JOURNAL OF VIROLOGY,Nov. 1994,p.7406-7417 0022-538X/94/$04.00+0
Copyright C 1994, American Society forMicrobiology
Localization and Putative Function of the
UL20
Membrane
Protein in Cells Infected with Herpes Simplex Virus
1
PATRICIAL.WARD,' GABRIELLA CAMPADELLI-FIUME,2 ELISAAVITABILE,2
ANDBERNARDROIZMANl*
TheMarjorieB. Kovler ViralOncology Laboratories, The Universityof Chicago, Chicago, Illinois 60637,1 and Sectionof Microbiology and Virology, Department of Experimental Pathology,
University of Bologna, Bologna, Italy2 Received 1 April 1994/Accepted 22 July 1994
TheUL20 proteinof herpessimplexvirus 1,anintrinsic membraneprotein,isrequiredininfected Vero cells
inwhichtheGolgiapparatusisfragmented forthetransportof virionsfrom thespacebetweenthe inner and outernuclearmembranesand for the transport offully processedcellmembrane-associatedglycoproteinsfrom thetrans-Golgitotheplasmamembrane.It isnotrequiredin the human 143TK- cellline,inwhich theGolgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a yl gene. (ii) Pulse-chase experiments revealed no detectablealteration inthemobilityoftheUL20 proteininpolyacrylamidegels. (iii)In bothinfected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with "-COP, a protein
associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular spacebut couldnotbe detected in theplasmamembrane.These resultsareconsistent with the hypothesis thatUL20 is acomponentof virion envelopesand membranes of virion transport vesicles and is selectivelyretained from the latter inaGolgicompartment.
The 152-kbp herpes simplexvirus 1 (HSV-1) genome
en-codes at least 15 membrane proteins. These include the glycoproteins gB, gC, gD, gE, gG, gH,gI,gJ, gK, gL, and gM,
thegeneproductsspecified bytheUL20, UL24,andUL34open reading frames, and the predicted gene product of open reading frame UL43 (1, 5, 6, 7, 14, 15, 17, 19, 20, 30, 33, 37, 45). Of theseproteins, morethanhalf, i.e., gC, gE, gG,gI,gJ, gM, UL20, UL24, and UL43,aredispensable for viral replication in at leastsome cells inculture (1, 4, 6, 16, 21, 26-29, 50). The function of thesedispensable membrane proteinsisof consid-erable interestinasmuch as their existence couldsuggestthat theyfunctiononlyincellsnotcultured in vitroorthatcultured
cellsexpressfunctions similartothoseof thedispensableviral proteins. Recent studies have shownthatatleastsome of the dispensableviralglycoproteins mayberequiredforapicalbut
notbasolateral entryinto polarized cells (43).Little is known about the functions of the UL24 and UL43 proteins. The function of the UL20 protein, however, has been studied in
somedetail and is ofconsiderable interest.Thus, viralmutants from which theUL20genehas been deletedmultiply and form small, at times syncytial plaques in some cell lines (e.g., 143TK-); they multiply but do notformplaques in other cell lines(e.g., Vero) (6).Adifferential featureof thetwocell lines is that the Golgi apparatus is fragmented and dispersed in infected Vero cells but not in infected 143TK- cells (8). Detailed studies of cells infectedwiththeUL20-virus revealed the following.
(i) In Vero cells infected with the UL20- virus, virions accumulatedbetweenthe inner andouternuclear membranes, and virus particles were absent from the space between
*Corresponding author. Mailing address: The Marjorie B. Kovler
ViralOncology Laboratories, The University of Chicago, 910 E. 58th St., Chicago,IL60637.Phone: (312) 702-1898. Fax:(312) 702-1631.
infected cells (6). In cells infected with wild-type virus, this
spaceisfilled with virions. The absence of extracellular virions
explained,atleast inpart,the absenceofplaqueformation in Vero cellmonolayer cultures.
(ii) The glycoprotein oligosaccharides of virions purified from infectedVero cellswereunprocessed. The oligosaccha-rides of the bulk of the viralglycoproteinsextracted from Vero cells infected with UL20- virus were fully processed in that theycontained terminal sialic acid residues. Nevertheless,the glycoproteinswerenottransportedtotheplasmamembranes. Incontrast,the glycoproteins associatedwith the membranes of 143TK- cells infected with the UL20- virus were fully processed and transported to the plasma membrane (3). In cells in which theGolgiapparatusisfragmentedanddispersed, UL20 protein appears to function at the point of entry of virions into the exocytic pathway from the outer nuclear membrane and in the egress of viral glycoproteins from the trans-Golgi.
In thisreportwe describe studiesdesignedto elucidate the distribution of theUL20 protein in the infected cellsas akey
steptowardanunderstandingof itsfunction. Asafirststepin the characterization of the protein, we constructed a fusion proteintoraise antiserumagainstUL20 protein.With the aid of this antiserumandanantibodytoaconstituent of theGolgi
aparatus, we determined the following: (i) theUL20 gene is regulated as a -Yl gene, and the protein can be detected in
infected-cell lysates by 6 h after infection; (ii) pulse-chase analysisofUL20revealednodiscerniblealterations inmobility in polyacrylamide gels; (iii) the proteinwas not expressedat
the cell surface but was present in virions purified from extracellular fluid and from the cytoplasm; and (iv) UL20 proteinwasfoundtobepresentinthe nuclearmembranes,in the Golgi apparatus, and dispersed in thecytoplasm butwas notdetected in theplasmamembranesof infected cells.
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UL20 LOCALIZATION AND FUNCTION IN HSV-INFECTED CELLS
ab
I
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UL
FF
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20
alpha 4p X
4
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UL20 - ,
-IS K PS/H
\
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I No _
_ UL20
21-!pRB4335
FIG. 1. Schematicrepresentations of the sequence arrangements of HSV-1 DNA and of the variousplasmids used in these studies.
