JOURNAL OF VIROLOGY, Dec. 1994, p. 7966-79730022-538X/94/$04.00+0Copyright C 1994, American Society for Microbiology

Entry and Release of Transmissible Gastroenteritis Coronavirus AreRestricted to Apical Surfaces of Polarized Epithelial Cells

J. W. A. ROSSEN,' C. P. J. BEKKER,' W. F. VOORHOUT,2 G. J. A. M. STROUS,3A. VAN DER ENDE,3t AND P. J. M. ROTTIERl*

Institute of Virology, Department of Infectious Diseases and Immunology,' and Department of Functional Morphology,Faculty of Veterinary Medicine,2 Utrecht University, 3584 CL Utrecht, and Laboratory of Cell Biology,

Medical School, Utrecht University, 3584 CX Utrecht,3 The Netherlands

Received 15 April 1994/Accepted 31 August 1994

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract ofpigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcineepithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supportsand infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enterthe cells from both sides. During further development of cell polarity, however, the entry became restricted tothe apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptoraminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at boththe apical and basolateral plasma membrane domains just after plating of the cells but that it becamerestricted to the apical plasma membrane during culture. To establish the site of viral release, the viral contentof the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining theamount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV waspreferentially released from the apical plasma membrane. This conclusion was confirmed by electronmicroscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. Theresults support the idea that the rapid lateral spread ofTGEV infection over the intestinal epithelia occurs bythe preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infectionof nearby cells through the apical domain.

Coronaviruses cause a wide spectrum of diseases in humansand animals. Respiratory and enteric diseases are most com-monly seen. The basis for these diseases appears to be themarked tropism of most coronaviruses for epithelial cells ofthe respiratory and intestinal tracts. For example, infectiousbronchitis virus, causing an infectious respiratory disease inchickens, infects epithelial cells of the trachea (1, 10, 31), andtransmissible gastroenteritis virus (TGEV), which causes anenteric disease in pigs, infects intestinal epithelial cells (12, 23,24). Evidently, epithelial cells are the primary target cells forcoronaviruses and are important in the pathogenesis of coro-navirus-induced diseases.

Epithelial cells form highly organized cell sheets that sepa-rate the external milieu from the organism's interior. Theirplasma membrane is divided into an apical domain, facing theexternal milieu, and a basolateral domain, facing the internalmilieu (e.g., the blood supply). These two domains differ inlipid and protein composition and are separated by tightjunctions. Transport of many proteins in epithelial cells ispolarized; i.e., the proteins are transported either to the apicalor to the basolateral plasma membrane (for reviews, seereferences 5, 9, 17, 18, 28, 33, and 34). For a number of viruses,it has been shown that entry into and release from epithelialcells are also restricted to one plasma membrane domain (fora recent review, see reference 40). Vesicular stomatitis virus(VSV), for example, uses the basolateral plasma membrane forboth entry and release (13, 30), while influenza virus can infect

* Corresponding author. Mailing address: Institute of Virology,Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31-30532462.Fax: 31-30536723.

t Present address: Department of Medical Microbiology, Universityof Amsterdam, 1105 AZ Amsterdam, The Netherlands.

from both sides but is released from the apical membrane (30).The polarized release of viruses from epithelial cells caninfluence viral spread. Basolateral release allows the virus toinfect underlying tissues and to spread through the body via theblood, causing a systemic infection. In contrast, apical releaseof viruses can limit viral spread by preventing the infection ofother than epithelial cells (36, 37).Most studies of the mechanisms of virus release from

polarized cells deal with viruses that bud at the plasmamembrane. Little is known, however, about the principlesunderlying the sorting of viruses assembled at intracellularmembranes. Coronaviruses bud intracellularly between therough endoplasmic reticulum and the Golgi apparatus in theintermediate compartment (21, 22, 39). From there, assembledviruses are transported in vesicles via the constitutive exocyticpathway to the plasma membrane, where they are released(38). An interesting question is whether coronaviruses arereleased from epithelial cells in a polarized fashion and, if so,where and how the specific sorting of the carrier vesiclesoccurs. In this study, we have investigated the polarity of entryand release of the porcine coronavirus TGEV in the polarizedepithelial cell line LLC-PK1, which is derived from the proxi-mal tubule of a porcine kidney (reference 17 and referencestherein). We found that both entry and release are restricted tothe apical surface.

