A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

0022-538X/92/063803-08$02.00/0

Copyright ©D 1992,American Society for Microbiology

A

Cellular

Function

Is

Required

for

Pseudorabies Virus Envelope

Glycoprotein

Processing and Virus

Egress

M. E. WHEALY,' A. K. ROBBINS,' F. TUFARO,' ANDL. W. ENQUISTl* DuPontMerckPharmaceutical Company, ViralDiseases Research, Experimental Station,

Wilmington, Delaware 19880-0328,' and Department of Microbiology, University of

British Columbia, Vancouver, Canada V6T1232 Received 15January 1992/Accepted 23 March 1992

ThemouseL-cell mutantgro29 is defective foregressof herpes simplex virustype 1 (HSV-1) virionsand is

significantlyreduced inHSV-1glycoproteinexport(B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report,we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctivesetof defects after infectionofgro29cells.Specifically,weidentifydefects in therateandextentof viral

glycoproteinexport, infectious particleformation, plaque formation, andvirusegress. Theinitialrateofviral

glycoprotein synthesiswasunaffected ingro29 cells,but theextentofexportfromtheendoplasmicreticulumto

theGolgi apparatuswas impaired and export through theGolgi apparatusbecameessentially blocked late in

infection.Moreover, by using asecretedvariant ofa viralmembraneprotein,wefoundthatexportfrom the

Golgiapparatus outofthecellwasalso defectiveingro29cells. PRVdoesnotform plaquesongro29 monolayers.

A lowlevel ofinfectiousvirus isformedandreleased early afterinfection, but furthervirus egressis blocked.

Taken together, these observationssuggestthat the gro29 phenotype involves eithermultipleproteinsor asingle

proteinused atmultiplesteps in viralglycoproteinexport andvirus egressfrom cells. Moreover,this host cell

protein is required byboth HSV and PRV forefficientpropagation in infected cells.

It is well established that the glycoproteins encoded by herpesviruses use the host cell secretory mechanism for

synthesis, processing, and sorting of the glycoproteins to

appropriate organelles (3, 6, 8, 9, 12, 13, 18, 22). Even

though we know these viral glycoproteins play important

rolesinthe viral life cycle,wedonotunderstand the precise processingandorderedassemblyeventsrequiredfor

forma-tion ofinfectiousvirus. Currently, therole ofthe hostcellas

wellasthevirusintheassembly andtrafficking of virions is

thesubject ofintensiveinvestigation.

Tounderstandthe role of thesecretorypathwayinthe life cycleof thisfamilyofviruses, Tufaroandcolleagues isolated

amurineL-cellmutant,gro29, whichisunableto supportthe propagationofherpes simplexvirustype 1 (HSV-1) (1, 21). Despite normal infection and viralgene expression, HSV-1

virions did not traverse the secretorypathway and instead

weretransportedtocytoplasmic vacuoles, wherethey

accu-mulatedasnoninfectiousparticles.Theinabilitytotransport

HSV-1virionsto the cell surface ofgro29cellswas

accom-panied byareduction in thetransportandprocessingof viral

glycoproteins, although the blockto glycoprotein transport was not as strong as the block to virus egress. It was

proposed that gro29 cells were able to transport and carry

outsomeof theprocessingeventsfor viralglycoproteinsbut

wereunabletoallow thetransportoflargercomplexes,such

as virions.

Pseudorabies virus(PRV)isdistantlyrelatedtoHSV-1 but sharesacommonlifecycleas analphaherpesvirus (3, 25). It

was of some interest to determine first whether the gro29 phenotypewas specificonlyto HSV-1 and second whether PRV was blocked in gro29 cells, to take advantage of the

uniquefeatures of PRVglycoproteinsto testcertain

predic-tions of the modelproposed byTufaro andcolleagues (1,21)

* Correspondingauthor.

andto determine moreprecisely the nature of the defect in

thesemutant cells.