Lines 1 and2, locations within thegenomeofHSV-1(F) of the BamHI
B', F', and T fragments and theUL20 and UL21openreading frames. Line3,sequencearrangementof plasmidpRB4327;adouble-stranded
oligonucleotide containing multiple cloning sites and BsteII cohesive
endswasinserted into theunique BsteII site between the UL20 and UL21 openreading frames. Lines 4 and 5,sequence arrangementof pRB4335;a
KpnI
fragment containing 1.9 kb of the HSV-1 BamHI Zfragment (containing the a4 promoter [alpha 4p] sequences) was
insertedinto the
KpnI
site ofpRB4327in the propertranscriptionalorientation to the UL20 gene. Line 6, sequence arrangement of pRB158,theBamHI Ffragment of HSV-1 containing the a-TIFgene
and its regulatory sequences. S, Sacl; RV, EcoRV; X, XbaI; H,
Hindlll;
&K,pknl;
PS,Pstl; Bs,BStell.MATERUILSANDMETHODS
Cells andviruses. The isolation andpropertiesofHSV-1(F) andHSV-2(G), theprototype HSV-1 andHSV-2 strainsused inourlaboratories,havebeen described elsewhere(13).Viral
stocks ofwild-typevirusesweremadeinHEp-2cells,andtiters were determined in Vero cells. The genetically engineered mutantsused in these studieswere asfollows.R7032 lacks the
entire
Us8
gene, which encodes glycoprotein E (32); it wasused inexperimentsinwhichitwasdesirabletoavoid reactivity ofimmunoglobulin G (IgG)with the viral Fc receptor (e.g., blackplaqueassaysandpulse-chaseexperiments). R7405(49) contains an epitope derived from glycoprotein B of human cytomegalovirus(CMV) (6)inserted into theUL20geneanda portionof theUL26.5genepromotersequences(25) oriented such that thispromoteris inthepropertranscriptional orien-tationtotheUL20gene.R7225 lacks355bpfrom the5' end of the coding domain of the UL20 gene. (6). All recombinants exceptR7225weregrown,andtitersweredetermined,in Vero
cells. Inasmuch asrecombinant R7225 doesnotformplaques inVerocells,itwasgrown,and titerswere determined,in143 thymidine kinase-minus (143TK-) cells originally obtained from Carlo Croce. Purified virionpreparationswereprepared
from infected BHK cells. The cell lines were maintained in
Dulbecco's modified Eagle's medium supplemented with
ei-
14-FIG. 2. Photograph of immunoblot of electrophoretically
sepa-ratedlysates of HEp-2 and 143TK- cells infected with the indicated viruses.The blotswereprobed with rabbitantiserum raised against the
UL20--p-galactosidase
fusionprotein. Mrsareinthousands.ther5% newborn calfserum(VeroorHEp-2 cells)or5%fetal calfserum (143TK- cells).
Plasmids. Figure 1 illustrates the construction of pRB4335 and pRB158; line 1 shows the sequence arrangement of the HSV-1genome,andline 2 shows thesequencearrangementof the HSV-1 BamHI B', F', and Tfragments and the orienta-tions of the UL20 and UL21 genes. A 1.99-kb PstI-SacI fragmentofpRB451 containing DNAsequencesspanningmap
units0.256to0.294(6)wascloned into thePstI andSacl sites of pGEM3Z (Promega). This plasmid was designated pRB4324. ThePstI andHindlll sites in the polylinker of this plasmid were destroyed by cleavage with PstI and HindlIl
restriction enzymes (New England Biolabs, Inc., Beverly,
Mass.) andtreatmentwith T4polymerase (U.S. Biochemical, Cleveland, Ohio) and the Klenow fragment of DNA poly-merase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). This cleaved plasmid was religated by using T4 ligase (NewEngland Biolabs),andadouble-strandedDNA
oligonu-cleotidecontaining multiple cloningsites and BsteII-compati-ble cohesive ends (5'GTAACGATATCTAGAAGCTTGGT ACCTGCAG3' and its complement 5'GTTACCTGCAGGG TACCAAGCTTCTAGATATC3')wasligatedinto theunique
BsteII site(Fig. 1,line3).Restrictionenzymecleavagesites in this polylinker wereEcoRV, XbaI, HindIII, KpnI, PstI, and BsteII. Insertion of this oligonucleotide destroyed the BsteII site at the 5' end, creatinganEcoRV site, butpreserved the BsteII siteatthe 3' end. Thisplasmidwasdesignated pRB4327 (Fig. 1, line 3). The 1.9-kb KpnI fragment from pRB178 (HSV-1 BamHI Z fragment cloned into pUC18) containing the x4promotersequencesandportionsofthe 5' transcribed noncoding sequencesof the (4 genestartingwith nucleotide
IS r
94-
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7408 WARD ET AL.
Size
Mr markers
-200
- 97 - 69
1 2 3 4 5 6
'-w _ _.
,
.-....INi.
- 46 - 30
Ig
Ig
- 21 - 14
w
N.l 3 6 6 9 12 12 15 15 18 18 24 h.post infection
-_ + + + - + - + - PAA
FIG. 3. Photograph of immunoblot of electrophoretically
sepa-rated lysates of Vero cells infected with 10 PFU of HSV-1(F)percell
in thepresence (+)orabsence (-) of 300 ,ugofphosphonoacetate
(PAA)perml. Replicate cultures of infectedcellswere harvestedat the indicated times after infection, the solubilized proteins derived
from equal numbers ofcells were separatedby electrophoresis and
transferred to nitrocellulose, and the blots were probed with the
polyclonal rabbit antiserum to UL20 protein. Mrs are in thousands.
N.I.,notinfected.
+33wasinserted into the KpnIsiteofpRB4327 such that the
ao4promoterwould bein thepropertranscriptional orientation totheUL20gene.Thisplasmidwasdesignated pRB4335 (Fig. 1, line 5).Inotherexperimentsweusedapromoterconsisting ofthe5' nontranscribed domain of the o4 genefused to the transcribed noncoding domain of the transcription initiation site and the5' transcribed noncoding domain of the
-Y1UL19
gene encoding the major capsid protein. This promoter is expressed throughoutthereproductive cycle of the virus and allows the accumulation oflarge amountsofprotein (23).