MATERUILS AND METHODS

Cells, viruses, and antisera. LLC-PK1 cells were maintainedat 37°C and 5% CO2 in plastic culture flasks (Nunc) inDulbecco's modified Eagle's medium (GIBCO Laboratories)containing 10% fetal calf serum, penicillin (100 U/ml), andstreptomycin (100 ,ug/ml). For the preparation of polarized cell

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TGEV IN POLARIZED EPITHELIAL CELLS 7967

monolayers, polycarbonate membrane filters attached to thebottom of plastic cups (Transwell inserts, 0.45 jum, 4.5 cm2;Costar Corp., Cambridge, Mass.) were placed into six-welltissue culture plates. Subsequently, confluent cell cultures in an80-cm2 culture flask were trypsinized and resuspended into 24ml of culture medium, and 2 ml of the suspension was addedper filter. Routinely, tightness of the monolayer was checkedby adding medium to the upper chamber up to a slightly higherlevel than in the lower chamber (6). The porcine kidney PD5cell line was kindly provided by Solvay Duphar B.V. (Weesp,The Netherlands).The Purdue strain of TGEV was used for all infections. We

used antibodies from ascitic fluids A36 and A40 obtained fromcats infected with feline infectious peritonitis virus (16). Theseantibodies cross-react with TGEV proteins, although theypoorly recognize the TGEV spike protein. Therefore, in someexperiments these antibodies were mixed with monoclonalantibody 995, specific for the S protein of TGEV, kindlyprovided by R. Meloen (Central Veterinary Institute, Lelystad,The Netherlands). Monoclonal antibodies G43, against por-cine aminopeptidase N, and CC1, against the mouse hepatitiscoronavirus (MHV) receptor, were kindly provided by H.Laude (Institut National de la Recherche Agronomique, Jouy-en-Josas, France) and K Holmes (Uniformed Services Uni-versity of the Health Sciences, Bethesda, Md.), respectively.TER measurements. Transepithelial resistance (TER) mea-

surements were done by using a Millicell ERS apparatus(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions. Measurements were performed by using LLC-PK1 cells grown on Transwell tissue culture inserts at roomtemperature in culture medium. TERs were measured overmonolayers of noninfected and TGEV-infected cells.

Infections. Filter-grown LLC-PK1 cells were infected withTGEV at a multiplicity of infection of 10 from either the apicalor the basolateral side at different time points after platingonto filters as indicated. Infection was allowed to take place for1 h at 37°C, after which the inoculum was removed. Cells werewashed with phosphate-buffered saline (PBS), and culturemedium was added. Infected cells were used for titrationassays, radioimmunoprecipitation assays, and electron micro-scopical studies.

Radioactive labelling and immunoprecipitation. For label-ling and immunoprecipitation of intracellular viral proteins,cells were labelled from 4 to 6 h postinfection (p.i.) byreplacing the apical medium by 1.3 ml of minimal essentialmedium without methionine (MEM-meth) and the basolateralmedium by 2.7 ml of MEM-meth supplemented with 50 jiCi ofL-35S in vitro cell labelling mix (Amersham). Subsequently,cells were rinsed with ice-cold PBS and solubilized in 300 ,ul oflysis buffer, which consisted of TES (20 mM Tris hydrochloride[pH 7.5], 1 mM EDTA, 100 mM NaCl) containing 1% TritonX-100, 1 jig of aprotinin per ml, 1 jig of pepstatin per ml, and100 jig of phenylmethylsulfonyl fluoride per ml. Nuclei wereremoved by centrifugation at 12,000 x g for 10 min at 4°C. Forlabelling and immunoprecipitation of viral proteins releasedinto the medium, cells were labelled from 6 to 7 h p.i. with 100jiCi of L-35S in vitro cell labelling mix as described above andchased for 2 h in MEM-meth containing 1 mM methionine.Media were collected and cleared by centrifugation for 10 minat 1,000 x g and 4°C. To the supernatants, 0.25 volume of afivefold-concentrated stock solution of lysis buffer was added.Viral proteins in the cell lysates and in the media wereimmunoprecipitated with 6 jil of a 1:1 mixture of ascites fluidA40 (undiluted) and monoclonal antibody 995 (diluted 1:100in PBS) by incubation overnight at 4°C. To each sample, 60 jilof a 10% (wttvol) suspension of formalin-fixed Staphylococcus