In this report,we show that PRV growth, like HSV-1, is

defective in gro29 cells. The specific modifications and

processing of certain PRV membrane proteins provided insight into which secretory organelle had been traversed duringexportthrough thesecretorypathway.We foundthat in gro29 cells, egress of infectious particles was partially

blockedearlyininfection andwascompletelydefective late

after infection.Moreover,infectedgro29cells hadadefectin exportof viralglycoproteinsfrom theendoplasmicreticulum

(ER) to the Golgi apparatus and did not secrete a

nonan-chored viral membrane protein. Taken together, these

re-sults suggest that gro29 cells have a defect in a cellular

functionrequiredfor ERtoGolgiapparatus exportaswellas

Golgi apparatustocell surface export.Thisdefectbecomes

rate limiting for egress of virus particles as infection

pro-ceeds. It is likelythatherpesvirus particle egressfromcells andglycoproteinmovement throughthesecretory pathway

share acommonmechanism.

MATERUILS AND METHODS

Cells and viruses. ThepropagationofL andgro29cellshas been describedpreviously (21). PK15 cells and the Becker strain of PRV (PRV Be) were prepared as previously

de-scribed (15). PRV1007 encodes a mutant gIll glycoprotein

geneinwhichaTAGstopcodonreplacesatyrosineatcodon 436. Theresultingtruncatedglll protein lacks the

carboxy-terminal transmembrane and anchoring domain. Further characterizationof thisvirushasbeen describedbySolomon

et al. (17).

Glycoprotein molecular mass nomenclature. Viral

glyco-proteinsglll, gll, andgp5Ohaveapparent molecularmasses

after infection of L cells different from those after infection

of PK15 cells. These differences are most likely due to differencesinthedegreeofglycosylation. In PK15cells, the

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3804 WHEALY ET AL.

PRVglycoproteins have the following apparent molecular masses: gIII precursor, 74 kDa; gIll mature, 92 kDa; gll precursor, 100 kDa; gll mature, 110 kDa; gll cleavage

products, 68 and 55 kDa; gpSO precursor, 46kDa; and gpSO mature, 55 kDa. In L cells, the PRV glycoproteins have apparent molecular masses as follows: glll precursor, 72 kDa; gIll mature, 82kDa; gll precursor, 92kDa;gII mature, 98kDa;gIlcleavageproducts, 66and 60kDa; gpSO precur-sor, 44kDa; andgpSO mature, 56 kDa. The PRV

glycopro-teins in gro29 cells have thesameapparentmolecularmasses asthose inLcells, except the gllcleavage products,which

are64and 54 kDa.

Fixation and stainingof PRVplaques. PRVplaqueswere

prepared under Methocel byusingPK15, L-cell, and gro29 monolayers. At 3 days postinfection, the Methocel was

removed, and the cells were fixed with 4% formalin for 30 min at room temperature. After 30 min, the formalin was

replaced with 0.5% crystal violet in 22% ethanol for 10 min, and the cellswerewashed withwater.

Growth curves. Single-step growth curves of PRV were

completed as described by Whealy et al. (23) with the following modifications: at 1 h postinfection, the inoculum

wasremoved from thecells, and the cellsweretreatedfor 1 minwith 40mM citric acid-10mM KCl-135 mMNaCl and replaced with Dulbecco modified Eagle medium (DMEM) containing 2% fetal calfserumpluspenicillinand

streptomy-cin.

Antibody reagents. The antisera used in these studies included a mouse monoclonal antibody, 6D8MB4, reactive withPRVgpSO(a kind gift from C.Whetstone, Ames,Iowa), agoatpolyvalent antiserum(labeled282)thatrecognizesthe native and denatured forms of the gIIIglycoprotein(14, 15),

andagoatpolyvalentantiserum(labeled 284)thatrecognizes

the native and denatured forms of thegII glycoprotein (14,

24).

Pulse-chaseexperiments to measurekinetics ofviral glyco-protein export. The kinetic analysis was done as described previously(16), except that infected cellswerepulse-labeled for 5 min rather than for2min. Techniquesfor immunopre-cipitation and autoradiography of viral glycoproteins have been described previously (4, 15).

Measurement ofgll oligomer formation by sedimentation through sucrosegradients. ThetechniquesformeasuringglI

oligomer formation by sedimentation throughsucrose

gradi-entswere asdescribedbyWhealyetal. (24), with modifica-tions as noted in the legend to Fig. 6. Briefly, L cells and gro29 cellswereinfected with10PFUofPRV Bepercell.At

5.5h postinfection, the cellswerestarved forcysteine. At 6 hpostinfection, cells were labeled for 5 min with

[35S]CYS-teine and then chased for 25 and 90 min with coldcysteine. Next, the monolayerswere solubilized in Triton X-100 and fractionated on sucrose gradients. Gradient fractions were immunoprecipitated with anti-gll serum (284 serum) and

analyzed on sodium dodecyl sulfate (SDS)-polyacrylamide gels.