Productionof the
UL20-0-galactosidase
fusionproteinand polyclonalrabbit antiserumtotheUL20 protein.The BamHI F' fragment of HSV-1(F) DNA was cloned into pUC18 as pGCF1053. The 670-bp BglII-BamHI fragment from pGCF 1053 was filled inwith the Klenow fragment of DNA poly-merase,ligatedwith12-mer EcoRIlinkers,and cloned into the EcoRI site ofpGEMtoyield plasmid pGCF1057. The EcoRI fragment from pGCF1057containing95% oftheUL20 codingsequencewascloned into the EcoRI site of thepEX-2vector
(Biochemia, Mannheim, Germany), which contains a
cro-Escherichia coli lacZ gene fusion. The recombinant plasmid pGCF1136wasintroducedintoPOP2136cells. Topreparethe
UL20-1-galactosidase fusionprotein, anovernightculturewas
diluted 1:100 and allowedtogrowat30°Ctoanopticaldensity
at600nmof0.1. The culturewasthenshiftedto42°C for 3 h. Pelleted cells from a 2-liter culture were sonicated, and the inclusionbodieswerepelleted, washedtwotimes in phosphate-buffered saline (PBS) containing 1% deoxycholate and 1%
Nonidet P-40, and subjected to preparative denaturing poly-acrylamide gel electrophoresis. The protein corresponding
totheUL20-1-galactosidase fusion proteinwas electroeluted
in electrophoresis buffer containing 0.01% sodium dodecyl sulfate (SDS). New Zealand White rabbits were inoculated subcutaneously with 200 jgof fusion protein emulsifiedwith complete Freund's adjuvant. The rabbits were boosted five
times at 2-week intervals with the same amount of fusion proteininincomplete Freund's adjuvant.Serumsampleswere
FIG. 4. Photographs of [35S]methionine-labeled proteins immuno-precipitatedwith the UL20 rabbit serum (lanes 1 to 3) and ofan
immunoblotof the identical immunoprecipitatesreacted with
UL20-,B-galactosidase antiserum (lanes 4 to 6). HEp-2 cells were mock
infectedorexposedto10 PFUofR7032(gE-)viruspercell. At 8 h
after infection, the mediumwasreplaced with 1 ml of mediumlacking methionine andserum.The cellswerelabeledfor 1 h with 50 iLCiof [35S]methionineat9or16h after infection. In the cultures labeled at
9 h afterinfection, the radiolabeled methioninewaschasedwithcold
methionine at37°Cfor anadditional 12 h. The cells labeledat 16h
afterinfectionwere harvestedimmediately after thelabeling period. Lanes1and 4cellspulse-labeled at9h;lanes 2 and5,cellspulsedat
9 handchasedfor 12h; lanes 3and6,cellspulsedat16 h.Celllysates
wereimmunoprecipitatedwiththeUL20 antiserum,heatedat37°Cfor 30min, separated by electrophoresisonadenaturing polyacrylamide
gel,transferredtonitrocellulose,andexposedtoX-rayfilm(lanes1 to
3). The identical nitrocelluloseblotwasthenprobedwith the UL20
antiserum(lanes4to6). Thesecondary antibodyused in the
immu-noblotreactswiththerabbitUL20antiserumused in the immunopre-cipitation (bandslabeledIg).SinceUL20 proteinaggregatesonboiling
inSDSand doesnot entergels (6),theimmunoprecipitated samples
werenotboiled. Asaconsequence,IgGpresentin thesampleswasnot completelysolubilized and appearsasmultiple speciesin the immu-noblot.
collected andanalyzed forreactivitywiththeUL20 protein in lysatesofcellselectrophoretically separatedindenaturing gels and transferred toanitrocellulose sheet.
Antibodies. The polyclonal rabbit antiserum to UL20was
usedatdilutions of1:1,000forimmunofluorescence, 1:100,000 forimmunoblotting,and 1:200 forimmunoprecipitations. The
mousemonoclonalantibodyto 1-COP,theGolgicoatprotein ofnon-clathrin-coated vesicles (2), wasprovidedbyT. Kreis. The CH-28 monoclonal antibody to glycoprotein B ofCMV andthe monoclonal antibodiesspecificforHSV-1glycoprotein D (H170) and for LCP4 (H640)wereprovided byL. Pereira. UL20
J. VIROL.
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UL20 LOCALIZATION AND FUNCTION IN HSV-INFECTED CELLS 7409
A
B
c
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FIG. 5. Photomicrographofmethanol-fixed andunfixed gE- virus (R7032)-infected Vero cells probed with rabbit
anti-UL20-4-galactosidase
serum orrabbitanti-gM serum. Forty-eight hours after infection, the cells were probed with rabbit antisera. The bound antibody was detected with
abiotinylatedgoatanti-rabbitantibodyfollowedby biotinylated horseradish peroxidase bound to avidin and by4-chloro-1-naphtholsubstrate. (A) Fixedcells; aviralplaquereactedwitha1:200 dilution of rabbitanti-UL20serum. (B) Unfixed cells; a viral plaque reacted with a 1:200 dilution ofrabbitanti-UL20serum. (C) Unfixedcells; a viralplaque reacted with a 1:200 dilution of rabbit anti-gM serum.
The rabbit anti-UL53 antibody was obtained from David Johnson. The goat anti-rabbit IgG fluorescein isothiocyanate
(FITC)-conjugated antibody was purchased from Sigma
Chemical Co., (St. Louis, Mo.). The goat anti-mouse IgG Texasred-conjugated antibody was purchased from Molecular Probes, Inc. (Eugene, Oreg.). Commercial antibodies were usedasrecommendedby the manufacturers.
Transfections. Vero or143TK- cells were seeded onto glass
coverslips and allowed to adhere. Transfections were done
essentially as previously described (31) except that 20 ,ug of
each plasmidwas used and cells were fixed for immunofluo-rescenceanalysis 30 h after transfection.
Polyacrylamide gel electrophoresis and immunoblotting. Infected-cell lysates wereseparated indenaturing gels
consist-ing of 12.5 or 15% polyacrylamide and 0.1% SDS, and the proteins were electrically transferredto nitrocellulose sheets. Thesheets were soakedat roomtemperaturefor 10 to 30 min in PBS containing 5% skim milk (Carnation) andthen were reacted at room temperature for 90 min with the UL20
polyclonal rabbit antiserum (1:100,000 dilution) in PBS
con-taining 1% bovine serum albumin (BSA). The blots were washedonceinPBS-5%milkfor 15 min, rinsed four times in
PBS, and reacted for 1 h in a 1:3,000 dilution of goat anti-rabbit orgoat anti-mouse antiserum conjugated to alka-line phosphatase. The blotswere washed as described above
and developed byusingreagents and protocols supplied in a
Bio-Rad Laboratories(Richmond, Calif.) kit.