aureus cells (BRL, Life Technologies, Inc.) and 30 jil ofOmnisorb (Calbiochem-Novabiochem Corp.) were added. Af-ter 30 min of incubation at 4°C, the immune complexes werewashed three times with TES containing 0.1% Triton X-100,the third time supplemented with 0.1% sodium dodecyl sulfate(SDS). Finally, immune complexes were resuspended in 25 jilof Laemmli sample buffer (62.5 mM Tris-HCl [pH 6.8], 2%SDS, 10% glycerol, 5% mercaptoethanol), incubated for 30min at room temperature, heated for 4 min at 95°C, and loadedonto an SDS-10% polyacrylamide gel.

In a control experiment, LLC-PK1 cells were infected withVSV (San Juan strain). At 4 h postseeding (p.s.), cells wereinoculated (multiplicity of infection of 20) from the apical andbasolateral sides for 1 h at 37°C. To slow down the cytopathiceffects (CPE), all further incubations were done at 32°C (41).Cells were labelled from 4 to 6 h p.i. with 300 jiCi of 3 Slabelling mix and chased for 2 h. Media were collected, andVSV proteins were immunoprecipitated as described above,using a polyclonal rabbit anti-VSV serum.

Inhibition of infection by aminopeptidase N antibodies.LLC-PK1 cells were incubated for 2 h at 37°C with serialdilutions of monoclonal antibody G43 against aminopeptidaseN (11) or a control monoclonal antibody, CC1, against theMHV receptor (43) prior to inoculation with TGEV (multi-plicity of infection of 1). Infection was monitored both byvisual inspection of cytopathic changes under a light micro-scope and by titration of released virus present in the mediacollected at 10 h p.i.

Titration of virus. The amounts of infectious TGEV parti-cles released into the media of infected cells were determinedby plaque assay on PD5 cells. Monolayers of cells wereinoculated with serial dilutions of the culture media in PBS.After 1 h, the inoculum was replaced by culture mediumcontaining 1.5% agarose. Cells were incubated at 37°C, andafter 2 days, plaques were counted. An endpoint dilution assaywas used to titrate virus in the case of the infection inhibitionexperiment. LLC-PK1 cells grown in 96-well plates wereinoculated with serial dilutions of the culture medium inDulbecco's modified Eagle's medium. The 50% tissue cultureinfectious dose values were determined by using the Spear-man/Kaerber relationship (20).

Localization of the TGEV receptor. Filter-grown LLC-PK1cells were fixed at 14 and 84 h p.s. with 3% paraformaldehyde,rinsed in PBS containing 50 mM glycine (PBG) for 10 min, andblocked for 30 min with 5% bovine serum albumin (BSA), 5%normal goat serum, and 0.1% BSA-C (Aurion) in PBG. Cellswere rinsed for 10 min with 0.1% BSA-C in PBG (PBG+) andincubated for 2 h from both the apical and basolateral sideswith the aminopeptidase N-specific monoclonal antibody G43(11) diluted 1,000-fold in PBG+. After three washes withPBG+, cells were stained from the basolateral side withfluorescein isothiocyanate (FITC)-conjugated goat anti-mouseimmunoglobulin G (1:200; Cappel) and from the apical sidewith tetramethyl rhodamine isothiocyanate (TRITC)-conju-gated goat anti-mouse immunoglobulin G (1:100; Protos Im-munoresearch) for 2 h. Finally, cells were washed three timeswith PBG+, and filters were cut from their holders andmounted in FluorSave reagent (Calbiochem). Fluorescencewas viewed in a Zeiss Axioplan microscope in conjunction witha confocal laser scanning unit (model MRC600; Bio-RadLaboratories). Optical sections were taken at intervals of -1jim parallel to the filters. For alternative observation of TGEVreceptor distribution, optical sections of -1-jim thickness weretaken perpendicular to the cell monolayer.

