Central European Journal of Biology

* E-mail: riabi_samira@yahoo.fr

Research Article

1Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives (LR99-ES 27), Faculté de Pharmacie de Monastir; Tunisie

2 Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP, EA 3064), Faculté de Médecine Jacques Lisfranc, Université de Lyon, 42023 Saint-Etienne cedex 02, France

† These two authors contributed equally to this work.

Samira Riabi1,2,*†, Rafik Harrath1,2,†, Imed Gaâloul1, Hind Hamzeh-Cognasse2, Olivier Délezay2, Mahjoub Aouni1, Bruno Pozzetto2

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

1. IntroductionPolarized membranes constitute the interface of a great number of viral pathogens for their penetration across mucosal epitheliums of various tracts. At the intestinal level, many viruses -including adenoviruses, reoviruses and enteroviruses- have been shown to interact with polarized enterocytes displaying tight junctions at their basolateral side [1].

Coxsackieviruses B (CV-B), which belong to the Enterovirus genus of the Picornaviridae family, have been particularly studied for their interaction with polarized cells of the intestinal mucosa. They comprise six serotypes (1 to 6) that share the same tissue tropism including gut, central nervous system, heart and pancreas. In the gut, they interact with at least one or

two of the following cellular receptors: coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) [2-5].

CAR is a 46-kDa transmembrane glycoprotein that functions as a primary receptor for CV-B and adenoviruses [3]. This cell surface molecule appears to be more efficient to support CV-B infection due to its capacity to induce conformational changes in the viral capsid resulting in the formation of A particles, an event common to all enteroviruses [3,4,6,7]. Functionally and structurally, CAR is a member of the subgroup of junction adhesion molecules belonging to the immunoglobulin superfamily [8,9], with a typical transmembrane region, a long cytoplasmic domain and an extracellular region composed of two disulfide-linked loops [4]. In polarized epithelial cells, CAR is closely associated with the tight

Cent. Eur. J. Biol. • 9(7) • 2014 • 699-707DOI: 10.2478/s11535-014-0305-2

699

Received 07 August 2013; Accepted 21 December 2013

Keywords: CaCo-2 cell line • Coxsackievirus and adenovirus receptor • Decay accelerating factor • Trypsin • Flow cytometry • Coxsackievirus B.

Abstract: A subset of coxsackieviruses B (CV-B) is able to initiate intestinal infection via the attachment to two cell surface proteins, decay-accelerating factor (DAF) and coxsackie adenovirus receptor (CAR). The aim of the present study was to investigate the expression pattern of these receptors in the polarized CaCo-2 cell line using flow cytometry. The expression of CAR-specific mRNA and proteins was analyzed by reverse transcriptase polymerase chain reaction and western blotting, respectively. Flow cytometry analysis was used to study the surface expression patterns of CAR and DAF. CAR and DAF were well detected at the surface of CaCo-2 cells by flow cytometry. Despite the fact that CAR was susceptible to the action of trypsin, a few amounts of the latter enzyme and a precise dilution did not impair its correct detection by flow cytometry. This technique was used to demonstrate that the density of cells did not influence the expression of CAR at the cell surface. CaCo-2 cells express high levels of CAR and DAF at their surface. Flow cytometry, if used adequately, represents a helpful tool for the study of the interactions between these cells and various viral targets.

© Versita Sp. z o.o.

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

junctions where it may be sequestered and inaccessible to virus, especially from the apical side [9,10].

A subset of CV-B also attaches to an accessory receptor called DAF (or CD55). DAF is a 70-KDa glycosylphosphatidylinositol-linked complement regulatory protein consisting of four extracellular short consensus repeats (SCR) [11,12]. DAF protects autologous cells from attack by complement proteins [13] and its expression on the cell surface is almost ubiquitous throughout the human body. DAF protein has been shown to only lead to virus attachment, “handing off” the virus to CAR and allowing viral entry to proceed [6,14,15]. In polarized cells, the interaction between some CV-B and DAF at the apical site of the cells has been shown to play a key role for giving access to CAR, the major receptor of CV-B associated to the tight junctions of the basolateral side of the cells [16].