Black plaque assay. The procedures for the black plaque assaywereessentially as previously described (24). Verocell
monolayercultures were exposed to 10to 50 PFU of R7032
(gE-)virus per
25-cm2
cellculture flask and incubatedat34°Cfor 48 h. The monolayers were either fixed in methanol to
permeabilize the cells or left intact. The monolayers were
incubated at roomtemperatureinblocking mediumconsisting
of medium 199supplementedwith 1% heat-inactivated horse serum(199v) for 1 h. Cellswere then incubated in asolution
containingtheappropriate primary antibodyat room temper-aturefor90 min. Foranalysis of UL20 expressionattheplasma membrane, infected cellswerereacted with eitherUL20 poly-clonal rabbit antiserumor anti-gM(ULlO protein) polyclonal rabbit antiserum (1:200 dilution). The monolayers were washed three times with medium 199supplemented with 1% fetal calfserum (199v), and incubated in a 1:500 dilution of biotinylated goat anti-mouse or goat anti-rabbit antibody at room temperature for 1 h. Monolayers were again washed three times and reacted with avidin boundtobiotin-conjugated
horseradish peroxidase (Vector Laboratories), and the color reaction wasdevelopedby using 1-chloro-4 napthol substrate aspreviously described (24).
Immunoprecipitations.Forpulse-chase experiments, radio-labeled, infected HEp-2 cell lysateswere solubilized in 200pul
ofimmunoprecipitation buffer(consisting ofamixtureof1% Nonidet P-40, 1% sodium deoxycholate, 10 p.M TPCK
[tolyl-sulfonyl phenylalanyl choromethyl ketone], 10 p.M TLCK
[cx-tosyl L-lysine chloromethyl ketone] in PBS), sonicated
briefly, andclarifiedby centrifugation for 20 min at4°Cin an
Eppendorfmicrocentrifuge. Fifty microliters ofa50%slurry of protein A-conjugated Sepharose beads (Sigma)was addedto theclarifiedlysatesonice and left for1htoremovematerials whichmightreactnonspecifically.The beadswereremovedby
centrifugation, andthe supernatantfluidswerereactedwith 1
p.l of UL20 antiserum overnight at 4°C. The immune com-plexeswererecoveredby addition of50 p.lofa50% slurry of protein A-Sepharose beads and incubationonicefor1h. The beadswerewashed four times withimmunoprecipitationbuffer and oncewith 100 mM NaCl-10 mM Tris-HCl, pH 7.4. The
immunoprecipitated proteinswere eluted from the beads by
addition of50p.l ofsamplebuffercontaining2% SDS and 5%
3-mercaptoethanol
andheating of the samples for 30 min at 37°C,electrophoretically separatedon adenaturing
polyacryl-amide gel, transferred to a nitrocellulose sheet, reacted with theUL20 antiserum,andexposed toX-ray film.
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7410 WARD ET AL.
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A. 7h. B. 16h C. HEp-2Cells D. 143TK-Cells
FIG. 6. Photographs ofbiotinylated, immunoprecipitated proteins. (AandB)Vero cellmonolayerswereinfected withHSV-1(F)virus(10PFU
percell), andthemonolayerswere exposedtoNHS-LC-biotinat7or16 hpostinfection. (CandD)HEp-2or143TK-cellswereinfected with
HSV-1(F)orR7405(UL20-CMV tagged,used with CMVantibody)virus(10PFUpercell),and themonolayerswereexposedtoNHS-LC-biotin
at16hpostinfection.All cellsweremaintainedat34°C. Surface-biotinylatedcellswerewashed, harvested,andlysedinimmunoprecipitationbuffer.
The celllysateswerereactedwith eitherUL20rabbitantibody (lanesUL20pol. Ab.),monoclonalantibodytothe CMVepitope (lanesCMVmon.
Ab.), monoclonal antibodytogD (lanes gDmon.Ab.),rabbitantibodytoUL53(lanesUL53pol. Ab.),ormonoclonalantibodytoICP4(lanesICP4 mon.Ab.). Allimmunoprecipitated samplesother thanUL20orUL53wereboiled beforeelectrophoresis.Theprecipitates containingUL20or
UL53 rabbitantiserumwereheatedat37°Cfor 30min priortoelectrophoresis.Theimmunoprecipitated proteinswereseparatedondenaturing
polyacrylamidegels(panelsA andB,12.5%gel; panelsC andD,11%gel),transferredtonitrocellulosesheets,and reacted withstreptavidin-horseradish peroxidase conjugate.Theprecipitated proteinswerevisualizedby usingthe ECLchemiluminescence detectionsystemfrom Amersham. Thearrowhead
inpanelB indicatesthepositionofUL20 migrationasdetectedbyimmunoblots(not shown). M.W.,molecularweight.
Forimmunoprecipitation of cell surfaceproteins, infected-cellmonolayerswerewashed withPBS andincubatedatroom
temperature in asolution of 75 jig of NHS-LC-biotin (Pierce Chemical Co., Rockford, Ill.)perml of PBS for 30min. The cells were rinsed with PBS, harvested, and lysed in immuno-precipitation buffer. Immunoprecipitations, electrophoresis, and transfer of the precipitated proteins were done as de-scribed above exceptthatgoatanti-mouseIgG-coatedagarose
beads (Sigma ChemicalCo.)wereusedtoimmunoprecipitate samplesincubated withmonoclonal antibodies and allsamples exceptthoseprecipitatedwithantibodies toUL20ortoUL53 wereboiledbeforebeing loadedonSDS-polyacrylamide gels.
Thenitrocellulosesheetswerethenblocked overnightat40C in asolution of5% milk inPBS, rinsed in PBS containing 0.1%
Tween 20, and reacted with streptavidin-horseradish peroxi-dase conjugate (1:1,500 dilution in PBS-0.1% Tween 20) (Amersham Life Sciences) for 1 h. The membranes were
washedextensivelyinPBS-0.1% Tween20 and developed by usingreagentsandprotocolsforchemiluminescence obtained from Amersham Life Sciences. The developed blots were
exposed toX-rayfilmtovisualize theprecipitated proteins. Immunofluorescence. Approximately
10'
Veroor143TK-cells were seeded onto sterilized glass coverslips in 24-well tissue cultureplates (Costar)and allowedtoattachovernight.
The cellswereexposedto5 PFU of R7032(gE-)viruspercell andfixed in ice-cold methanolat14to16hafter infection. The coverslipswerefirstblocked for 30minatroomtemperaturein PBS containing 1% BSA and then reacted with primary antibody for 1 h at room temperature, washed in PBS, and reacted with the appropriate fluorescein-orTexas red-conju-gated secondary antibody for 1 h atroom temperature. The coverslipcultureswererinsedagaininPBS, mountedonglass
microscope slideson adropof90%glycerolin PBScontaining 1 mgofp-phenylenediamineperml. and examinedin aZeiss confocal fluorescence microscope. Digital images of the fluo-rescentprofileswereacquired by usingsoftwareprovidedwith theZeiss confocalmicroscopeandprinted byaCP210 Codon-icsdigital printer.