Electron microscopy. LLC-PK1 cells grown on filters for 5days were inoculated with TGEV from the apical side for 1 h.

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J. VIROL.7968 ROSSEN ET AL.

The cells were then washed three times with PBS to removeresidual inoculum virus, and culture medium was added. Whenindicated, brefeldin A (Boehringer Mannheim) was added to aconcentration of 6 ,ug/ml. At 9 h p.i., cells were rinsed with PBSand fixed for 24 h in 2% paraformaldehyde-0.5% glutaralde-hyde in 0.1 M phosphate buffer (pH 7.2). Filters were thentreated for 1 h with 1% osmium tetroxide, block stained for 1h with 2% uranyl acetate, and cut from their holders. Afterdehydration in acetone, the filters were embedded in Durco-pan epoxy resin (Fluka). Ultrathin sections of 45 nm were cuton a Reichert UltracutS and stained for 2 min with Reynoldslead citrate (27). Sections were observed and photographed ina Philips CM10 electron microscope at 80 kV.

hps 16 24 48A B A B A B

EGTA

S- 4"i jl -i

72 95 95A B A B A B

A W N

*:-.

RESULTS

Cells and TER. LLC-PK1 cells were originally derived fromthe proximal tubule of a pig kidney. Their epithelial charac-teristics and susceptibility to TGEV have been describedelsewhere (19). In initial experiments, we found that TGEVinfected up to 100% of the cells and that the virus was releasedinto the medium from 6 h p.i., giving titers of 106 PFU/ml at 9h p.i. CPE were observed at around 12 h p.i. (results notshown).To study the polarity of entry and release of TGEV in

epithelial cells, the cells must form tight monolayers. Thetightness of the monolayer can easily be assessed by measuringthe TER. Under our experimental conditions, the TER wasfound to increase quite rapidly to a value of about 100 f1cm2during the first day after plating cells onto filters. After 4 to 5days, it reached a steady-state value of 250 to 300 f1cm2 (resultsnot shown), similar to that found by others (reference 17 andreferences therein). Routinely, tightness of the monolayer waschecked by adding medium to the apical compartment up to aslightly higher level than in the basolateral compartment (6).No leakage of culture medium from the apical to the basolat-eral compartment occurred from 4 to 8 h p.s., indicating that bythis criterion LLC-PK1 cell monolayers were already com-pletely tight from 4 to 8 h p.s. (results not shown).

During TGEV infection of filter-grown cells, minimalchanges in the TER were observed during the first 10 h p.i.Thereafter, the resistance decreased, but the difference in thelevel of the culture media was maintained until 12 h p.i.,indicating that the monolayer is intact until 10 to 12 h p.i.Consequently, all further experiments were performed be-tween 2 and 10 h p.i.

Polarity of virus entry. (i) Synthesis of viral proteins. Todetermine if the entry of TGEV in LLC-PK1 cells is restrictedto a specific membrane domain, we analyzed virus-specificprotein synthesis in filter-grown cells infected from the apicalor basolateral side at different times p.s. The cells were labelledwith 35S labelling reagent, and intracellular viral proteins wereimmunoprecipitated with a mixture of ascites fluid A40 andmonoclonal antibody 995. Figure 1 shows that the virus couldinfect the cells from both sides at 16 and 24 h p.s. After 24 to48 h of growth, the infectability through the basolateralmembrane decreased and the entry of TGEV became re-stricted to the apical side. To exclude the possibility that theseobservations were due to the inability of the virus to pass thefilters and reach the cells at later time points p.s., cells grownfor 95 h on filters were treated with EGTA prior to infection.This treatment opens the tight junctions between the cells. Asa consequence, intercellular transport of viruses becomespossible and plasma membrane proteins, e.g., proteins func-tioning as viral receptors, are redistributed over the wholesurface. Cells treated with EGTA immediately before inocu-

