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0022-538X/93/063644-05$02.00/0

CopyrightX)1993, American Society forMicrobiology

An

Immortalized Human Fibroblast

Cell Line Is

Permissive

for Human

Cytomegalovirus Infection

TERESA COMPTON

Departmentof MedicalMicrobiologyandImmunology, Universityof

Wisconsin-MadisonMedical

School, Madison,

Wisconsin

53706-1532

Received 21 December1992/Accepted21March1993

Human foreskin fibroblasts (HFF)wereimmortalized via retrovirus-mediatedgenetransferofthe E6andE7

genes ofhuman papillomavirus type16. An immortalized fibroblast (IF)cell linewhichwasmorphologically

akin to the parental cell line was isolated. The IF cell line was evaluated for permissiveness to human

cytomegalovirus (HCMV) infection after the IF cell linesurpassed the normalpassage limitation ofdiploid

fibroblasts. Western immunoblot analysisofrepresentative HCMV-encodedimmediate-early (72-kDa),early

(gB), andlate(gH)geneproductsdemonstrated that the IF cell lineproducedtheseproteinsanalogoustothose

produced bytheparental 11FFcells. SimilarquantitiesofinfectiousviruswereproducedintheIFand 11FFcell

lines as determined in one-step growth curve experiments. Compared with the HFFcells, morphologically

identical plaqueswereproducedinthe IFcellline inapproximately10to12days postinfection.Thesefindings indicate that fibroblast cell lines immortalized with transforming genes of human papillomavirus retain

complete permissivenesstoHCMVinfection and supportplaqueformation. TheIFcelllinewillbe usefulfor

future genetic analysisofHCMV.

Human cytomegalovirus (HCMV) is a significant human

pathogen responsible for severe clinical manifestations in

immunosuppressed individuals (1, 15, 17, 25, 29, 30).

Mo-lecular characterization of HCMV has proven challenging becauseof itslonglifecycleand limited hostrangeinvitro.

Secondary culturesof human diploid fibroblasts from

vari-ousoriginsarepredominatelyusedtopropagateHCMV,and these cell lines are uniquely capable ofproducing plaques.

Other celllines, such as humanbrain endothelial cells (24, 28),human umbilical vein endothelial cells(14, 31,33, 38),a

teratocarcinoma cell line (23), cultures of human

macro-phagesandmonocytic cell lines(16, 39), andhuman

astro-cytomacelllines(2, 21),arealso knowntobepermissivefor theproductionofHCMV; however,noneofthese cells lines

fullysupport plaque formation.Apermanently growingcell line which is permissive for productive HCMV infection

andsupportsplaqueformation wouldbeanextremely useful

and convenient tool to study HCMV infections in vitro. It

hasbeen demonstrated thattheE6 andE7genesofhuman

papillomavirus (HPV)type 16 have the capacity to immor-talize primary human keratinocytes, the permissive host

for HPV infections (13). In the present study, the E6 and

E7 genes of HPV type 16 along with the neomycin

resis-tance marker were stably introduced into the genome of

human foreskinfibroblasts (HFF)andanimmortalized

fibro-blast (IF) cell line was isolated. The resultant cell line is

thus far immortal in its growth properties and remains permissive to productive HCMV infection and plaque

for-mation.

HFF cells were purchased from Clonetics, San Diego,

Calif., and propagated as previously described (8, 27). An

amphotropicretrovirus-packaging cell line which produces the recombinant virus LXSN16E6E7 as described by

