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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 628-640, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4841

Mechanism of Restriction of Normal and Cystic Fibrosis Transmembrane Conductance Regulator–Deficient Human Tracheal Gland Cells to Adenovirus Infection and Ad-Mediated Gene Transfer

Florence Gaden, Laure Franqueville, Saw See Hong, Valérie Legrand, Catherine Figarella and Pierre Boulanger

Laboratoire de Virologie et Pathogénèse Virale, Faculté de Médecine RTH Laennec, Lyon; Transgene SA, Strasbourg; and Groupe de Recherche sur les Glandes Endocrines, Faculté de Médecine, Marseille, France

Address correspondence to: Pr. Pierre Boulanger, Laboratoire de Virologie et Pathogénèse Virale, Faculté de Médecine RTH Laennec de Lyon, 7, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail: Pierre.Boulanger{at}laennec.univ-lyon1.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CF-KM4 (cystic fibrosis transmebrane conductance regulator–deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that {alpha}[2->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base– and Arg-Gly-Asp (RGD)-integrin–independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

Abbreviations: adenovirus, Ad • Coxsackie-Ad receptor, CAR • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • Dulbecco's modified Eagle's medium, DMEM • electron microscopy, EM • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • fast performance liquid chromatography, FPLC • heparan sulfate glycosaminoglycans, HS-GAG • multiplicity of infection, MOI • phosphate-buffered saline, PBS • plaque-forming units, PFU • post-infection, pi • Arg-Gly-Asp, RGD • sialic acid, SA • sialoglycoproteins, SAGP • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A number of studies, performed in vitro and in vivo, have shown that transferring the gene for the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) to the cells of the airway epithelium was feasible, using an adenovirus (Ad) vector (1–4). However, the Ad-mediated transduction of the cftr gene in well-differentiated airway epithelium was inefficient in vitro (5), as well as in vivo, in patients with CF (4). Several factors were responsible for the low efficiency of cftr gene transfer (2): (i) nonspecific inflammatory reactions (6) and immune response to the Ad-CFTR vector (7, 8) have been reported; (ii) airway epithelial cells lack high-affinity receptors for Ad (9), or these receptors have a basolateral localization, a position which is detrimental to their accessibility to Ad-CFTR vectors (10); (iii) in vivo, other mechanical factors, like bronchial mucus (11), or local bacterial infections, could negatively influence the effective binding of Ad vectors to the surface of epithelial cells, and the subsequent delivery of the therapeutic gene; (iv) a combination of these different mechanisms, or/and intrinsic properties of differentiated airway epithelial cells and cellular factors acting at different steps of the viral cycle, could constitute barriers to Ad infection of recipient cells.