RESULTS

Plaque-forming efficiency of PRV Be on PK15, L, and gro29

cells. Banfield and Tufaro(1) demonstrated that HSV-1 was unable to form plaques on gro29 cells. We measured the plaque-forming efficiency of PRV on swine kidney fibro-blasts (PK15 cells), the parent murine L-cell line, and the gro29mutant. The titer of PRV Be stock prepared on PK15 cellswasdetermined for PK15, L, and gro29 cells. Plaques

werefixed, stained, and counted. A typical dilution series is

PK1 5 CELLS

!

I

S.1

LCELLS

IL. .. .. ..ix X, > v

,. ,1

4/ IX}

;ti.4-:_ .

[image:2.612.336.552.79.416.2]

gro29CELLS |

FIG. 1. Plaque-forming efficiencyof PRVonPK15, L,andgro29

cells. Serial 10-fold dilutions of PRV(10-2dilution[topleft ineach dish]through 10-7dilution[bottom rightineachdish])wereplated

on PK15, L and gro29monolayers and allowedtoadsorb for 1 h. Afteradsorption, the inoculumwas replacedwith Methocel. At 3 dayspostinfection,the Methocelwas removed,and the cellswere

fixed with 4% formalin and stained with 0.5% crystal violet, as

described in Materials and Methods.

shown inFig. 1. Theplaque-formingefficiencies of this stock

onthevarious lineswere 1.0 (PK15), 1.3 x 10-1 (L cells),

and>1.7 x 10-6(gro29cells).PRVformsplaquesonLcells about10-fold lessefficientlythan onPK15 cells. Itisclear,

however, that PRV, like HSV, does not form plaques on

gro29 cells.

Production of infectious virus by gro29 cells. We deter-minedthe extentofviral propagation in L cells and gro29

cellsby comparingsingle-step growth curves of intracellular

andreleasedinfectious virions (Fig. 2).Animportant part of theexperimental protocolwas alow-pH citrate washat 1 h

postinfection to remove any virus that had not penetrated the cells. Thus, any infectious virus measured after that

pointmust be newly formed virus.

When Lcellswereinfected with PRV, intracellular infec-tiousviruswasdetected between3 and5hpostinfection and infectious virus begantobe releasedextracellularly between 5 and 7 hpostinfection. Incontrast, when gro29 cells were infectedwith PRV, infectious virus particles were detected

intracellularly between 5 and 7 h postinfection and were releasedtothe media between 7and 9hpostinfection.From

the results in Fig. 2, we conclude that in gro29 cells, the J. VIROL.

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L

Cells

I.. 106

010 4

CL 10

102'

.1

10 15 20 25 31

Hours Post-infection

gro29

Cells

107.

106.

105.

E

0I IL

0D 103

102

10

0 5 10 15 20 25 30

Hours Post-infection

FIG. 2. Single-step growth curves of PRV Beon L and gro29

cells. The cellswereinfectedatamultiplicityofinfection(MOI)of

5 andwereincubated at37°C. At1 hpostinfection, the cellswere treatedwithlow-pH citrate,asdescribedinMaterialsand Methods.

At 1,3, 5, 7, 9, 11, and 25 hpostinfection, plateswereharvested, andthevirus titers of the cell and mediumfractionsweredetermined separatelyandplotted.

LCELLS gro29CELLS

0 45 90 120 240 0 45 90 120 240

A 92

-68-=W 0

opmed

46

-

92-

6846

-C

68

-46

-FIG. 3. Kinetic analysis of PRV glycoprotein synthesis and modification in Land gro29 cells at4 h postinfection. Cellswere

infectedwith PRVatanMOI of 10. The cellswerepulse-labeledat

4 h postinfection and chased for the time (in minutes) indicated above each lane.The cellswerelysed andimmunoprecipitated with

glll-specific antiserum (A), gll-specific antiserum (B), or a

gp5O-specific monoclonal antibody (C). Immunoprecipitates were

re-solvedon a10%polyacrylamide gel and visualized by fluorography. Themolecularmassstandards (inkilodaltons)are indicatedat the

left ofeach panel.

appearance of infectious PRV particles in the media is delayed and thequantityisreduceddramaticallyin

compar-isontovirusproduced bytheparental Lcells. Wenotethat the rate of intracellular accumulation and the release of infectious virus from cells are indistinguishable for L and

PK15 cells(data notshown).