Virion preparation. Virions were purified essentially as
describedby Szilagyi and Cunningham (47). Briefly, medium containing virions was centrifuged at 1,000 x g for 30 min. Virionswerethenpelleted by centrifugationfor 2 hat80,000
x g, resuspended in Dulbecco's modified Eagle's medium
lacking phenol red,andlayeredontoa5to15%Ficollgradient made in the same medium. Bands containing the virion particleswerecollectedbysidepuncture,diluted with medium lacking phenol red, pelleted by centrifugation (80,000 xgfor 2 hat4°C), gently resuspendedin 200
RI
ofPBS,and storedat 21`.-J. VIROL.
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UL20 LOCALIZATION AND FUNCTION IN HSV-INFECTED CELLS
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FIG. 7. Photographs ofbiotinylated,immunoprecipitatedproteins. HEp-2 cells were infected with HSV-1(F) (lanes 1 and 3) or with UL20- virus R7225 (lanes 2 and4) and reacted with biotin at 16h postinfection. Lanes 1 and 2, infected cell monolayerswerewashed and exposedtoNHS-LC-biotinas described in the legendtoFig. 6. The surface-biotinylated cellswerewashed, harvested, and lysed in immunoprecipitation buffer. The cell lysates were reactedwith anti-UL20 rabbit serum. Lanes 3 and 4, infected cells were harvested, washed,and disrupted in0.4%Nonidet P-40. The cytoplasmic extracts werethenexposed to 300 ,ug of NHS-LC-biotin per ml for15min at roomtemperature.Thereaction was quenched by the addition of100 mMTris-HCl,pH7.5,and thecell lysateswerereacted withanti-UL20 antiserum.Immunoprecipitated, biotinylated proteins wereseparated by electrophoresis and transferred to nitrocellulose blots, and the proteinswerevisualized as described inthelegend toFig.6.
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-80°C.Afraction of thevirionpreparationwasprocessed for electron microscopic observation in order to monitor the extent ofpurification.
RESULTS
Production of UL20 antiserum. NewZealand White rabbits were inoculated subcutaneously with
UL20-3-galactosidase
fusion protein that had been electroeluted from denaturing
polyacrylamide gels as described in Materials and Methods.
The resulting antiserum was reacted with electrophoretically
separated infected-cell proteins that hadbeen transferred to
nitrocellulose sheets. The results are shown in Fig. 2. The antiserum reacted withasingle proteinband withanapparent Mr of approximately 24,000 in lysates of HEp-2 or 143TK-cells infected with HSV-1(F). An identical pattern was ob-served forlysatesof infected Vero cells(datanotshown).The
specificityofthe antiserumwasdemonstratedby itsreactivity
with a single protein band with lysatesof HSV-1(F)-infected
cells butnotwithlysates of mock-orR7225(UL20-)-infected
cells (Fig. 2). The antiserum did not react with infected-cell
polypeptidesfromlysatesof cellsinfected withHSV-2(G)(Fig.
2).
Requirements and timing ofexpression of the UL20 gene. Verocellswereexposedto 10 PFUofHSV-1(F)percell and incubated at37°Cinmedium199vinthe presenceorabsence
ofphosphonoacetate (300
jig/ml;
Sigma). At thisconcentra-tion, phosphonoacetate totallyabolishes viral DNAsynthesis.
Atintervals the cellswere harvested, solubilized indisruption
buffer containing 2% SDS and 1% ,-mercaptoethanol, incu-bated at 37°C for 20 min, subjected to electrophoresis in a
denaturing polyacrylamide gel,transferred to anitrocellulose
sheet,andreacted with theanti-UL20rabbitserum.The results
(Fig. 3) indicate that theUL20 protein accumulated inreadily
detectable amounts by 6 h postinfection, andno appreciable
change in the amounts ofprotein was detected beyond 9 h
postinfection. The observation that the synthesis of UL20
proteinwasdiminished butnotabolishedbyphosphonoacetate
suggests thattheUL20 genewasregulated as a
yl
gene.1 2 3 4 5
FIG. 8. Photographs of silver-stained or immunoblotted electro-phoretically separated virion polypeptides. Virions were prepared from the extracellular fluidofHSV-1(F)-infected BHKcells. Virion polypeptideswere denatured inabuffer containingSDS and 1-mer-captoethanolandwereseparatedon adenaturing polyacrylamide gel. Theproteinswereeither silver stained(lanes1, 2,and3)ortransferred
to nitrocellulose sheets and reacted with the anti-UL20rabbit
anti-serum (lanes 4 and 5). Virion protein designations (center) and molecularweightsize markers(in thousands)(sides)areindicated.H., heavy; L.,light.