N- _ 4 _ _ _

M- 4b2 u x*

FIG. 1. Virus-specific protein synthesis in LLC-PK1 cells infectedwith TGEV from the apical or basolateral side. Cells were infectedfrom the apical (lanes A) or basolateral (lanes B) side at the indicatedtimes p.s., labelled from 4 to 6 h p.i. with 50 ,uCi of 35S labelling mix perculture, and lysed at 6 h p.i. Two cultures were treated with 30 mMEGTA for 15 min just before infection at 95 h p.s. (hps). Viral proteinswere precipitated from the cell lysates with polyclonal antiserum A40in combination with monoclonal antibody 995. Proteins were separatedin an SDS-10% polyacrylamide gel. The positions of the viral spike(S), nucleocapsid (N), and membrane (M) protein are indicated.

lation had similar amounts of newly synthesized viral proteinswhether they were infected through the apical or the basolat-eral side (Fig. 1). The preferential apical entry of TGEV intoLLC-PK1 cells was confirmed by using an indirect immunoflu-orescence assay to detect intracellular viral proteins (data notshown).

(ii) Inhibition of TGEV infection by aminopeptidase Nantibodies. The ectoenzyme aminopeptidase N has been iden-tified by Delmas et al. (11) as a receptor for TGEV. Thisprotein is also present in LLC-PK1 cells (32). To determinewhether TGEV uses aminopeptidase N as its receptor in oursystem, we performed an infection inhibition experiment. Priorto inoculation with TGEV, LLC-PK1 cells were incubated withmonoclonal antibody G43, known to recognize aminopepti-dase N (11). As judged from the release of progeny virus intothe culture medium, the infection was inhibited in a concen-tration-dependent manner (Fig. 2). Accordingly, no or onlyminor CPE was observed after treatment with the G43 anti-body at a dilution of 57 or lower, while at higher dilutions, theinhibitory effect of the antibody ceased concomitant with anincrease in CPE (results not shown). In contrast, no inhibitionwas observed when the cells were preincubated with monoclo-nal antibody CC1 (Fig. 2), directed against the receptor forMHV (43). Thus, the entry of TGEV into LLC-PK1 cells ismediated by the aminopeptidase N protein.

(iii) Localization of the TGEV receptor. To determine theplasma membrane distribution of the TGEV receptor, filter-grown LLC-PK1 cells were examined in a confocal laserscanning microscope, which allows optical sections to be cuteither in the horizontal plane (XY section; Fig. 3A, B, E, andF) or in the vertical plane (XZ section; Fig. 3C, D, G, and H).

TGEV IN POLARIZED EPITHELIAL CELLS 7969

9 =

Foc

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0

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C70

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*.

FITC TRITC

14 hp.

- G43cc1

1 2 3 4 5 6 7 9 10 11 12 13 14Antibody dilution (log 6)

FIG. 2. Inhibition of TGEV infection with an antibody againstaminopeptidase N. Prior to inoculation, cells were incubated for 2 h at37°C with serial dilutions of a monoclonal antibody to aminopeptidaseN (G43) or, as a control, a monoclonal antibody to the MHV receptor(CC1). Media were collected at 10 h p.i. and assayed for the presence

of infectious viral particles, using an endpoint dilution assay on

LLC-PK1 cells. TCID50, 50% tissue culture infectious dose.

Uninfected cells were incubated apically and basolaterally withantibody G43 and then incubated with an FITC-conjugatedantibody from the basolateral side and a TRITC-conjugatedantibody from the apical side. When cells were fixed at 14 hp.s., the TGEV receptor was detected both on the apical side(TRITC channel; Fig. 3B and D) and on the basolateral side(FITC channel; Fig. 3A and C). In contrast, in cells fixed at 84h p.s., the receptor was detected only on the apical side (Fig.3F and H), not on the basolateral side (Fig. 3E and G). Similarresults were obtained in the reverse experiment, in whichFITC- and TRITC-conjugated second antibodies were usedapically and basolaterally, respectively (data not shown).