Kinetics of viral glycoprotein export at 4 h postinfection.

Since infectious particle formation and release of extracel-lular virus were aberrant ingro29 cells, we next examined thesynthesisandexportof critical viralglycoproteins. PRV offers a unique set ofglycoproteins for this analysis since their modification andprocessingareindicative of the secre-toryorganelletheyhave traversed during export(3, 11, 16, 17, 22, 24).

We measured the rate and extentof synthesis and

proc-essing byapulse-chase protocolusedpreviously(16). Since

a small burst of infectious particles appeared to be made early,but further infectiousvirusproductionwasblocked in gro29 cells, experimentsweredoneat4and 6 hpostinfection

to examine an early and a later time point during the

infection. The results of the 4-hanalysisareshown inFig.3.

The pulse-chase profile forglycoprotein gIll in Lcells is

shown onthe left side ofFig. 3A. Thepredominant protein

made in the5-minpulse (lane0)was theprecursor

synthe-sized in the rough ER. This form of the glycoprotein was

already fully glycosylated with high-mannose

oligosaccha-rides and was sensitive to endoglycosidase H (data not

shown). By 45 min of chase (lane 45), the precursor was

converted to the diffuse, more slowly migrating species

characteristic of mature gIII. This form is known to be resistant to endoglycosidase H, indicating that the

high-mannose oligosaccharides have been converted to complex

carbohydrates (10). The 5-min pulse sample from infected

gro29 cells was essentially identical to that seen in L cells

(lane 0). This similarity indicated that the primary rate of

synthesisofgIIIwasunaffected ingro29 cells and thatthere

was nodefect in the earlyeventsof PRV infectionorin the

addition of high-mannose oligosaccharide moieties to the

newlysynthesizedpolypeptides. However,while the parent

L cells couldsupportefficientexportof thegIll precursorto

the Golgi cisternae forfurtherprocessing, gro29 cells were

markedly defective. There was little, if any, chase of the precursoruntil 90minpostlabeling,andmostof themassof precursorneverchased tothemature form, suggestingthat

these species had not been exposed to the processing

en-zymesresident in the peripheral Golgicisternae.

a-- cells

-*- media

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3806 WHEALY ET AL.

Further evidence that gro29 was defective in PRV glyco-protein export was obtained from a kinetic analysis of glycoprotein gIl synthesis and processing (Fig. 3B). ThegII glycoprotein is cleaved by a cellular protease located in the trans-Golgi apparatus or trans-Golgi network, and this

proc-essing occurs only after the protein has formed oligomers

and has been exported out of the ER (22, 24). Asobserved forgIll, therewas nodifferencein the 5-min pulse profilefor

gIl in either Lorgro29 cells: the 92-kDa gIl precursor was

synthesized in equal abundance in both. The smaller

gIl-related polypeptides are incomplete translation products,

and their intensities and general profiles are also

indistin-guishable in thetwocell lines. By 45 min of chase (lane 45)

in the Lcells, the mature uncleaved 98-kDaspecies, as well as the 66- and 60-kDa protease cleavage products, was apparent. Significantly, the time and extent ofgIl protease

cleavage were delayed in gro29 cells. Processing was only detected after 90 min of chase, and the apparent molecular massesoftheproductswere 64and55kDa. Whilethere was astrongblockin gIl processing, somemoleculeslabeled at 4 h postinfection can traverse the secretory pathway to the

trans-Golgi ortrans-Golgi network.

One modification that has been difficult to assess in

previous studies using HSV is theextent of 0-linked

glyco-sylation in gro29 cells during infection. Previous

observa-tions of HSV-1 gD suggested, but did not establish, that 0-linked glycosylation was impaired during infection (1).