Hal-bert et al. (13) was provided by Denise Galloway (Fred

Hutchinson Cancer Research Center, Seattle, Wash.). The

genomeofthisvirus containsaneomycinresistance marker,

theE6 andE7genesoftype16 HPV flanked bythe Moloney murine leukemia virus long terminal repeats. The E6-E7

recombinant stock produced by thepackagingcell linewas

usedtoinfect 50%confluentmonolayersofpassage11HFF cells. Briefly, 1 ml of virus stock plus 3 ml of complete medium (Dulbecco modified Eagle medium plus 10% fetal bovine serum) containing 4 ,ug of Polybrene per ml was

addedtothe monolayersfor 2 h and further incubatedwith additionalPolybrene-containing medium for 5 h.Fresh

com-plete mediumwasadded overnight. On day2,thecellswere

tyrpsinized and plated in limiting dilutions. On day 3, the

mediumwas supplemented with 200 ,ug ofG418 per ml to

select neomycin-resistantcolonies. A kill curve analysis of

the HFF cells indicated that 200 ,ug of G418 per ml was

appropriatefortheHFF cell line(datanotshown). Approx-imately 2 weeks later, individual neomycin-resistant colo-nieswereevident and 12cloneswereselectedandexpanded.

In thestudy shown here, asingle neomycin-resistant clone

thatwasmorphologicallyverysimilartotheparentalcellline

waschosenfor furtheranalysis. The IF cell linewas

main-tained under selectivepressureand has beenpassagedupto

50 times, whereas normal HFF cells reach senesence

be-tweenpassages23 and28.

One-step growthcurveanalysiswasperformedtoevaluate

the IF cell line forpermissivenessto HCMV infection. The

AD169 strain of HCMV was used for these studies and

propagated in HFFcellsaspreviouslydescribed (8, 27).For

thesingle-cycle growth analysis, 10-cm2 culture dishes of IF (passage 40)orHFF(passage 15) cellswereinfected with104

PFU of HCMVperdish (multiplicity of infection [MOI] = 0.1). Following a 90-min adsorption period, the inoculum

wasremovedandmediumcontaining 2% fetal bovineserum

(maintenancemedium) wasaddedto eachplate. The plates

wereincubatedat37°C andharvestedondays 2, 5, 7, 9, and

13 postinfection. On the indicated day postinfection, one

dish from each cell line was collected for plaque titration

analysis. Plaque titrations were performed as described

previously (8). As shown in Fig. 1, virusproduction peaked

in both the HFF and IF cell lines approximately 7 days postinfection.Theyieldofinfectious viruswasgreaterthan

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_~~

HFF SIF

+HCMV Mo +HCMV Mo

Days Post-infection:

Immediate Early (72 kDa)

0 3 6 9 12 15

Days post-infection

FIG. 1. One-step growthcurveanalysis of HCMV production in HFF and IF cell lines. HFForIF cellswereinfected with HCMV (AD169) at an MOI of 0.1. After adsorption for 90 min at 37°C, monolayers werewashed with serum-free medium and incubated with maintenance medium. Viruswasharvestedasindicated inthe text,and titersweredetermined by plaque assays onHFF

mono-layers.

10-fold higher from the IF cells in the results of the experi-ment presented here. In repeat experiments, the yield of AD169wasgenerally higher from the IF cells by 10-fold than

that from the HFF cells. It isnotknownwhether this slight

difference is strainspecificorbiologically significant. Highly

similargrowthcurves were also obtained whenonly extra-cellularvirus was recovered, although the titers were

ap-proximately100-fold lower(datanot shown).

HCMVgeneexpressioninpermissive fibroblasts is char-acterizedby sequential expressionof the viralgenomethat is

temporally regulated anddesignated into three classes:

im-mediate-early(IE), early,and late(34, 36).Thesynthesisof the major IEprotein, a 72-kDa protein, andtwo glycopro-teins, gB and gH, was examined in HFF and IF cells at

various times postinfection byWestern immunoblot

analy-sis. The major IE gene product, a 72-kDa protein, was

detected witha polyclonal antiserum raised against

recom-binant-derived, purified72-kDaprotein (26).Thisantiserum,

which recognizes both the 72-kDa (IE1) and 86-kDa (IE2) proteins, was agenerous gift fromJay A. Nelson, Oregon