Theelectrophoretic mobilityofpulse-labeled UL20 protein is not altered after a chase. The majority of membrane-associatedproteinsencoded in the HSV-1 genome have been showntobemodifiedbyglycosylation.Althoughthepredicted
amino acid sequence of theUL20openreadingframe present in HSV-1 strain 17 does not contain consensus sites for N-linkedglycosylation(30),we wereinterestedto seewhether there was any change in the electrophoretic mobility of the UL20 protein in a pulse-chase experiment. In this series of
experimentswe usedHEp-2cells inasmuch asearlier studies
have shown that Vero cells contain an endogenous protease which may alter the electrophoretic mobilityof viralproteins
duringachase(35). HEp-2cells grownin 25-cm2 tissue culture flaskswere exposedto 10 PFU of R7032
(gE-)
per cell. At8 or15 h after infection,themediumwasreplacedwith 1mlof thesame mixture butwithout methionine or the serum sup-plement. The cellswere pulse-labeledateither 9or 16 h after infection with50,uCiof[35S]methionine
for1hat37°C.Inthe cultures labeledat9h,thelabelingmediumwasthenreplaced
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FIG. 9. Confocal,digitalimagesofinfected Vero(a, b,andc)or143TK-(d,e,andf)cells reacted with antibodiestoUL20(green fluorescence)
andj-COP(redfluorescence).Cellsweregrownonglass coverslips, infectedwith 3 to 5 PFU of R7032(gE-)virus percell,andfixedin ice-cold
methanolat14 to16h after infection. Thecoverslipswerereacted with theprimary antibodiesfor 1.5 hat roomtemperature, washed inPBS,and subsequentlyreacted with theappropriate fluoresceinorTexasred-conjugated secondary antibodyfor 1 hat roomtemperature.(aandb) Split imagesof Vero cells double stained with antibodies to
3-COP
(a;Texasredfluorescence) andtoUL20(b;FITCfluorescence). (c) Overlayof imagesinpanels a and b.(dande) Split imageof 143TK- cells double stained with antibodiesto,B-COP(d;Texasredfluorescence)andtoUL20 (e;FITCfluorescence). (f) Overlayofimagesinpanelsd ande.Theimageswerecapturedwith softwareprovidedbyZeiss with theinstrument andprintedbyaCodonics CP210printer.Theimageinpanelc wasattenuatedtoreduce theintensityoffluorescence.with medium containing normal levels of unlabeled methio-nine, andthe cells were reincubatedat37°Cfor an additional 12 h. The cells labeled at 16 hafterinfectionwere harvested
immediately after the labelinginterval. The
[35S]methionine-labeledimmunoprecipitated proteinsareshown in Fig. 4, lanes 1to3.Therelative migration of UL20 protein was not altered at 12h after the
[35S]methionine
pulse(lane2),norwasthere evidence ofanyalteration when cells were pulse-labeled at 16 hafterinfection (lane 3). Furthermore, the migration of UL20 protein was also unaltered in pulse-chase experiments done with cells treated with monensin from the time of infection(data not shown), suggesting that UL20 is not modified by
0-linkedglycosylation (22). The identification of the
[35S]me-thionine-labeled species as UL20 was verified by reacting the nitrocellulose sheet with the UL20 polyclonal serum (Fig. 4, lanes4 to6).The two moreslowly migrating species in lanes4 to6represent reactivity of the UL20 antiserum presentin the
immunoprecipitationreactionwith the alkaline
phosphatase-conjugated anti-rabbit antibody used in the immunoblot. We shouldnotethat (i) theduration of thepulsewas notexcessive giventhe slow rate ofglycosylationof viralproteinsininfected cells and (ii) the anti-rabbit alkaline phosphatase-conjugated secondary antibody used in the immunoblot does not detect the light chain of IgG present in the immunoprecipitation reactions (data notshown).
UL20 is not detected at the plasma membrane. Earlier studies have shown that the UL20 protein is associated with cellular membranes. To determinewhetherUL20was present intheplasmamembrane,two series ofexperimentsweredone. In thefirst,unfixed Vero cells infected withgE- (R7032) virus wereexposedtorabbitanti-UL20 polyclonal antibodyandthen to biotinylated goat anti-rabbit immunoglobulin followed by avidinbound tobiotinylated peroxidase (ABCcomplex). Ina parallel experiment, a replicate infected-cell culture was ex-posed to rabbit anti-gM polyclonal antibody (5). The proce-dure, designatedthe blackplaquetechnique (24),detectedthe J.VIROL.
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UL20 LOCALIZATION AND FUNCTION IN HSV-INFECTED CELLS 7413
FIG. 10. Digital, unprocessedimages ofanR7032-infected Vero cell reacted with antibody to UL20 and anti-rabbit IgG conjugated to FITC. Cellswereinfected,fixed, and stainedasdescribedinthe legend to Fig. 8. Twelve0.5-jimsectionsthrough the z axis werecaptured and printed by a Codonics CP210 printer. The section in the top left panel is from the basolateral side of the cell.
presence of abundant amounts of gM (Fig. 5C) but not of UL20 proteins on the surface of infected cells (Fig. 5B). The UL20 protein does react with the antibody in the same system butonly after fixation of the cells with methanol, which renders the cells permeabletothe antibody(Fig. 5A).
Inthe second series of experiments, surface proteins from cells infected with wild-type virus were biotinylated at 16 h after infection as described in Materials and Methods. The cellswereharvested andlysed,and the celllysateswerereacted with the rabbitanti-UL20polyclonal antibodyorantibodies to CMVepitope for detection of CMV-tagged UL20. Antibodies totheviralglycoproteinDwereincluded as apositive control for surfaceprotein expression. The anti-UL53 rabbit polyclonal serum or anti-ICP4 monoclonal antibody was included as a
negative control,since these proteinsare notexpressedatthe
cell surface. Theimmunoprecipitated proteinswereseparated
ondenaturingpolyacrylamide gelsandtransferredto
nitrocel-lulose sheets. The sheets were reacted with streptavidin-horseradish peroxidase conjugate and developed by chemilu-minescence (ECL).TraceamountsofUL20weredetectedby chemiluminescence in immunoprecipitates from the surfaces of infected 143TK- and
HEp-2,
butnotVero, cells maintainedat37°C(datanotshown).Arepeatof thisexperimentat34°C
failed to yield detectable amounts of UL20 protein on the surfaceof infected Vero,143TK-,orHEp-2cells(Fig.6, lanes
UL20 pol. Ab.), whereas large amounts of gD were readily
detectable at both 7 and 16 h postinfection (Fig. 6A and B, lanes gDmon.Ab.). Furthermore, UL20 couldbebiotinylated ifinfected cellswerefirstdisrupted with NonidetP-40(Fig. 7, lane 3), while UL20 was not detected on the surface of wild-type or UL20 virus-infected cells or on UL20 virus-infected cells that had beendisrupted(Fig.7,lanes1, 2, and4).
Two comments regarding these experiments are
appropri-ate.First,weusedagE-virus in the first series ofexperiments to avoidnonspecific interaction of gE with the Fc portion of the IgG molecule. Since the absence of UL20proteinon the surface of infected cells could be due to the absence of gE,
wild-type virus (gE+) was used in the second series. As
expected, gEwas immunoprecipitated nonspecifically to vari-ousdegrees,dependingoncell type and rabbit serum (Fig.6).
Inthis instance, discrimination between specificand nonspe-cificbindingwas feasible and in fact reinforcedourconcerns regarding the interaction of gE with the Fc domain of IgG. Second,wesuspect that thetraceamountsofUL20detectedon the surface of 143TK-orHEp-2 cells incubatedat37°C(data
notshown)wasdue to the presence of virionsatthecellsuface, sincewecouldnotdetect anyUL20proteinatthecell surface of cells maintainedat34°C. Theegress of virionsfrom cells is delayed at 34°C (18). We cannot exclude leakage of the infected cells incubated at 37°C, however, neither ICP4 nor
UL53 (both internal proteins) was labeled with biotin and
precipitated from the surface of infected HEp-2 or 143TK-cells maintained at34°C (Fig. 6C andD). We conclude from these studies that the plasma membrane of infected cells containsatmostsmallornegligibleamountsofUL20protein.