The PRV gpSO glycoprotein can be used to determine whetherthe gro29 mutantis defective in 0-linked modifica-tions, since this viral glycoprotein has no N-linked sugars

butis modified by 0-linked glycosylation (11, 22). 0-linked glycosylation ispredictedtooccurinitially in thetransitional compartment ofthe ER, and the final complex sugar addi-tionsarecompletedinmedial andtrans-Golgi compartments

(19). Addition of0-linked sugars and the subsequent addi-tion of terminal sialic residues are accompanied by charac-teristic decreasesin electrophoretic mobility. Previously,we have shown that the initial addition of 0-linked sugars resulted in a shift in the apparent molecular mass of gpSO intermediate between the precursor and mature forms (22). Since thisformis detectable only in thepresenceofbrefeldin

A(22),ourinterpretation is that the protein had reached the

transitionalcompartmentof theERbuthad notproceededto the Golgi apparatus.

The kinetics ofgpSO synthesis andprocessing is shown in

Fig. 3C. There was no difference in precursor synthesis in either L or gro29 cells, as indicated by the identical 5-min

pulselanes(lanes 0).Lcells supported thecomplete

conver-sion of thegp5Oprecursor tofully modifiedgp5Oby 45min of chase. However, gro29 cells were delayed considerably in the efficiency and extent of gpSO processing. Since with

gro29 cells we were unable to detect the form of gp5O intermediate between the precursor and mature form

char-acteristic of protein accumulating in the transitional com-partment, we conclude thatmost of thegp5Osynthesized at 4hpostinfectiondid not reach the transitional compartment. Moreover, because a small quantity of gp5O was able to evadethisblock and mature properly, it appeared that once

gp50budded from the ER, it was capable of traversing the

transitional compartment efficiently enroute to the Golgi

organelle,whereit was subsequently modified. It is apparent that a fraction of the newly synthesized gpSO larger than those ofgllandglllwasprocessed, suggesting that the PRV

glycoproteins maybe differentially affectedin gro29 cells. These pulse-chase results indicated that gro29 cells have

anearlyblock inthe secretorypathway. Because precursor

CELLS 0 15 30 45 60' 90 120

MEDIA 15 30 45 60 90 120

LCELLS

92

gro29CELLS _{am o A:,.}

.

92

[image:4.612.332.553.77.266.2]

-68

FIG. 4. Kineticanalysis of a secretedformofgIll produced by

PRV1007. L andgro29 cellswere infectedwithPRV1007 at anMOI

of10, pulse-labeledat 6 hpostinfection,and chased forthe time(in

minutes) indicated above each lane. glll-specific antiserum was

used to immunoprecipitate gIll from both the cellandthe medium

fractions. Immunoprecipitates were resolved on a 10%

polyacryl-amide gel and visualized by fluorography. The molecular mass

standards(in kilodaltons) areindicated tothe left ofeachpanel.

glycoproteinsaccumulated and did notreceive typical

post-translational modifications, it was likely that export out of the ER was defective. However, this block was not complete

since some precursor glycoproteins escaped the ER block

and were processed in the Golgi apparatus. As measured by protease processing ofgll, some fractionof the glycoprotein

traveled as far as the trans-Golgi ortrans-Golgi network. It was, therefore, of interest to determine whether gro29 cells were defective in export from the Golgi apparatus to the cell surface. We determined this defect by analyzingexport of a

secreted form of the normally membrane-anchored glll

glycoprotein.

The glll gene encoded by PRV1007 has a stop codon in

place of tyrosine at codon 436 of the mature protein and

consequently expresses a truncated protein lacking a trans-membrane and anchoring domain. This mutantgIll protein contains the same modifications as the wild-type glllbut is

secreted into the medium and is notfound in virus envelopes (17). Since we can measure the rateand extent of

glycopro-teinprocessing as well as the rate and extent of secretion of the mature protein into the medium, we can deduce the

kinetics of export from the Golgi apparatus to the cell surface and out of the cell.

The results of thekinetic analysis for L and gro29 cells are shown in Fig. 4. In Lcells, the gIII precursor was synthe-sized in the5-min pulse period, and by 45 min of chase, the mature form was clearly detectable in the media. Theresults

with gro29cells were noteworthy. While theglllprecursor was madenormally and a small proportion received mature modifications, no glll-specific protein could be detected in the media, even after 120min. Either the secreted form of

glll was blocked in export from the Golgi apparatus to the media, or it was degraded rapidly after leaving the Golgi apparatus. We conclude from these results that at 4 h postinfection, the movement of newly made viral glycopro-teins through the secretory apparatus effectively has stopped.