The CaCo-2 cell line, which spontaneously forms relatively well-differentiated in vitro epithelial monolayers, exhibits several characteristics of the human intestinal cells from which it derives and mimics the properties of the cells that line the colonic crypts [17]. These cells have been extensively used to study the mechanisms of entry of CV-B at the intestinal site [9,16,18]. When polarized, CaCo-2 cells express CAR and DAF at their basolateral and apical side, respectively. Whereas DAF is resistant to the action of trypsin, CAR has been shown to be trypsin-sensitive [19], which could impair the detection of this receptor in trypsinised non-adherent cells. The goal of the present work was to characterize the expression of CAR and DAF on CaCo-2 cells by flow cytrometry. We applied this technique to the estimation of the expression of CAR and DAF on these cells as a function of cell density.

2. Experimental Procedures2.1 Cell linesThe CaCo-2 cell line (human colon adenocarcinoma cell line) was kindly provided by the department of Immunity of mucous membranes and pathogenic agents (GIMAP, Faculty of medicine Jacques Lisfranc, Saint-Etienne, France). These cells were cultured in Dulbecco’s modified Eagle medium (DMEM-F12, PAA laboratories, Les Mureaux France) supplemented with 10% foetal bovine serum (FBS) (GIBCO-Invitrogen, Cergy Pontoise, France) and an antimicrobial solution containing 10,000 units mL-1 of penicillin, 10 mg mL-1 of streptomycin and 25 μg mL-1 of amphotericin B (Sigma-Aldrich, Lyon, France).

The KB cell line, derived from a human squamous carcinoma, and the HeLa cell line, derived from a

human cervical cancer, were grown in RPMI 1640 medium (Sigma-Aldrich) containing 10% FBS, 2 mM L-1

L-glutamine (Sigma-Aldrich) and the antimicrobial solution as described earlier. These cell lines are not polarized and were used as positive controls known to express high levels of CAR and DAF, and to be permissive to CV-B. The cells were incubated at 37°C in an atmosphere of 5% CO2.

2.2 Detection of CAR gene by RT-PCRTo verify the expression levels of CAR mRNA in CaCo-2 cell line, total RNA was extracted using guanidinium thiocyanate-phenol chloroform, as described previously [20]. Total RNA were treated with DNase I amplification grade (Invitrogen, Carlsbad, CA, USA) to eliminate contaminated genomic DNA. One µg of RNA was converted to cDNA by reverse transcription, using Moloney murine leukemia virus reverse transcriptase and oligo(dT)15 primers (Promega, Charbonnières les Bains, France). Then, the cDNA was used as template to amplify the entire coding region of the CAR gene with primers 5’-AGGAGCGAGAGCCGCCTAC-3’ and 5’-ACGGAGAGCACAGATGAGACA-3’. The length of the expected product was 1170 bp. The PCR protocol consisted of an initial denaturation step for 5 min at 95°C followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and extension for 60 s at 72°C; the final cycle had a prolonged extension time of 10 min at 72°C. A control PCR with primers targeting a 983-bp fragment of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) housekeeping gene (5’-TGAAGGTCGGAGTCAACGGATTTGGT-3’ and 5’-CATGTGGGCCATGAGGTCCACCAC-3’) was performed in parallel to verify that similar amounts of cDNA were provided in each preparation. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide and photographed under UV light.