Polarity of virus release. To investigate whether the releaseof TGEV from the epithelial cells is polarized, we comparedthe amounts of infective virus in the apical and basolateralmedia. Apical and basolateral media of TGEV-infected cellswere assayed for viral progeny by using a plaque titration assayon PD5 cells. Table 1 shows that 30-fold more PFU hadaccumulated in the apical medium than in the basolateralmedium.To confirm these data in an independent assay and to

determine the kinetics of cell polarization, cells were infectedfrom the apical side at different times after seeding andlabelled with 35S labelling reagent. Subsequently, culture me-

dia were collected, an aliquot from each was taken for plaquetitration, and viral proteins were immunoprecipitated from theremaining part. The release of radiolabelled viral proteins waspolarized from the earliest infection time point on, i.e., whenmeasured between 11 and 13 h p.s. (Fig. 4A). As was generallythe case (e.g., Fig. 4B), the efficiency of labelling early afterseeding was somewhat lower than at later times. Probably, thecells need some time to settle and become metabolically active.The results were further confirmed by analyses of infectivi-

ties in the same media. Again the release of infectious TGEVparticles from cells was polarized when measured between 11

84 hp:

FIG. 3. Localization of the TGEV receptor on the plasma mem-brane of LLC-PK1 cells by confocal microscopy. Filter-grown LLC-PK1 cells were fixed at 14 and 84 h p.s. (hps) and incubated from theapical and basolateral sides with a monoclonal antibody against thereceptor of TGEV (G43). Subsequently, cells were incubated from theapical side with a TRITC-conjugated second antibody (B, D, F, and H)and from the basolateral side with an FITC-conjugated second anti-body (A, C, E, and G). Panels A, B, E, and F show XY sectionsthrough the cells, i.e., parallel to the filter. Panels C, D, G, and H showXZ sections through the cells, i.e., perpendicular to the XY sections.The apical region of the cells is oriented topmost. Note that panels Ato D and E to H are sets of images each taken from the same cells.

and 13 h p.s. Calculations reveal that for the three infectiontime points (4, 8, and 12 h p.s.) shown in Fig. 4A, 95, 83.7, and95.9%, respectively, of the infective virus particles were shedinto the apical medium.

In the course of our studies, we noticed that an unidentifiedcellular protein (-250 kDa) was nonspecifically coimmunopre-cipitated from basolateral media of LLC-PK1 cells. We took

TABLE 1. Release of infective virus particles from TGEV-infectedaLLC-PK1 cells into apical and basolateral media

Titer (PFU/ml)cMediumb

2.5 h p.i. 9 h p.i.

Apical 1,100 4.1 x 106 96.9Basolateral 145 1.3 x 105 3.1

a Cells were infected from the apical side 5 days after seeding.b Media were collected at 9 h p.i.cPlaque assays (n = 6 for each time point) were performed on PD5 cells.d Relative to the total amount of infective virus particles released.

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7970 ROSSEN ET AL.

A

hps 4 8 12 16A B A B A B A B

S- ..- _... _ .11

N- -_d_

B

analyzed by electron microscopy. In Fig. 5A, a large number ofC viral particles can be seen attached to the apical plasma

membrane. In contrast, hardly any viral particles can be seenhps 4 between the cells (Fig. 5A) or between the cells and the filter

A B (Fig. 5C). Figure 5B shows an enlargement of part of the apicalmembrane with some bound viral particles. It appeared thatviruses bind with some preference to microvilli. To ascertainthat the viral particles observed at the apical plasma membranewere newly synthesized virions and not residues of the inocu-lum, cells were infected and then treated from 2 h p.i. with 6 ,ugof brefeldin A per ml. Brefeldin A blocks the transport ofproteins from the endoplasmic reticulum to the Golgi complexand has been shown to prevent the release of coronaviral

G - particles (22a). Figure 5D shows a representative part of theG, apical plasma membrane of a brefeldin A-treated cell. As

illustrated by this figure, no viral particles attached to the

N/NS - apical membrane were observed under these conditions, al-though such particles were produced. Additional evidence thatthe viruses seen in association with the apical membrane werenewly synthesized particles came from an experiment in whichthe cells were inoculated at 16 h p.s. from the basolateral side.