Most of the published work on airway epithelial cells and Ad vectors has focused on the first step of attachment of Ad virions to cell receptors. We present here a study of the virus–cell attachment and postattachment steps following Ad infection of bronchial airway cells, using two immortalized human tracheal submucosal gland serous cell lines recently isolated and characterized, named MM-39 and CF-KM4 (12, 13). MM-39 are serous cells from a healthy subject (12), whereas CF-KM4 are serous cells from a patient carrying a {Delta}F508-{Delta}F508 mutation (13). We show that both MM-39 and CF-KM4 cells are poorly permissive to subgenus C serotypes 2 (Ad2) and 5 (Ad5). Both cell lines do not express the high-affinity receptor Coxsackie-Ad receptor (CAR) at their surface, but express high levels of integrins. The efficiency of cell surface attachment, endocytosis, endosomal release of virions, and their traverse of the nuclear pores were studied in both cell lines. We provide data suggesting that Ad5 attach to MM-39 cells through heparan sulfate glycosaminoglycans (HS-GAG), and to CF-KM4 cells through {alpha}[2->6]-linked sialic acid residues (SA) of oligosaccharide moiety of sialoglycoproteins (SAGP), two types of molecules which have been previously reported to act as alternative receptors for Ad5 (14–16) and Ad37 (17), respectively. The limiting factor in Ad infection apparently resided at the early steps of virus–cell attachment and entry. Genetic insertion of a basic stretch of lysine residues into the fiber knob, aimed at retargeting Ad5 vectors to acidic surface molecules, significantly improved the efficiency of gene delivery in MM-39, but not in CF-KM4 cells. However, both cell lines were transduced with a significantly higher efficiency (20- to 50-fold) using capsid-modified Ad5 vectors retargeted to integrin molecules via fiber-inserted RGD peptide ligands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
HeLa cells and HEK-293 embryonic kidney cells (abbreviated 293) were obtained from ATCC (Rockville, MD)and maintained as monolayers in Dulbecco's modified Eagle's medium (DMEM; Gibco-Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (FCS; Sigma, St. Louis, MO), penicillin (100 U/ml), and streptomycin (100 µg/ml; Gibco-Invitrogen) at 37°C and 5% CO2. SV40-transformed human tracheal gland cell lines, MM-39 (normal) and CF-KM4 (CFTR-deficient), had been isolated and characterized in previous studies (12, 13, 18). They were maintained as monolayers in DMEM-Ham's F12 (DMEM-F12) supplemented with 1% Ultroser G (Gibco-Invitrogen), penicillin (200 U/ml), streptomycin (200 µg/ml), and epinephrin (3 µM; Sigma), on collagen-I-coated flasks (Biocoat; Becton-Dickinson, Franklin Lakes, NJ). Insect cells used for recombinant Ad protein production were Spodoptera frugiperda cells (Sf9 subclone). Sf9 cells were propagated in TNM medium (Gibco-Invitrogen) and cultured as monolayers with 10% FCS, penicillin, and streptomycin as mentioned above, and maintained at 28°C (19–21).

Ad and Ad5 Vectors
Wild-type (WT) Ad serotypes 2 and 5 (subgroup C) were propagated on HeLa cells. E1-deleted recombinant viruses were propagated on 293 cells. Virus stocks were purified by using conventional methods (22). Replication-competent Ad vector Ad5Luc3 (23) harbored the firefly luciferase gene (luc) under the control of the SV40 early promoter inserted in the E3 region of the Ad5 genome. Replication-deficient Ad5LacZ had its deleted E1 region ({Delta}E1) replaced by a ß-galactosidase expression cassette, with the lacZ bacterial gene under the control of the Ad2 major late promoter and terminating with the SV40 polyadenylation signal. Genetic manipulations of the fiber gene were performed as previously described (24–26). Ad5LacZ-FiWT carried WT fibers. Ad5LacZ-FiK7 and Ad5LacZ-Fi408K7 were two fiber knob–modified recombinant Ad5 with a stretch of seven lysine residues (K7) fused to the carboxy-terminus of their fibers (Ad5LacZ-FiK7). Ad5LacZ-Fi408K7 contained an additional Ser-to-Glu substitution at position 408. Two recombinant Ad5 expressing the gfp reporter gene, Ad5GFP-R7-knob and Ad5GFP-R7-RGD, carried a short fiber shaft (7 repeats instead of 22) terminated by the knob or the RGD peptide, respectively. They have been described and characterized in detail elsewhere (27). All cloning steps were perfomed by using standard molecular biology protocols (28), and the fiber-modified Ad5LacZ-FiK7 and Ad5LacZ-Fi408K7 virions were generated according to a strategy previously described (25). The overall structure and properties of our Ad virions and Ad vectors is summarized in Table 1, and detailed steps of genetic construction will be communicated upon request.