Health SciencesUniversity,Portland. HFForIFcellswere

cultured in 35-mm-diameter dishes and infected with HCMV

atanMOIof 3. On the indicatedday postinfection, the cells were harvested by scrapingwith a rubber policeman,

pel-leted by centrifugation, washed with phosphate-buffered

saline (PBS), and suspended in glass-distilled water. After

disruption by sonication, the cell extract was treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and resolved in triplicate on

SDS-7.5% PAGEgelsfollowedbytransfer to nitrocellulose

membranes essentially asdescribed elsewhere (6).The effi-ciencyof transferwasmonitoredbyPonceau Sstaining.The

nitrocellulose membraneswere blocked in 10 mM Tris(pH

7.4)-150

mM NaCl plus 5% dried milk, 1% bovine serum

albumin, and 0.1% Tween 20 for 60 min at 37°C and then

subjected to three successive washes in 10 mM Tris (pH

7.4)-150mM NaCl-1%dried milk-0.1% Tween 20(WB) or

WB supplemented with 0.1% SDS (for polyclonal

antibod-ies).To detect theIEprotein,theanti-IEserum wasdiluted

Early (gB)

Late

(gH)

[image:2.612.65.304.69.251.2]

1 3 5 7 9 9 1 3 5 7 9 9

FIG. 2. Synthesis of the major IE protein, gB, and gH as a function of time in HFForIFcells. HFForIFcellswereinfected withHCMV (AD169)atanMOI of 3. After adsorption for 90minat

37°C, the inoculum was removed and maintenance medium was added.Ontheindicatedday postinfection, the cellswereharvested, lysed, treated with SDS-PAGE sample buffer which contained 2-1-mercaptoethanol, resolved in triplicate by SDS-PAGE (7.5% acrylamide), and transferred to nitrocellulose asdescribed in the

text. The 72-kDa IE protein, gB, and gH were detected with monospecificantibodies followedby horseradish perosidase-conju-gated anti-species second antibody and detected with chemilumines-cence asdescribed in thetext.

inWBplus 0.1% SDS and incubated with the membrane for

60 min at room temperature. After the membrane was

washed, the primary antibodywasdetected with horseradish

perioxidase-conjugated goat anti-rabbit serum (Pierce).

Chemiluminesence (ECL; Amersham) was used to detect the antibody conjugates as describedby the manufacturer,

and the membraneswereexposedtoX-OMAT film(Kodak), generally for 10-30 s. As shown in Fig. 2, the major IE

proteinwasevident at 1 day postinfection inboth the HFF and IF cells. Thesteady-statelevels of the 72-kDaIE protein

in HFF cells declined after 3 days postinfection, afindingin

agreementwith that ofStenbergetal. (35).Incontrast, the levels of the IE protein remained fairly constant inthe IF

cells until 9days postinfection.

GlycoproteinBwaschosen as ageneral representativeof

anearlygeneproduct sincesynthesisofgBis detectable in

the presence of inhibitors ofDNAreplication (5). The gB glycoprotein is contained within thegC-I envelope

glycop-roteincomplexand is also referredtoasgpl30/55 (3, 9, 11,

12, 20). A monoclonal antibody (27-78) specific for glyco-proteinBwaskindly provided byWilliam J.Britt, University

ofAlabama, Birmingham. Purified 27-78immunoglobulinG

109

108

i07 106

105

104

103

102

*1141*

-72 kDa

Igpl30/116

-gp55

-gp86

HFF EF

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wasprepared from ascitic fluidsbyprotein A chromatogra-phy. As described above, lysates of HFF or IF cellswere

transferredto nitrocellulose membranes and thegB protein

was detected with 27-78 immunoglobulin G (0.25 ,g/ml)

diluted in WB followed by horseradish peroxidase-conju-gated goat anti-mouse serum and with chemiluminesence.

Synthesis of the gB protein was detectable on day 3 and

remained evident in both cell lines forthe duration of the

experiment (Fig. 2). Inaddition, processing andproteolytic cleavage of the gB biosynthesis products appeared to be

identicalin the IFcells and HFF cells.