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gro29

MUTATION AND PRV MATURATION 3807

9ro29 CELLS 0 1530 45 60 904 -2

w

68

-46

c

92

68

-46- 46

FIG. 5. Kinetic analysis of PRV glycoprotein synthesis and

modification in L and gro29 cells at 6 h postinfection. Cells were

infectedwithPRVatan MOI of 10. The cellswere pulse-labeledat

6 h postinfection and chased for the time (in minutes) indicated aboveeachlane. The cellswerelysed and immunoprecipitatedwith

gIll-specific antiserum (A), gIl-specific antiserum (B), or a

gp5O-specific monoclonal antibody (C). Immunoprecipitates were

re-solvedon a10% polyacrylamide-SDSgel and visualized by

fluorog-raphy.The molecular mass standards(in kilodaltons) are indicated

tothe leftofeach panel.

Kineticanalysisof PRV glycoprotein synthesis andexport6 h after infection. In the previoussection, we determined the

kinetics of viral glycoprotein synthesis at 4 h postinfection

and noted at least two characteristic defects in viral glyco-protein export in gro29 cells. To determine whether these

export blocks remained constant throughout infection, we analyzed the pulse-chase kinetics at 6 h postinfection (Fig.

5).

Thekinetics ofgIII andgpSOsynthesis and processingare

shown in Fig. 5A and C, respectively. These data are essentially identical to those seen at the 4-h time point. However, the 6-h pulse-chase profile for gIl was noticeably

differentingro29 cells. Processing of the precursor afterthe

5-min pulse was complete within 15

min

of chase, but

protease processingwascompletelyblocked, even atchase times aslong as 120min. Thus, by comparing Fig. 3 and4,

we concluded that the same defect in the rateand extent of export from the ER to the Golgi apparatus occurred at

both 4 and 6 h postinfection. However, export to the

trans-Golgi or trans-Golgi networkwas defective at 6h, as deduced by the lack of proteolytic processing of the gll glycoprotein at 6 h. An alternative view is that gll export

maynotbedefectivebut ratherthat the cellularproteasethat

processes

gIl

is active at 4 h but not 6 h postinfection in gro29 cells.

Oligomer

formation is normal in gro29 cells. Pulse-chase results at both 4 and 6 h

postinfection

indicated that gro29

cells could

support

normal precursor viral glycoprotein

synthesis

inthe

ER,

but theseprecursors accumulated. This

finding suggested

that exit from the ER was defective. To

analyze

this defect

further,

wemeasuredthe formation of

gIl

oligomers.

We have demonstrated

previously

that the

gIl

glycoprotein

is

synthesized

initially

in theERas a monomer

and

subsequently

is converted toanoligomer, presumably a

dimer

(22, 24).

Thedimer

form,

butnotthe monomeric form,

is

transported

to the

Golgi

apparatus where it receives further modifications and

proteolytic

processing. Therefore,

one

explanation

for the defect in

gIl

export from the ER in

gro29

cells may be that

oligomer

formation is defective. To determine whether

gIl

is

capable

of forming oligomers in

gro29

cells,

we carried out a

pulse-chase

analysis at 6 h

postinfection

followed

by

sucrose

gradient

fractionation as

describedin Materials and Methods(Fig. 6). In both L cells and the

gro29

cells,

the

gIl

precursor sedimented

predomi-nantly

asa monomer at25

min

ofchase, and by90

min

after

synthesis,

most of thesemonomers were converted to

oligo-mers. We conclude that

gIl

oligomerized

normally in the

mutant cells and that the lack of

oligomer

formation cannot accountfor the ER

export

defect ingro29 cells. In addition, these results enable us to confirm and extend the previous observations of the defective protease processing of

gII

in

gro29

cells. In the L-cell

gradient

profile,

protease cleavage

products

were

clearly

visible in the dimer region of the

gradient,

whereas no such

cleavage

productswere detected in the dimer

region

in the gro29 profile.