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  		TABLE 1 Genotype and fiber structure of Ad vectors*

 
Ad Titration
The concentration of infectious viral particles in CsCl-purified stocks of Ad was determined by plaque assays on 293 cell monolayers, according to conventional methods (27), and the infectivity titers expressed as plaque-forming units per ml (PFU/ml). The concentration in physical particles (PP) was determined from the protein concentration of CsCl-purified virus stocks, using the Bradford protein assay (BioRad, Hercules, CA), with bovine serum albumin (2x crystallized BSA; BioRad) as the standard. The number of PP was calculated from the total protein content of the sample, and the mass of 2.91 x 10-16 g per single virion, i.e., 2.91 mg per 1013 virions (29). The infectivity index represented the ratio of infectious to physical particles (PFU:PP), and usually ranged between 1:25 to 1:50 for WT Ad5. For propagation of Ad virus clones, we used a multiplicity of infection (MOI) ranging from 2–10 PFU/cell, corresponding to 50–500 PP/cell. For probing cellular functions (e.g., attachment, endocytosis, vesicular escape), the MOI used varied from 500–10,000 PP/cell.

Recombinant Baculoviruses and Soluble Ad Proteins
Except for Ad2 and Ad5 hexon proteins, which were isolated from Ad-infected HeLa cells, the other Ad major capsid proteins (penton base and fiber), their mutants and isolated domains (e.g., the fiber knob domain) were isolated in native and soluble form from AcNPV-infected Sf9 cell lysates, and purified according to conventional protocols adapted to fast performance liquid chromatography (FPLC) (30). The genetic constructions of recombinant Autographa californica Nuclear Polyhedrosis Viruses (AcNPV) expressing various Ad proteins have been described in detail elsewhere (20, 21, 26, 31). Protein samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting using the required antibodies, as already described (26). Ad protein concentration was estimated by the intensity of Coomassie blue staining of protein band in SDS-gel, measured by scanning at 610 nm in an automatic densitometer (REP-EDC; Helena Laboratories, Beaumont, TX), using a range of known BSA concentrations for calibration.

Fluorescent Probes, Immunoreagents, and Flow Cytometric Analysis
Chemical probes. Fluorescein isothiocyanate labeled (FITC)-dextran with an average of molecular weight of 70,000 (FD-70) was purchased from Sigma.

Fluorescent Ad5 (Cy3-Ad). Ad5 virions were conjugated with the fluorophore carbocyanine dye (Cy3; FuoroLink-Ab Cy3 labeling kit; Amersham Biosciences, Little Chalfont, UK), according to the manufacturer's protocol.

Antibodies. 1D6.14, an anti-Ad5 fiber knob, CAR-blocking monoclonal antibody (mAb), was obtained from David Curiel (University of Alabama at Birmingham, Birmingham, AL) (27, 32). MAb 2Hx2, directed against a subgroup-specific epitope of hexon protein (33), was obtained from the ATCC. The anti-CAR mAb RmcB (34) and E1–1 (35) were provided by J. Bergelson (Children's Hospital, Philadelphia, PA) and S. Hemmi (Institute of Molecular Biology, University of Zürich, Zürich, Switzerland), respectively. The anti-human HLA class I mAb (clone W6/32) was from DAKO. Mouse monoclonal anti-human cytokeratin-14 (IgM; Sigma clone CKB1) and cytokeratin-18 (IgG1; Sigma clone CY90) were detected using TRITC-conjugated goat anti-mouse IgM-m chain (Cat.#31664; Pierce, Rockford, IL), and anti-mouse IgG (Cat.#31661; Pierce), respectively. The R-PE–conjugated anti-human {alpha}M integrins mAb (anti-CD11b/Mac-1; clone ICRF-44), and the FITC-conjugated anti-human {alpha}Vß3 integrins mAb (anti-CD51/CD61; clone 23C6) were from BD Pharmingen (Franklin Lakes, NJ). The FITC-conjugated anti-human {alpha}Vß5 integrins mAb (clone P1F6), and the FITC-conjugated anti-human {alpha}5 integrins mAb (anti-CD49E; clone SAM-1) were from Chemicon International (Temecula, CA). When required for negative controls, irrelevant mAb were anti–HIV-1-Nef (TG-0020, IgG2 subclass; Transgene SA, Strasbourg, France), and anti–HIV-1-CA (EPIclone-5001, IgG1 subclass; Cylex Inc., Columbia, MD).