Synthesis of glycoprotein H (gH) is not evident in the absence of viral DNAsynthesisand is thereforea

represen-tative of lategeneproducts (5).To detect gH,apolyclonal

antibody (no. 6824)wasgeneratedin arabbit after

immuni-zation withtwopeptidesderived from thegHsequence(10). Briefly, two peptides consisting of C-SYRSFQQLKAQD SLGQQPTT and C-SDLYTPCSSSSGRRDHSLERLTR

(amino acids 135 to 155 and 517 to 538, respectively)were

synthesized (kindly providedbyJohnson andJohnson,San

Diego, Calif.)andcoupledtoasinglekeyhole limpet

hemo-cyanin carrier by using a heterobifunctional cross-linking

reagent (sulfo-SMCC; Pierce). The peptide conjugate (500 ,ug)was injectedinto aNew Zealand Whiterabbit, and the animalwasboosted every4weeks. Ahigh-titer polyclonal

antiserumwasobtained. Theno.6824 antiserumrecognizes

the gp86 component of the gC-III complex (12) which containsthegHhomologof HCMV(10).To confirmthat this

reagentwasspecific for gH,amonoclonalantibody specific

forgH(14-4b)wasalsoprovided byWilliam J. Britt and used

to immunoprecipitate gH from infected cell extracts. The

immunoprecipitate was then transferred to nitrocellulose andpositivelydetected withtheno.6824antibody (datanot shown). The anti-gH serum was diluted in WB plus 0.1% SDS and detected as described above. In both cell lines, glycoprotein H was not readily detectable until 5 days postinfection and remained evident through 9 days

postin-fection(Fig. 2). Ingeneral, the kinetics ofexpressionofIE, early, and lategene products wasvery similar in both cell lines.

While the kinetics of detection of the three proteins

studied here were similar, levels of all three HCMVgene

productsappearedslightly higherin the IFcells. In particu-lar,the IEproteinwasdetectable until 9days postinfection,

while levels ofIE proteindeclined inthe HFFcellline after 3days postinfection.Todeterminewhether the IFcellswere more efficiently infected by HCMV, immunofluorescence

analysis was performed to examine the number of

IE-positivecellsas afunction oftime. Inthisexperiment,HFF

or IFcells cultured on glass coverslipswere infected with

HCMV (MOI = 3) for various periods of time. At the

indicated time, the cellswere fixed with 3%

paraformalde-hyde inPBS for20 min and then permeabilized with 0.1%

TritonX-100for 10min. The fixed monolayerswerewashed

withPBS plus 0.2% gelatin, and the cellswerereactedwith

the anti-IE antibody described above for 60 min at room temperature. Thecoverslipswere then washed three times

for 5 min each time with PBS-gelatin, and the primary antibodywasdetectedwithrhodamine-conjugatedgoat anti-rabbitserum(Kirkegaard&PerryLaboratories, Inc.,

Gaith-ersburg, Md.) for 60 min at room temperature. The wash steps were repeated, and the coverslips were mounted in

Gelvatol as described previously (7). The percentage of IE-positive cellswas determined, andthe data are summa-rized in Table 1. As shown in Table 1, the number of

IE-positivecellswasnotsignificantlygreaterintheIF cells

TABLE 1. Number ofIE-positiveHFForIFcells

as afunction of time

% of cellspositivefor IEproteinattheindicated

Cell time postinfectiona

line

18 h 36 h 72 h

HFF 66.1 95 33

IF 70.3 100 47

a Cells were fixed and stained at the indicated hour postinfection. A

minimum of 100IE-positive nucleiwerecounted in each sample.

than in theHFF cells. This result indicates that the IF cell line is not more efficiently infected than the parental fibro-blasts. These data and the results of the Western blot analysis (Fig. 2) suggest that synthesis of the major IE protein is sustainedlonger in the IF cells than in HFF cells, asituation which may be due to the presence of the HPV E6 andE7proteins.