DISCUSSION

The

gro29

cell linewasselected forits inabilitytosupport

propagation

ofHSV-1

(21).

In the present experiments, we

showthat this

inability

tosupportpropagationistruealsofor

PRV,

a distant relative of HSV-1. We were able to extend

and define the

gro29

defect in PRV propagation bystudying

the

synthesis

and modifications of three PRV glycoproteins

as well as

by conducting

single-step

virus growth

experi-ments. It is

apparent

that

gro29

cells are able to support

normal

synthesis

of viral

glycoprotein

precursors but that

these precursors accumulate in the ER. This early export

block is not

complete,

and someprecursors enter the Golgi

apparatus,

as demonstrated

by

addition of complex sugars and

protease

processing

ofthe

gIl

glycoprotein.

It is

note-worthy

thatastheinfection

proceeds

ingro29 cells, protease

processing

of the

gII

glycoprotein

becomes less efficient. Sincethe cellular

protease

is

thought

toreside inalateGolgi

compartment,

perhaps

the trans-Golgi network, gro29 cells may also be defective in entrance to late Golgi

compart-ments. A schematic

representation

of these blocks at 4

(early)

and 6

(late)

h

postinfection

is shown in Fig. 7.

Export

in

gro29

cells is defective at multiple steps,

sug-gesting

that the defect is due either toasingle mutation in a function

required

at

multiple

steps in the export pathway or

to

multiple

mutations

affecting

avarietyoffunctions. There

isa

precedent

forthe former

hypothesis.

In yeastcells, the

secl8

mutation defines afunction requiredat multiplesteps

in the

export

pathway

(7).

Similarly,

the N-ethylmaleimide-sensitive fusion

protein

in mammalian cells is required in fusionofER-derived and

Golgi-derived

transportvesicles(2,

5).

Ifthis

hypothesis

iscorrect, the gro29 mutation would affect either a common step in transport vesicle formation

L CELLS

0 1 5 30 45 60 90 120

A

92

-

6846

-B

92- a __

-VOL.66, 1992

1w

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4W.- 4040.

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3808 WHEALY ET AL.

FRAU iCJw4S

)iMER MONOMER

34 A.6 76 610 l.2l 3s3 1,4 1i 16 1.7 19

_ _- _

-gro29 CELLS

92

68 X

46

-K0 OAS*SE

92-1 "w ft a___4i

_-** .:

FIG. 6. PRV gIl dimer formation in L and gro29 cells. Cells were infected at an MOI of 10. At 6 h postinfection, the cells were pulse-labeled and chased for 25 or90min as indicated. The cellswere solubilized in 1% Triton X-100 and fractionatedbysedimentation

througha5to15%sucrosegradient containing0.1% Triton X-100. The fractionswereimmunoprecipitatedwithagIl-specificantiserum.The immunoprecipitates were resolved on a 10% polyacrylamide-SDS gel andvisualized by fluorography. The positions ofmolecular mass standards (in kilodaltons)areindicatedtotheleft of thepanel.The relativepositionsof theexpectedmonomersand dimers ofgllareindicated

atthe topof thefigure.

from ER and Golgi compartments or a common step in

transport vesicle fusion to intermediate compartment or

trans-Golgi network compartments. Moreover, since such functions wouldmostlikelybeessential, thegro29mutation

mustbe leaky. Indeed, Tufaro has demonstrated that unin-fectedgro29 cells contain at least 35% of normal secretory activityfor endogenous proteins (20). This level ofactivity clearly is not sufficient to handle the increased demand for

sorting and export of viral glycoproteins as the infection

proceeds.

Astrikingphenotype ofgro29 cells is the block in HSV-1 and PRV plaque formation and virus egress. Single-step

growth curve studies of PRV in gro29 cells indicate that

infectiousparticlesformatearlytimesafter infection andare

slowly releasedfromcells, but thisprocessisblockedasthe

infection proceeds. These observations are consistent with

the hypothesisthatacellular function becomes ratelimiting

forviral egressduring herpesvirus infection. It maybe the case that the decrease in host cell protein synthesis that accompanies infectionbyboth PRV andHSV results inthe

disappearance ofan already labile host protein involved in

glycoprotein transport, processing, or virus egress. This

resultcouldexplainthenearly complete blocktosecretion of glycoproteins and viralparticlesasthe demand foracritical protein(s)increases during infection.