2.3 Western blot analysisConfluent 25 cm2 tissue culture flasks containing the cell lines were washed three times with phosphate buffered saline (PBS). Cells were harvested by scraping in 3 ml of PBS and centrifuged in 15 ml polypropylene tubes at 1500 rpm for 15 min. Supernatants were removed and the cell pellets were treated for 1h at 4°C in lysis buffer containing 20 mM L-1 Tris, 150 mM L-1 sodium chloride, 5 mM L-1 EDTA, 1% Triton-X100, 25 mM L-1 sodium fluoride, 1 mM L-1 phenyl methylsulphonyl fluoride, 1 mM L-1 sodium metavanadate, 10% glycerol and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Meylan, France). After removal of cell nuclei by centrifugation, protein

700

S. Riabi et al.

concentrations were measured by the Bradford protein assay. Equal amounts of protein (15 µg) from each cell lysate were diluted with sodium dodecyl sulphate (SDS) loading buffer, heated for 5 min at 95°C and loaded on a 10% SDS-polyacrylamide gel. The separated proteins were then electrotransferred to a nitrocellulose membrane first treated with 50 g L-1 non-fat dried milk in Tris buffered saline-Tween buffer in order to block the non-specific binding sites. The blot was then incubated with either monoclonal mouse anti-human CAR antibody (clone CAR 3C100, Santa Cruz Biotechnology, Inc, Heidelberg, Germany) diluted 1:3 in PBS, or monoclonal mouse anti-CD55 antibody (clone IA10, BD Biosciences Pharmingen, San Diego, CA, USA) diluted 1:1000 in Tris buffered saline-Tween buffer containing 0.01 g mL-1 bovine serum albumin, for 1h at room temperature with gentle shaking. After extensive washing, an alkaline phosphatase-coupled goat anti-mouse IgG secondary antibody (Sigma-Aldrich) diluted 1:1000 in Tris buffered saline-Tween containing 0.01 g mL-1 bovine serum albumin was applied for 1h at room temperature with gentle shaking. The bands were incubated for 5 min with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate for revelation.

2.4 Detection of CAR and DAF receptors by flow cytometry

To optimize the expression of CAR and DAF receptors and to determine the effect of trypsin on CAR expression, confluent CaCo-2 cell monolayers in T25 flasks were rinsed with PBS and detached by incubation with one of the following solutions: trypsin-EDTA (Sigma-Aldrich), accutase (PAA Laboratories), alfazyme (PAA Laboratories), Hank’s buffer (cell dissociation buffer enzyme free, GIBCO-Invitrogen) or 1 mg mL-1 trypsin (GIBCO-Invitrogen) diluted 1:2 to 1:10 in Hank’s buffer. After 5-10 min at 37°C, adherent cells were scraped, washed with DMEM-F12 containing 10% FBS in order to stop the activity of the different enzymes, and collected by centrifugation. The cells were then incubated for 30 min at +4°C with one of the following mouse monoclonal antibodies: anti-CAR (clone RmcB, Millipore, Molsheim, France) or anti-CD55 (clone IA10, BD Biosciences Pharmingen, Pont de Claix, France); a mouse isotype IgG (BD Biosciences Pharmingen) was used as control. After two washing steps with PBS, the cells were stained for further 30 min at +4°C with the secondary antibody, a goat anti-mouse fluorescein isothiocyanate-conjugated (FITC) antibody (BD Biosciences Pharmingen), washed again twice in PBS and resuspended in 0.5 ml of PBS. The flow cytometry analysis was performed using a FACS Calibur apparatus and CellQuest™ Pro software (Becton Dickinson, Pont de Claix, France)

2.5 CAR and DAF expression according to cell differentiation

To obtain different stages of cell growth, CaCo-2 cells were seeded into 6-well format Transwell® inserts at various dilutions (0.5 to 0.03 106 cells per well). Cells were visually controlled to be evenly subconfluent or confluent, as measured by the transepithelial electric resistance (TEER) between the apical and basal compartments with a EVOM ohm voltmeter (World Precision Instruments Inc, Gentaur, Paris, France) using a STX2 manual electrode. A value of 100 Ω.cm2

was chosen to indicate a significant TEER. At different stages of confluence, the cells were disrupted from their support according to the procedures described earlier, pelleted at 1200 rpm for 5 min in PBS and stained with one of the following mouse monoclonal antibodies: anti-CAR (clone RmcB, Millipore) or anti-CD55 (clone IA10, BD Biosciences Pharmingen). After incubation with a goat anti-mouse FITC-conjugated antibody (BD Biosciences Pharmingen) as described above, the cells were washed twice with PBS, resuspended in 500 µl PBS and analyzed by flow cytometry; KB cells were tested as control using the same protocol.