Flow cytometry. For cytofluorometric analysis, subconfluent cells were rinsed with phosphate-buffered saline (PBS), detached with PBS containing 1 mM Na2 EDTA, and resuspended in aliquots of 106 cells per 0.1 ml of PBS containing BSA at 0.1 mg/ml. Cell samples were reacted with 1 µg of the different fluorescent antibodies for 30 min on ice and in the dark. When unlabeled primary antibodies were used, the cells were pelleted, washed, and reacted with 1 µg PE-conjugated anti-mouse goat secondary antibody (Jackson ImmunoResearch, West Grove, PA). After rinsing in PBS, the cell samples were analyzed in a DAKO Flow Cytometer (Galaxy Flow Cytometry System; DAKO, Glostrup, Denmark).

Cellular Ligands and Cell Surface Molecule Modifications
Lectins. Triticum vulgaris (WGA), Sambucus nigra (SNA) and Maackia amurensis (MAA) agglutinins, all purchased from Sigma, were used at concentrations ranging from 1 to 200 µg/ml.

Monosaccharides, polysaccharides and mucopolysaccharides. Synthetic sialic acid (N-acetylneuraminic acid, type IV-S; concentration range of 3–20 mg/ml), heparin (from bovine intestinal mucosa; 0.25–50 µg/ml), and mucin (type I-S, from bovine submaxillary glands; 0.2–5 mg/ml) were also from Sigma.

Peptides. GRGDS (Sigma) was used at 0.5–5 mM, and lactalbumin hydrolysate (Difco tissue culture grade; Becton-Dickinson) was used as control, nonspecific peptides at the same wt:vol (31).

Enzymes. Subtilisin (subtilopeptidase A type VIII, from Bacillus licheniformis), and heparinase (heparinase III, from Flavobacterium heparinum) were both purchased from Sigma and used at 0.2–2 U/ml. Neuraminidase from Vibrio cholerae (Boehringer Mannheim, Mannheim, Germany), neuraminidase with single {alpha}[2->3] specificity and neuraminidase with dual {alpha}[2->3] and {alpha}[2->6] specificity (Cat.# N7271 and # N5521, respectively; Sigma) were used at a concentration range of 100–500 mU/ml.

Ad–Cell Binding Assays and Binding Competitions
Parameters of the Ad5-cell binding reaction. Attachment of Ad to MM39 and CF-KM4 cells was performed for 1 h at 0°C, using aliquots of 2.5 x 105 cells grown as monolayers in 6-well plates, and [14C]-valine labeled Ad5, in conditions previously described (22). The virus sample used in these experiments had a titer of 1.9 x 1012 physical particles per ml, and a specific radioactivity of 103 cpm for 1.5 x 108 particles. The apparent number of receptors for Ad at the cell surface and their relative affinity for the virions were derived from Scatchard plots (36). Parallel experiments were performed with HeLa cells used as controls.

Cell binding capacity of Ad5 vectors. Confluent cell monolayers were incubated with aliquots of Cy3-Ad5 vectors (4 x 104 particles per cell) at 0°C for 1 h. After rinsing, cell-adsorbed fluorescent signal was quantitated in fluorescence microscopy using an Axiovert 135 microscope (Zeiss, Germany) equipped with an AxioCam videocamera, and a quantitative image analysis program. The cell-binding index, expressed as arbitrary units, was determined by using the formula: number of positive, fluorescent cells per field x mean fluorescence intensity per cell.