M - - Again, viral particles were clearly observed only at the apicalplasma membrane (data not shown). From these electronmicroscopical results, it can be concluded that TGEV isreleased apically and that no virus accumulates between thecells or between the cells and the filter.

hps 2-5 5-8A B A B

8-11A B

of_

11-14A B

.. 4....X

FIG. 4. Analysis of TGEV proteins released apically and basolat-erally from LLC-PK1 cells. (A) Cells were infected with TGEV fromthe apical side at the indicated hours p.s. (hps), labelled from 6 to 7 hp.i. with 100 ,uCi of 35S labelling mix per culture, and chased for 2 h.Apical (lanes A) and basolateral (lanes B) media were collected, andviral proteins were precipitated with polyclonal antiserum A40 incombination with monoclonal antibody 995. Proteins were separated inan SDS-10% polyacrylamide gel. Note that the indicated times are thetimes at which the infection was started. (B) Uninfected LLC-PK1 cellswere labelled at the indicated times with 300 puCi of 35S labelling mixper culture. Subsequently, apical (lanes A) and basolateral (lanes B)media were collected and subjected to the same immunoprecipitationprocedure and analysis as described for panel A. (C) LLC-PK1 cellswere infected with VSV at 4 h p.s., labelled from 4 to 6 h p.i. with 35Slabelling mix, and chased for 2 h. Apical (lanes A) and basolateral(lanes B) media were collected, and viral proteins were immunopre-cipitated with a polyclonal anti-VSV serum.

advantage of this observation and used the protein as a markerto monitor the development of polarity in the release of acellular protein. As Fig. 4B illustrates, the protein appeared inboth media early after seeding of the (uninfected) cells,indicating that the monolayer was not intact or that the releaseof the protein was not yet polarized at this time. Soon,however, the protein started to appear with rapidly increasingpreference in the basolateral medium. As an additional con-

trol, we examined the release of VSV, which has been shownpreviously to bud specifically from the basolateral membraneof epithelial cells (13, 30). We found that also in LLC-PK1cells, the virus was predominantly released from the basolat-eral side. This polarity was again established soon after platingof the cells (Fig. 4C).

Electron microscopical studies. To exclude the possibilitythat these results had been biased by trapping of basolaterallyreleased viral particles between the cells or between the cellsand the filter, ultrathin sections of apically infected cells were

DISCUSSION

We have investigated the interaction of TGEV with epithe-lial cells in an in vitro system. Our data demonstrate thatTGEV enters LLC-PK1 cells only through the apical plasmamembrane. As has also been found for some other viruses (8,13, 14), the polarized entry of TGEV correlates with thepolarized distribution of its receptor, the ectoenzyme amino-peptidase N. Confocal laser scanning microscopy showed thatthis protein was present at both the apical and basolateralplasma membrane domains just after plating of the cells ontoa filter support but that it became restricted to the apicalmembrane during culture. This finding is consistent with theobservation that the polarity of entry of TGEV into filter-grown LLC-PK1 cells was not immediately evident but devel-oped as a function of time after seeding of the cells. Thekinetics of this process appeared to be delayed with respect tothe development of tight junctions. While the latter wereformed within 4 to 8 h p.s., it took at least 24 to 48 h for TGEVentry to become polarized. Presumably, this is the time re-quired before the basolateral membrane has become devoid ofreceptor molecules, either by normal turnover or by theirredistribution through transcytotic delivery to the apical side.A similar asynchrony between polarization of cell surfaceproteins and development of tight junctions by LLC-PK1 cellshas been reported for alkaline phosphatase and -y-glutamyltranspeptidase (26).Our virological, biochemical, and electron microscopical

data clearly revealed that TGEV is preferentially releasedfrom the apical side. This is advantageous for a virus thatpenetrates the cells also from the apical side. Apical exit ofvirus into the lumen of the gut allows rapid infection ofadjacent cells in the intestine. The apical release of TGEV wassuggested earlier by Pensaert et al. (23, 24) in a study ofisolated ileum and jejunum loops from infected pigs. Byelectron microscopy, many virus particles were detected in thelumen, especially around the microvillar border, before celldegeneration was first detected.The selective release of TGEV from the apical membrane