To determine whether the IF cell line could supportplaque formation, HFF or IF cells cultured in 35-mm-diameter dishes were infected with 100 PFU of HCMV and incubated asdescribed above. Onday 12postinfection,themonolayers

werefixed with25% formalin and stained with0.05% meth-ylene blue. As shown inFig. 3, uninfectedmonolayers of IF and HFF cellswerevery similar in appearance. In infected

monolayers of IF and HFF cells, morphologically similar plaques were evident in the IF cells. Although the virus stock was grown in HFF cells,-comparable numbers of plaques were obtained in the IF cell line. Infectious virus

wasrecovered fromasingle plaque producedinthe IFcells,

as evidenced bysuccessfulreinfection of either HFForIF monolayers (data not shown). This finding suggests that

substantial adaptationto the IF cell line isnot requiredfor furthergrowth andpropagation.

The results of this study indicate thatdiploidcultures of HFFimmortalized with thetransforminggenesof HPV type 16retain theabilitytosupportproductive HCMV infection, including plaque formation. These data also demonstrate

thatthe E6 and E7 genes have thecapacitytoimmortalizea

celltype-fibroblasts-otherthan thepermissivecell

type-epithelial cells-for HPV infection. One-step growthcurve

experimentsanddetection of HCMV-encodedproteins

dem-onstrate that there were no significant differences in the general growth properties of HCMV in the IF cells. The

synthesisofthemajor IEprotein appeared tobe sustained longer in the IF cells, which likely explains the slightly

highertiters of virusproduced in these cells.

Herpesviruses,including HCMV,areamenable to genetic

manipulationviahomologous recombination (32). Strainsof viruscontaining whole-gene deletions or specific mutations

areextremely useful for functional analysis of the proteins encoded bythese genes. There are now several reports of the construction and production of deletion strains of

HCMV; however,in eachinstance, nonessential regions of the HCMV genome have been removed (4, 18, 19, 22, 37). The lack ofapermanently growing cell line which supports plaque formation has prevented the production of stains of virus with deletions in essential gene products, since a stably

transfected, complementing cell line which provides the gene product to be removed in trans is required. The

developmentofanimmortalized cell line which is permissive for HCMV infection and plaque formation represents a

major technical advancement that will be useful for future

genetic analysisofHCMV.

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MOCK

+HCMV

HFF

pll

IF

p41

FIG. 3. Morphological comparison of uninfectedandinfectedHFF and IFcell monolayers. HFF (passage 11; pll) or IF cells (passage 41;

p4l)wereeither mock infectedorinfectedwith100PFUofHCMV.Afteradsorption for 90 min at 37°C, the monolayers were overlaid with

agarose-containing medium and furtherincubated at37°Cfor12days. After fixationwith25%buffered formalin,the cell monolayers were

visualizedbystaining with0.05%methylene blue, observed, and photographed on a Televol inverted microscope. Magnification, x200.

I acknowledge Neil Cooper and Bonnie Bradt of The Scripps ResearchInstitute,LaJolla, Calif.,forassistancein theproduction

of theanti-gHantiserum.Sherry Colemanprovided experttechnical assistance. I thank my colleagues Curtis Brandt and Miroslav

Malkovskyin theDepartmentof MedicalMicrobiology and

Immu-nology, University of Wisconsin, forcriticalreadingof the

manu-script.

These studieswereaidedby grants IRG-35-341 from the Ameri-can Cancer Society and U01A131494 from the Midwest STD Re-searchCenter.

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http://jvi.asm.org/

Figure

FIG. 1.withHFFmonolayerstext,(AD169) One-step growth curve analysis of HCMV production in and IF cell lines
FIG. 3.visualizedp4l)agarose-containing Morphological comparison of uninfected and infected HFF and IF cell monolayers

References

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