3. Results and Discussion3.1 Expression of CAR and DAF on CaCo-2 cellsPreliminary experiments were conducted to confirm that the CaCo-2 cell line used in this study expressed correctly both CAR and DAF receptors. KB and HeLa cells were used as positive controls known to express high levels of CAR and DAF. The results show that CaCo-2 cells expressed CAR at the same level as control cells. The expression of this receptor was demonstrated by (i) the amplification of specific CAR mRNA (Figure 1a), which revealed a band of 1170 bp. The control PCR was performed with primers that amplify a 983-bp fragment of the G3PDH housekeeping gene (Figure 1b) and (ii) the intensity of the 46 kDa band revealed by immunoblotting analysis using a CAR specific monoclonal antibody (Figure 1c). Similar findings were observed for DAF expression, as illustrated by the band of 70 kDa specific of this receptor after immunoblotting analysis (Figure 1d). These results clearly show that the three cell lines express similar levels of both receptors.

3.2 Detection of CAR and DAF receptors on CaCo-2 cells by flow cytometry

The second step of this study consisted of quantifying the levels of CAR and DAF expression on the CaCo-2 cell line through flow cytometry analysis. The KB cell line was used as control. In order to perform flow

701

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

cytometry analysis, the cells need to be detached from their culture support. Various treatments were tested to detach cells, including trypsin-EDTA solution, enzymes like accutase and alfazyme, Hank’s buffer or trypsin diluted in Hank’s buffer were performed to ascertain the effect of detachment method on the expression of CAR and DAF receptors. Representative histograms are depicted in Figure 2 and a summary of the results is given in Table 1.

Hank’s buffer was found able to efficiently detach KB cells with no effect on the expression of both DAF (Figure 2c) and CAR (Figure 2d) receptors. In contrast, this buffer was not sufficient to dissociate the CaCo-2 monolayer, nor were other buffers containing accutase or alfazyme (Figure 2e). When high amounts of trypsin were used in association with EDTA to dissociate the CaCo-2 monolayer, the DAF receptor was correctly detected, however the expression of CAR receptor was effected by enzymatic dissociation protocols (Figure 2a). As illustrated in Figure 2b, a correct detection of the CAR receptor on CaCo-2 cells was rendered possible by using a low concentration of trypsin (1 mg mL-1 diluted 1:4 in Hank’s buffer); this concentration of trypsin was found to detach CaCo-2 cells efficiently without impairing the detection of CAR at the cell surface.

3.3 CAR and DAF expression according to the differentiation of CaCo-2 cells

In order to obtain different stages of cell growth, CaCo-2 cells were seeded at various dilutions (0.031 to 0.5 106 cells/well) and grown for 10 days. The detection of DAF and CAR was determined on the surface of the cells by flow cytometry after cell detachment with diluted trypsin as described in the previous section. The results depicted in Figure 3 show a high expression of both DAF and CAR whatever the cell density measured by the TEER of the monolayer before cell dissociation.

Table 1. Results of the expression of CAR and DAF receptors in CaCo-2 and KB cells using the different cell detaching methods. (+): expression of the receptor; (-): no expression of the receptor.