To explain both the glycoprotein export defect and the virusegressdefect,weproposethat thetwoprocessesshare

acommon mechanism. Thus, if gro29 is defective in

trans-portvesicleformationorfusion, itwould followthat

herpes-virusegressalsousessimilarmechanismsasparticles travel

from thenucleustothe cellsurface.Whealyetal. (22) have

proposed a model for the assembly and egress of PRV invoking primary envelopment at the inner nuclear mem-brane, de-envelopment asthe capsid leaves the ER, and a secondary envelopment at the transface of the Golgi

appa-ratus, similartothe envelopment pathway used byvaccinia

virus. In this model, the gro29 defect could affect

de-envelopment at the ER, secondary envelopment at the

trans-Golgi, orfusion of theGolgitransportvesicle with the

plasma membrane(Fig. 7).

An alternative possibility is that virus infection may

change the character of thesecretoryorganellessothatthey canfunctionduringviralmaturation,possibly bythe

synthe-sis ofnewER-orGolgi-resident proteins.Thisvirus-induced

change may not occur ingro29 cells because of its already

inefficientsecretory capacity, resultingin the observed gly-coproteinexportand virusegressphenotypes.Further work

isnecessaryto distinguish thesepossibilities.

We have also noted apparent differences in the secretion ofproteinsinHSV-infectedversusPRV-infectedcells. It has been demonstrated previously that by 18 h postinfection,

secretion of human growth hormone, a nonglycosylated

protein, was not seriously impeded in HSV-infected gro29 cells, despite the apparent lack of virion egress (20). This lack of impedance was explained by assuming that the

requirements for intracellular movement of virions and

nonglycosylated proteins were different. In the present study,we observed that a soluble form ofaviral

glycopro-teinwasblocked insecretion. Thisfindingsuggeststhat viral

glycoproteins are affected to a larger extent than are

non-glycosylatedcellularproteinslate inalphaherpesvirus infec-tion. Perhaps after lytic infection, improperly glycosylated

viral glycoproteins are produced that are less soluble and

therefore accumulate aberrantly in the gro29 secretory

or-ganelles. In contrast, proteinslike human growth hormone thatdonotrelyonglycosylation for their efficienttransport

would be solubleandtherebyremain mobileastheytraverse

theinfected-cellsecretoryorganelles. It is alsopossiblethat

PRVcauses a more rapid andextensive shutdown of

trans-portcapacity than does HSV in infected gro29 cells. Addi-tional studiesare neededto resolve these issues.

The gro29 block of both PRV and HSV-1 is a striking

observation. The mutation(s)present ingro29cells affectsa

cellular function required for efficient production of virus

FRA_ iONS

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2 3 5 6 7 8 9 10 ,1 2 3 _

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FIG. 7. A schematicrepresentation of PRVglycoprotein export andvirus egress in gro29 cells. The general model has been described

previously (22).The toppanelshowseventsearlyafterinfection,and the bottompanel shows events late after infection of gro29 cells. Blocks in theproposed pathwaywerededucedbydeterminingthe extent of PRVglycoproteinmodifications,as described in the text. At both early and latetimes, there isapartialblock of PRVglycoproteinexportfrom the ER to theGolgi apparatus (indicated by the dotted X). Early in

infection,there is a secondblocktoexportfromlate Golgi compartments(possible the trans-Golgi network) to the cell surface (indicated by the solidX).Inaddition,late afterinfection,thereisathird block from lateGolgicompartments to the trans-Golgi network (indicated by the solidX).Virus egress is blockedonlylate afterinfection,and thelocation of this block is unknown (indicated by question mark). The cisternae of theGolgiapparatusarelabeledC, M,and Tfor thecis,medial, and trans compartments, respectively. The trans-Golgi network is indicated

by the vesicleleavingthetrans-Golgi apparatus andmovingtothecell surface. Viralglycoproteins are designated as follows: " indicates

proteinswithonlyERmodifications, and t indicatesproteinswithGolgiapparatusmodifications.

progeny of these tworather diverse alphaherpesvirusesyet has only a marginal effect on uninfected cell growth in culture. Theseobservations suggestthatgro29definesa new

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