UL20ispresentin extracellularvirions. Inasmuch asUL20 is required to facilitate transport of virions from the space between the inner and outer nuclear membranes to the extracellular space, it was of interest to determine whether UL20isphysically associatedwith virions. Intheseexperiments
virionswere collected from extracellular fluid and banded in FicollgradientsasdescribedbySzilagyiandCunningham(47).
Thisprocedure allowed the differentiation between noninfec-tious (light) particles and infectious (heavy) particles. The
electrophoretically separated virion proteins obtained as
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FIG. 11. Digital, unprocessed imagesof Vero cellstransfectedwithplasmids containingtheUL20openreadingframeasdescribedinMaterials and Methods. The cellswerereacted withantibodytoUL20and anti-rabbitIgG conjugatedtoFITC.Cellswerefixedinmethanolandstained30 hafter transfection.
transferred tonitrocellulose sheets and stained with the anti-UL20 polyclonalrabbit antiserum. The results shown inFig.8 indicate that UL20 was present in extracellular virions. We should note that the electrophoretic profile of the virions presentedinFig.8 (lanes2and3)differs from thatpublished previously by this laboratory. The problem stems from the observation that while boiling in SDS is essential for the solubilization ofmanyvirionproteins,itcausestheaggregation
of integralmembrane proteins with multiple transmembrane domains (see reference 44 and references therein; 46). As a
consequence,UL20 proteininpreparationsboiled in SDS does not enterthegel. Conversely, failuretoboil results in electro-phoretic profilesof virionproteins in whichsomeproteinsare underrepresented.
Localization ofUL20 proteinin infected cells.Theobjective of theseexperimentswastodetermine the sites oflocalization of UL20 in the infected cells by immunofluorescence with selectedreagents. Two series ofexperimentsweredone.
In the first series, the cells were infected with gE- virus (R7032)topreclude nonspecificfluorescencecaused by inter-actionofIgGwithFcreceptors.The infected cellswerestained
withthe rabbitanti-UL20 serumaloneordouble stained with
theUL20antiserum andamonoclonalantibodytothe Golgi-derived vesicle coat protein 1-COP. Since in the course of
thesestudies it becameapparentthat UL20 protein colocalizes in partwiththeGolgi-and intermediate compartment-associ-atedprotein 1-COP,the studies focusedprimarilyoninfected Vero andhuman143TK- cells. The selection of thesecellsfor
our studies was based on the observation that the Golgi
apparatusis fragmentedand dispersedin infected Vero cells but notin infected 143TK- cells(8).
The results are shown in Fig. 9 and 10. In Fig. 9 the photographs show individual Vero (a) and 143TK- (d) cells reacted with the anti-,-COP monoclonal antibody and anti-mouseIgG conjugatedtoTexasred. Figure9b andeshow the same cells reacted withantibodytoUL20 and anti-rabbit IgG
conjugated toFITC. Figure 9c and f show the twoimagesof each cellsuperimposed.These results indicate that thatUL20
is present in nuclear membranes and that the strongestsignals
in the cytoplasmic domains of both Vero and 143TK- cells were colocalized with
p-COP.
Moreover, the distribution ofp-COP
wasconsistent with thefragmentation oftheGolgi in Verocells butnotin 143TK-cells. To make thecoincidenceofthe n-COP and UL20 colocalization more visible, the UL20
antibody fluorescence in Vero cellswas diminished (Fig. 9b
andc). Figure 10shows sections of another infected Vero cell
reacted withUL20antibodyandanti-rabbit IgGconjugatedto FITC. The results showastrongly fluorescentringaround the nucleus which extends into but doesnotfill thecytoplasm.
On the basis of the association ofUL20with membranes(6)
and its localization in infected cells, we conclude that this protein is present in nuclear membranes and in various cyto-plasmic structures, including all or portions of the Golgi
apparatus. Asnoted above, UL20 is notpresent in detectable amounts ontheinfected-cell surface.
In asecond series ofexperiments, Vero cellswere cotrans-fected withaplasmid containingthe entirecodingsequenceof UL20 driven by either the (4 or the ot-y promoter (see
MaterialsandMethods)and withasecondplasmid containing
the ao-TIF gene and itsregulatory sequences.Figure 11 shows that in the absence of viral infection,the fluorescence due to the UL20 protein was diffused thoughout the cytoplasm and J.VIROL.
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UL20 LOCALIZATION AND FUNCTION IN HSV-INFECTED CELLS
Extracellular
S
aeGolgi
c,Q
UL20% N f
Extracellular Space
Procesed
~~Uu_ eqoligosaccharides
Golgi
Cytoplasm ,.: -, Q..
Cvtoplasm
Unprocessed
oligosaccharides
Nucleus
FIG. 12. Schematic representation of the distributionofUL20, virions, and cellular membrane-associated glycoproteins in Vero and 143TK-cellsinfectedwithwild-type and UL20- virus. The right panel shows the events occurring in UL20- virus-infected Vero cells. From the bottom of thediagramup,capsidsbecome envelopedattheouternuclear membrane and accumulate in the space between the nuclear membranes. The oligosaccharidesonthe glycoproteins in virions and those contained in the nuclear membranes are not processed (3). However, the glycoproteins associated withmembranesdo reachtheGolgi apparatus and are fully processed, but they are retained in the trans-Golgi compartment (i.e., in a compartment after addition of the terminal sialic acid) and are not transported to the plasma membrane. The thin dashed lines represent unprocessed oligosaccharides, the Golgi stacks are shorter to emphasize that they are fragmented and dispersed, and the thicker dashed lines representprocessedoligosaccharides.Theleftpanel represents 143TK- cells infected with wild-type virus. In these instances the glycoproteins associated with cellular membranes transit from the trans-Golgi to the plasma membrane, and the virions enter the exocytic pathway through transportvesicles formed at the outer nuclear membranes. The dots lining all membranes except theplasma membrane represent UL20 protein, which does notreach the plasma membrane. The Golgi here is shown to be intact, as would be expected in infected 143TK- cells. Note that the samegeneralpatternofproteinand virion distribution would occur in 143TK-cells infected with UL20- virus, except that UL20 protein would beabsent, or in Vero cells infected with wild-type virus, except that the Golgi apparatus would be fragmented and dispersed.
included the perinuclear space. It is noteworthy that the amountofUL20 fluorescence detected in the transfected cells was ashighorhigher than thatseenin infectedcells, possibly because the promoters fused to the UL20 coding sequences were strongerthan the natural promoter of the gene.