+ CaCo-2 cells KB cells

Trypsin-EDTA CAR-/DAF+ CAR-/DAF+

Hank’s buffer CAR-/DAF+ CAR+/DAF+

Accutase/alfazyme CAR-/DAF+ CAR-/DAF+

Trypsin diluted 1:4 in Hank’s buffer CAR+/DAF+ CAR+/DAF+

Figure 1. Expression of different receptors by RT-PCR (a and b) and Western blot analysis (c and d) in Caco-2, HeLa and KB cell lines. Panel a: mRNA of CAR visualized by a band of 1170 bp. Panel b: mRNA of G3PDH (used as control) visualized by a band of 983 bp. Panels c and d: immunoblotting of CAR (46 kDa, panel c) and DAF (70 kDa, panel d) proteins using specific antibodies to these molecules.

70 KDa

Caco-2 KB HeLa

46 kDa

Caco-2 MW HeLa KB

1170 bp

KB HeLa Caco-2 M M KB HeLa Caco-2

983 bp

a b

c d

70 KDa

Caco-2 KB HeLa

46 kDa

Caco-2 MW HeLa KB

1170 bp

KB HeLa Caco-2 M M KB HeLa Caco-2

983 bp

a b

1170 bp

KB HeLa Caco-2 M

1170 bp

KB HeLa Caco-2 M M KB HeLa Caco-2

983 bp

a b

c d

702

S. Riabi et al.

Similar results were obtained for KB cells (data not shown). These results suggest that the level of cell differentiation in polarized cells does not influence the expression of both receptors.

Figure 2. Flow cytometry analysis of CAR and DAF expression on Caco-2 (a, b and e) and KB (c and d). The solution used for detaching the cells from their support was trypsin-EDTA for panel a, trypsin diluted 1:4 in Hank’s buffer (final trypsin concentration of 0.25 mg/ml) for panel b, Hank’s buffer for panels c and d and accutase or alfazyme for panel e. X axis: fluorescensce intensity (arbitrary units), Y axis: cells number. Peak: fluorescence intensity corresponding to the maximum of cells; height: increasing number of cells; area: total fluorescence. The Peaks are shifted to the higher fluorescence region relative to the control (left peak) suggesting that cells evidently express CAR (Panel b and d) or DAF (Panel c). Panel a and e: the right Peak is shifted to the higher fluorescence region relative to the control (left peak) suggesting that cells express DAF; for CAR expression, Fluorescence intensity is very low.

0

10

20

30

40

50

60

70

80

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04

FL2-H

Cou

nts

c

0

10

20

30

40

50

60

70

80

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04

FL2-H

Cou

nts

a

0

10

20

30

40

50

60

70

80

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04

FL2-H

Cou

nts

0

10

20

30

40

50

60

70

80

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04

FL2-H

Cou

nts

+ Hank’s buffer +Trypsin- EDTA

+ trypsine diluted 1:4 in Hank’s buffer

+ Hank’s buffer

CaCo-2 cells

KB cells

d b

iC CAR DAF

e

+accutase/alfazyme

4. DiscussionThe expression of specific receptors is an important determinant of virus tropism and cell susceptibility to

703

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

Figure 3. Variation of the fluorescence intensity (arbitrary units) and transepithelial electrical resistance (expressed in Ω.cm2) according to the CaCo-2 cell density. (a): Variation of the fluorescence intensity of CAR (closed lozenges) and DAF (open squares) receptors measured by flow cytometry according to the CaCo-2 cell density. (b): The transepithelial electrical resistance (triangles) measured by an epithelial voltohmmeter (EVOM) according to the CaCo-2 cell density.

