank Alan Shaw (Merck Company) for providing unpub-lished gE sequence data on the VZV-Oka strain and Shiro Nii (OkayamaUniversity) plus the Japan Society for the Promotion of Science. Thisresearch was supported by NIH Grants RO1-AI36884, RO1-AI22795,F31-DC00201, T32-GM07337, and T32-AI07260.

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FIG. 8. Recombination PCR mutagenesis. Epitope mapping and tag-ging by recombination PCR mutagenesis were developed in this lab-oratory (Duus and Grose, 1996; Hatfield et al., 1997). In this protocol,two additional codons (3B3.2) were inserted into the mAb 3B3 epitopeto produce plasmid gL 3B3.13 from plasmid gL 3B3.11. (Also see Fig. 3.)

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