DISCUSSION
The studiespresented in this report show the following.
(i)Antiserum raisedagainstachimeric protein consisting of the,-galactosidase gene fusedto95%of thecoding squences of theUL20protein reacted withapolypeptidewithanMrof approximately 24,000. This is consistent with the predicted molecular weight of the protein and with the results of previous studies with epitopically tagged proteinaswellaswith antipeptide antisera usedto immunoprecipitate UL20 (Mr
21,000)from infected cells (6, 29).
(ii) Addition of phosphonoacetate to cells infected with
HSV-1(F) did not block synthesis of UL20. The protein
accumulatedand wasdetectedby immunoblotting beginningat 6 hafter infection.Weconclude thatUL20,aprotein dispens-able for viralreplicationin cells inculture,isregulatedas aY1 gene. Our studiesare atvariance withaprevious report which suggested that the UL20 protein is encoded by an essential gene and isexpressed asearlyas2 to 4hpostinfection (29).
(iii) The predicted amino acid sequence of the UL20 protein encodedby HSV-1 strain 17 (30) doesnotcontain consensus
sites associated with N-linked glycosylation, and pulse-chase analysis of UL20 didnotreveal anysignificant alterations in the relative electrophoretic mobility of the protein in either the presence (Fig. 4) or absence(datanotshown) of monensin.
(iV) UL20 could not be detected at the cell surface as measured either by antibody staining of unfixed cells by the blackplaque assay(Fig. 5) orby immunoprecipitation of cell surfaceproteins (Fig. 6 and7). These results were surprising
since all of the membraneproteins that have been showntobe virion associated have also been detected in the plasma
membrane (5, 17, 19, 37,39). WhetherUL20 containsspecific
retentionsignals topreventit fromreachingthe cell surface is unknown.
(V) UL20was present in virions isolated from extracellular medium.
(vi) Immunofluorescence analyses of infected cells double stained with antibodies toUL20 and
a-COP
proteinrevealed thatUL20ispresentin acytoplasmiccompartmentinvolved in the exocytic pathway. In addition, UL20 was detected in aperinuclear ring, possibly the nuclear membrane. Of
signifi-canceis thefinding thatincertain cell types(VeroandHEp-2)
infected with HSV-1, the Golgi becomes fragmented and dispersed, and there is aconcomitant redistribution of
Golgi-resident enzymes and ofa Golgicoatprotein (8).This redis-tribution is a late event in the viral replicative cycle and
possibly reflects a disequilibrium between anterograde and
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7416 WARD ET AL.
retrogradetransportcaused byanoverwhelminginflux of viral
glycoproteins and virions into the exocytic pathway (8). Our results showed that regardless of cell type, the distribution of UL20 closely approximated that ofthe Golgi-associated
pro-tein.
The results reported here raise several key issues with
respect to viral egress and the role of UL20 in this process. While it is generally agreed that capsids assemble in the nucleus andareenvelopedattheinnernuclear membrane (38, 39), theprecise events whichgovernviriontransportbetween thesite ofenvelopment and theextracellularspace have been
the subject of much debate (reviewed in reference 40). The results obtained from several studies support the model, proposed on the basis of electron microscopic studies by Schwartz and Roizman (42), that virions are transported throughthecytoplasminmembrane-boundvesicles (9,22, 38, 40, 48) as would be expected for macromolecules that are
destined for export from the cell. Vesicularly transported proteinsfollowadefault pathway from the endoplasmic
retic-ulum through the Golgi to the plasma membrane unless the proteins contain specific retention signals (reviewed in
refer-ences 34, 36, and 41). That virions and viral glycoproteins utilizethenormal cellular exocytic pathwayhas been suggested by the observations that viral glycoprotein processing and maturationaswellasvirionexocytosisare impaired inmutant
cells defective in Golgi enzymatic activities or in competent
cellsexposedtoagentswhich disruptthis pathway (10, 11, 22). Although in cells infectedwith theUL20- virus, virion
exocy-tosis wasblocked inapre-Golgicompartment,viral glycopro-teinswere transportedatleastto the trans-Golgi inasmuchas
they contained terminal sialic acid residues (3). These obser-vations suggest that the vesicles which transport virions may
have anorigin different from that of vesicles which transport
viralglycoproteins (3, 6). The apparent presence ofUL20 in
the nuclear membrane is consistent with the requirementfor UL20- to facilitate the exocytosis of virions from the space
betweenthe innerandouternuclearmembranes. The
pheno-type of the UL20- virus, aswell as that ofavirus carrying a
temperature-sensitive mutation in the gHgene and
character-ized byretention of infectiousvirus containinggHin infected cells maintained at the nonpermissive temperature (12),
sug-gests that viral egress may not occur by default, as would
normally be expected for vesicularly transported macromole-cules, but ratheras a directedseries ofevents.
Theresults ofourstudies andthe considerations cited above lead ustoproposethe model illustratedin Fig. 12. According
to this model, UL20 is present in the nuclear membranes, possibly in theendoplasmic reticulum (although this remains tobeproven), in the Golgiapparatus,and,as a consequenceof envelopment, also in virion envelopes. The UL20 present in transportvesiclesisretained insome Golgicompartment and does not reach the plasma membrane. Finally, the UL20 protein present in virions remains associated with the virion envelope throughout its transit through the cytoplasm. The UL20 protein is required for exocytosis of virions in infected
VeroandHEp-2cellsand, mostprobably, in infected cellsin
which the Golgi apparatus is fragmented and dispersed throughout thecytoplasm. Conceivably thegeneencodingthe
UL20 proteins may have evolved to compensate for the fragmentation of the Golgiapparatus.
ACKNOWLEDGMENTS
We thank L. Pereira, D. Johnson, and T. Kreis for the gifts of invaluableimmunologicreagents.
Thestudiesatthe Universityof Chicagowereaided bygrantsfrom the National Cancer Institute (CA47451) and the National Institute
for Allergy and Infectious Diseases (AI24009), by the U.S. Public
Health Service, and by an unrestricted grant from Bristol-Myers
Squibb Program inInfectious Diseases.The studies attheUniversity of Bologna were aided by grants from Consiglio Nazionale delle Ricerche, Target Project in Genetic Engineering, Target Project on Biotechnology, Associazione Italiana per la Ricerca sul Cancro
(AIRC), and ProgettoAIDS-Istituto Superiore diSanita.
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