(a) (b)

TEER (Ω.cm²)

0

50

100

150

200

250

300

350

0,031 0,062 0,125 0,25 0,5 Cell density

TEER

Fluorescence intensity

0

50

100

150

200

250

300

350

0,031 0,062 0,125 0,25 0,5 Cell density

density

CAR DAF

704

S. Riabi et al.

infection by viruses [21]. In the case of enteroviruses and notably of CV-B, the fecal-oral route is the usual way for the virus to invade the host before reaching its target organs. So, the crossing of the epithelial barrier of the intestinal tract is, in most enteroviral infections, an obligate step that implicates complex interactions. The CAR molecule is the main receptor of CV-B; however, in polarized cells of the intestinal tract, CAR is preferentially expressed at the basolateral side of the tight junctions where it seems inaccessible to viral particles present in the lumen of the intestine. In the case of DAF-dependent strains of CV-B, elegant studies performed on CaCo-2 cells demonstrated the participation of different pathways mediated by the binding to DAF molecules, located at the apical side of the cells, to open the tight junctions, which permits the virus to reach its entry receptor [10,16]. The lumen crossing of DAF-independent strains of CV-B at the intestinal level is less clear [22] and needs further work.

Flow cytometry is a useful tool for analyzing the expression of receptors on the cell surface; it was notably used to characterize enterovirus binding molecules on different cells [23-26]. The aim of this work was to apply flow cytometry analysis to the study of the CV-B receptor expression at the CaCo-2 cell surface. Cytometry analysis requires the cells to be detached from their support. However, in contrast to DAF, CAR has previously been shown to be partially hydrolyzed by trypsin [19], which impairs its detection when high concentrations of trypsin is used to detach cells. Experiments conducted on HeLa cells determined that a trypsin concentration of 0.33 to 0.5 mg mL-1 was sufficient to digest most or all of the CAR molecules expressed on confluent cells within 15 min [19]. The results of the present study highlight that a managed trypsin treatment is able to both detach the CaCo-2 cells and preserve the detection of CAR by flow cytrometry (Figure 2b). Interestingly, the detection of CAR on KB cells did not need similar precautions (Figure 2d), which is probably due to the faculty of these cells to adhere less strongly to their support. It is also worthwhile to note that DAF was detected easily on both cell lines whatever the trypsin concentration, confirming its known resistance to trypsin.

To control that CAR was effectively expressed at high level by CaCo-2 cells, we verified the presence of specific mRNA and protein in these cells, by comparison to KB or HeLa cells. No difference in the expression of these markers was noticed between the different cell lines (Figure 1). Both markers were tested because one study related to the expression of CAR in kidney of newborn mice has shown that mRNA could be detected despite no protein expression [27].

Finally, flow cytometry experiments were used to study the influence of cell density on the expression of CAR and DAF at the surface of polarized cells. The interest for this feature was suggested by the fact that, by contrast to DAF, the expression of CAR at the surface of some cells is regulated by the organ development through life (for a review, see [22]), and that its expression was shown to increase with the cell density in other cellular models: RD cells, often considered as CAR-negative, can express this receptor when cultured after multiple passages at high cell density [28]; similarly, a close relationship has been reported between CAR expression and the density of cultured human endothelial cells derived from the umbilical vein [29,30]. By contrast to these examples, we observed no relationship between the density of CaCo-2 cells and the expression of either CAR or DAF (Figure 3a); actually, the restoration of tight junctions, demonstrated by a high transepithelial electrical resistance, was not associated with an increased expression of both receptors (Figure 3b). Sparse cultures with little or no intercellular contacts show relatively the same level of surface expression of CAR and DAF than in cells highly confluent with maximal intercellular contacts. Functionally and structurally, CAR is a member of the subgroup of junction adhesion molecules and part of a larger protein complex in the tight junction of the cell and might function as a cell–cell adhesion molecule [31,32]. We speculate that CAR functions as a receptor that signals homophilic cell-cell contact and thereby may regulate, either directly or indirectly, contact-mediated cellular responses such as, differentiation with stabilized expression of surface receptors. Cell-cell contact causes CAR to be trapped at the cell surface through homophilic binding, which leads to local accumulation of CAR at intercellular contact sites. Clustering of CAR then triggers signaling to be either auto-regulated or to regulate other receptors.