J. VIROL.

A

B

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FIG. 5. Electron microscopical analysis of TGEV-infected filter-grown LLC-PK1 cells. Cells were infected with TGEV from the apical side at5 days after seeding on filters. At 8 h p.i., the cells were fixed and embedded, and sections were cut for observation in an electron microscope. (A)Overview of a cell with viral particles attached to the apical plasma membrane (arrows) and vesicles with viral particles present inside the cell (V).The filter can be seen at the bottom. (B) Magnification of part of the apical plasma membrane shown in panel A with viral particles attached tothe membrane. (C) Magnification of part of the basolateral plasma membrane. The filter is seen at the bottom, and no viral particles are observed.(D) Apical plasma membrane of apically infected cells treated with brefeldin A from 2 h p.i. No viral particles are observed at the membrane, butsome are visible inside the cell (arrows). Calibration bars: 0.8 ,um (A), 0.2 pum (B), 0.4 p.m (C), and 0.5 p.m (D).

7971

I.,.,,

7972 ROSSEN ET AL.

domain of LLC-PK1 cells appeared already between 11 and 13h p.s., i.e., long before the polarity in the entry of the virus wasevident. At that time, both VSV and a cellular marker proteinwere predominantly released from the opposite side, i.e., thebasolateral membrane. Clearly, both the apical and basolateraltransport pathways of the cells are established rapidly. Thesefindings are consistent with electron microscopical observa-tions with influenza virus and VSV in MDCK cells, whichshowed that the exit of these viruses becomes polarized as soonas cell-cell contacts or cell-substrate contacts are established(29).

Coronaviruses are assembled intracellularly at pre-Golgimembranes (21, 22, 39). Little is known about the sorting andrelease in polarized epithelial cells of other enveloped virusesthat assemble at intracellular membranes. The few examplesstudied indicate that they are also asymmetrically released (fora recent review, see reference 40). Herpes simplex viruses,which are assembled by using membranes of the nucleus andthe Golgi complex (see reference 15 and references therein),are preferentially released from the basolateral plasma mem-brane (35). Studies with the bunyaviruses Punta Toro virus (7)and Rift Valley fever virus (2), assembled by budding in theGolgi complex, showed that these viruses are also selectivelyreleased from basolateral membrane domains.There are several possible mechanisms for the sorting of

intracellular budding viruses to the apical or basolateralplasma membrane domain. Coronaviruses and bunyavirusesare transported to the cell surface by vesicular transport.Virus-containing vesicles may be directed by the same mech-anisms that are responsible for the targeted delivery of soluble,secreted polypeptides. Newly synthesized secretory proteinsare often released in a polarized fashion (4, 40), but how thisoccurs is still unknown. One possibility involves their interac-tion with a membrane-bound receptor, which is itself targetedto a specific destination. This would be analogous to thetargeting of lysosomal enzymes to lysosomes by way of areceptor which recognizes the mannose-6-phosphate modifica-tion on these enzyme molecules (25). Importantly, in theosteoclast, a polarized cell which secretes large amounts oflysosomal enzymes into an apical lacuna, the mannose-6-phosphate receptor has been shown to colocalize with lysoso-mal enzymes along the secretory pathway to the apical mem-brane, suggesting its role in targeting (3). Assuming that theTGEV receptor is directly targeted to the apical membrane,TGEV virions might also be transported under the guidance oftheir receptor. The human equivalent of the TGEV receptor,human aminopeptidase N, is indeed directly sorted to theapical plasma membrane domain when expressed in MDCKcells (42). Another possible mechanism for the apical sorting ofTGEV involves the spike protein. In coronavirus-infected cells,not only is this protein incorporated into virions, but a certainamount is independently transported to the plasma membrane.It is conceivable that TGEV virions are cosorted into specificvesicles together with free spike proteins. In this way, the spikeprotein might confer specific targeting information to thevesicles. It will therefore be of interest to investigate thetransport behavior of independently expressed TGEV spikeprotein in LLC-PK1 cells.

ACKNOWLEDGMENTSWe thank Harry Vennema and Dirk-Jan Opstelten for helpful

discussions. We are very grateful to H. Laude and K. Holmes forproviding the antibodies directed against the TGEV receptor and theMHV receptor, respectively. We also thank A. Legendre for helpfulcomments and careful reading of the manuscript. Ton Ultee andWillem Hage are acknowledged for technical assistance with the

electron microscopical and confocal laser scanning microscopicalstudies, respectively.

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