5. ConclusionsTaken together, the results reported in this study illustrate that, even if flow cytometry is an excellent tool for analysing the expression of cell receptors, the possibility of receptor degradation by trypsin, as exemplified by CAR, must be taken into consideration when this enzyme is used to detach adherent cells. In the model of CaCo-2 cells expressing CAR, we show that a gentle trypsin treatment is able to detach adherent cells without impairing the expression of CAR in flow cytometry, suggesting that the detached cells can be tested readily by this technique without waiting hours

705

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

for CAR regeneration. This finding may have interesting practical implications for studying the complex interactions between CV-B and intestinal polarized cells. In the field of cancer gene therapy using adenoviruses as vectors, which also use CAR to enter into their target cells, the use of flow cytometry to measure the expression of CAR at the surface of colon carcinoma cells [26] must take into consideration the possible

alteration of this receptor after cell trypsinisation. In this model, it would be interesting to verify whether a gentle trypsin treatment would allow detaching the cells without altering the receptor integrity. This finding may have also interesting clinical implications, especially when verified before the use of recombinant adeno-associated virus vectors for gene therapy to correct some human gene mutations [33].

[1] Greber U.F., Gastaldelli M., Junctional gating: the Achilles’ heel of epithelial cells in pathogen infection, Cell Host & Microbe., 2007, 2, 143-146

[2] Mapoles J.E., Krah D.L., Crowell R.L., Purification of a HeLa cell receptor protein for group B coxsackieviruses, J.Virol., 1985, 55, 560-566

[3] Bergelson J.M., Cunningham J.A., Droguett G., Kurt-Jones E.A., Krithivas A., Hong, J.S., et al., Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5, Science., 1997, 275, 1320-1323

[4] Tomko R.P., Xu R., Philipson L., HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses, Proc. Natl. Acad. Sci. U S A., 1997, 94, 3352-3356

[5] Selinka H.C., Huber M., Pasch A., Klingel K., Aepinus C., Kandolf R., Coxsackie B virus and its interaction with permissive host cells, Clin. Diagn. Virol., 1998, 9, 115-123.

[6] Huang Y., Hogle J.M., Chow M., Is the 135S Poliovirus Particle an Intermediate during Cell Entry? J. Virol., 2000, 74, 8757–8761.

[7] Carson S.D., Chapman N.M., Tracy S.M., Purification of the putative coxsackievirus B receptor from HeLa cells, Biochem. Biophys. Res. Commun., 1997, 233, 325–328

[8] Aurrand-Lions M., Johnson-Leger C., Wong C., Du Pasquier L., Imhof B.A., Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members, Blood., 2001, 98, 3699-3707

[9] Cohen C.J., Shieh J.T., Pickles R.J., Okegawa T., Hsieh J.T., Bergelson J.M., The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction, Proc. Natl. Acad. Sci. U S A., 2001, 98, 15191-15196

[10] Coyne C.B., Bergelson J.M., CAR. A virus receptor within the tight junction. Adv. Drug. Deliver. Rev., 2005, 57, 869-882.

[11] Hafenstein S., Bowman V.D., Chipman P.R., Bator Kelly C.M., Lin F., Medof M.E., Rossmann M.G., Interaction of Decay-Accelerating Factor with Coxsackievirus B3, J. Virol., 2007, 81(23), 12927-12935.

[12] Williams P., Chaudhry Y., Goodfellow I.G., Billington J., Powell R., Spiller O.B., et al, Mapping CD55 function. The structure of two pathogen-binding domains at 1.7 A. J, Biol. Chem., 2003, 278, 10691–10696

[13] Koretz K., Brüderlein S., Henne C., Möller P., Decay-accelerating factor (DAF, CD55) in normal colorectal mucosa, adenomas and carcinomas, Brit. J. Cancer., 1992, 66, 810-814

[14] Shieh J.T., Bergelson J.M., Interaction with decay-accelerating factor facilitates coxsackievirus B infection of polarized epithelial cells, J. Virol., 2002, 76, 9474–9480.

[15] Shafren D.R., Bates R.C., Agrez M.V., Herd R.L., Burns G.F., Barry R.D., Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment, J. Virol., 1995, 69, 3873-3877

[16] Coyne C.B., Bergelson J.M., Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions, Cell., 2006, 124, 119-31

[17] Grasset E., Pinto M., Dussaulx E., Zweibaum A., Desjeux J.F., Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters, Am. J. Physiol., 1984, 247, C260-267

[18] Coyne C.B., Shen L., Turner J.R., Bergelson J.M., Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5, Cell Host & Microbe., 2007, 2, 181-192

[19] Carson S.D., Limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) on HeLa cells exposed to trypsin, FEBS Letter., 2000, 484, 149-152

[20] Chomczynski P., Sacchi N., Single-step method of RNA isolation by acid guanidinium thiocyanate-

References

706

S. Riabi et al.

phenol-chlorophorm extraction, Anal. Biochem., 1987, 162, 156-159

[21] Boulanger P., Philipson L., Membrane components interacting with non-enveloped viruses, In: Lonberg-Holm K., Philipson L. (Eds.), Virus receptors, Part 2, Animal Viruses., Chapman & Hall, New York, USA, 1981, pp. 117-139

[22] Freimuth P., Philipson L., Carson S.D., The coxsackievirus and adenovirus receptor, Curr Top Microbiol., 2008, 323, 67-87

[23] Mbida A.D., Pozzetto B., Sabido O., Akono Y., Grattard F., Habib M., et al., Competition binding studies with biotinylated echovirus 11 in cytofluorimetry analysis, J. Virol. Methods., 1991, 35, 169-176

[24] Martino T.A., Petric M., Brown M., Aitken K., Gauntt C.J., Richardson C.D., et al., Cardiovirulent coxsackieviruses and the decay-accelerating factor (CD55) receptor, Virology., 1998, 244, 302-314

[25] Triantafilou M., Wilson K.M., Triantafilou K., Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method, Cytometry., 2001, 43, 279-289

[26] Zhang N.H., Song L.B., Wu X.J., Li R.P., Zeng M.S., Zhu X.F., et al., Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo, Cell Cycle., 2008, 7, 925-933

[27] Honda T., Saitoh H., Masuko M., Katagiri-Abe T., Tominaga K., Kozakai I., et al., The coxsackievirus-adenovirus receptor protein as a cell adhesion

molecule in the developing mouse brain, Mol. Brain. Res., 2000, 77, 19-28

[28] Shafren D.R., Williams D.T., Barry R.D., A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells, J. Virol.,1997, 71, 9844-9848

[29] Carson S.D., Hobbs J.T., Tracy S.M., Chapman N.M., Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density, J. Virol.,1999, 73, 7077-7079

[30] Vincent T., Pettersson R.F., Crystal R.G., Leopold P.L., Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells, J. Virol., 2004, 78, 8047-8058.

[31] Fanning A.S., Mitic L.L., Anderson J.M., Transmembrane proteins in the tight junction barrier, J. Am. Soc. Nephrol., 1999; 10, 1337–1345.

[32] Philipson L., Pettersson R.F., The coxsackie-adenovirus receptor-a new receptor in the immunoglobulin family involved in cell adhesion, Curr. Top. Microbiol. Immunol., 2004, 273, 87–111.

[33] Bainbridge W.B., Smith A. J., Barker S.S., Robbie S., Henderson R., Balaggan K., Viswanathan A., Holder G.E., Stockman A.,Tyler N., Petersen-Jones S., Bhattacharya S.S., Thrasher A. J.., M.R.C.P., F.R.C.P., Fitzke F.W., Carter B.J., Rubin G.S., Moore A.T., Ali R. R., Effect of Gene Therapy on Visual Function in Leber’s Congenital Amaurosis, N. Engl. J. Med., 2008, 358, 2231-2239.

707