Binding competition experiments. Ad5Luc3 was used at a MOI of 10 to 100 PFU/105 cells. Cellular and viral ligands were used in 103- to 104-fold excess over the theoretical number of their cell surface receptors or viral binding sites. Aliquots of confluent cells (5 x 105), grown as monolayers in 24-well plates, were incubated with various concentrations of cell surface ligand used as Ad competitor or enzyme for 2 h at 37°C in PBS. The excess of reagent was eliminated by washing cells with cold PBS, after which Ad5Luc3 was added and the cells further incubated for 30 min at 0°C. Unadsorbed virions were eliminated by washing in cold PBS, and cells transferred to 37°C and postincubated in prewarmed culture medium for a further 20 h at 37°C. Because all reactions were performed at 0°C, a temperature which inhibits the Ad endocytosis and viral entry, the level of luciferase expression has been shown to be directly related to the efficiency of virus attachment to the cell plasma membrane (31). When ligands of Ad virions were used (e.g., Ad-directed mAb or sialic acid), Ad5Luc3 was reacted with the desired ligand for 2 h at room temperature, then the mixture cooled down to 0°C and added to the cell monolayers, and the cells processed as described above.

Endocytosis Assays
Cell samples (105 cells) were incubated in PBS containing 1 or 5 mg/ml of FITC-dextran (FD-70) for 1 h at 37°C. Cells were analyzed by flow cytometry as described above. Endocytosis competition assays were performed using recombinant WT penton base protein, penton base mutant R340E (20), GRGDS, or control lactalbumin peptides as competitors. Ad5Luc3 was incubated with the cell monolayers at 0°C for 30 min to allow for cell attachment, then unadsorbed virions eliminated by washing the cells with cold PBS. Competitor dissolved in precooled PBS was then added, and cells incubated at 0°C for a further 15 min. The cells were then transferred to 37°C for 1 h, PBS was replaced by culture medium prewarmed at 37°C, and the cultures maintained at this temperatrure for 20 h. The cells were then harvested and processed for luciferase expression assays.

Endosomolysis Assays
The effect of Ad on endosomolysis was assayed by the degree of Ad-mediated augmentation of the inhibition of cell protein synthesis by a toxin, as a result of their concomittant vesicular release (37, 38). Ricin A lectin (RcA; ricin agglutinin of 120,000 mol wt; Sigma) was used at concentrations ranging from 0–5 µg of RcA per sample of 105 cells, grown as monolayers. Cells were preincubated with RcA in the presence or absence of Ad virions for 1 h at 37°C, in methionine- and cysteine-free culture medium. Ad inoculum was added at a constant input of 500 PP/cell (~ 10 PFU/cell). [35S]-methionine and [35S]-cysteine (> 1,000 Ci/mM; PRO-MIX; Amersham) was added at 15 µCi per 105 cell sample, and incubation further proceeded for 2 h at 37°C. Cells were then rinsed with culture medium, detached from the support, and dissolved in 0.2 N NaOH, 1% SDS. Cellular proteins were precipitated by addition of 10 vol trichloroacetic acid (TCA) at 10%, and retained on GF/C glass filters. RcA-induced inhibition of protein synthesis was evaluated from TCA-precipitable radioactivity, determined by scintillation counting in a liquid scintillation spectrometer (LS 6500; Beckman, Fullerton, CA).

Ad-Mediated Gene Transduction Assays
Ad5 vector, carrying the luc, lacZ, or gfp reporter gene, was added to treated or mock-treated cells, in a final vol of 200 µl in culture medium and at an MOI ranging from 0–10 PFU/cell. After incubation for 30 min at 0°C with intermittent rocking, the cell monolayers were washed with cold medium to remove unadsorbed virus. Prewarmed medium was then added and the cells transferred to 37°C for 20 h. The cells were then harvested, and the efficiency of Ad-mediated gene delivery was estimated by the level of reporter gene expression. Luciferase activity was assayed in cell lysates at 20 h after infection, using luciferase substrate solution (Promega, Madison, WI) in a Lumat LB-9501 luminometer (Berthold Bioanalytical, Germany). The results were expressed in relative light units (RLU) per cell aliquot, or per mg of whole protein present in cell lysates (31, 39). ß-Galactosidase activity was determined using a galactoside substrate (Galacton-Star; Clontech, Palo Alto, CA) which, upon ß-galactosidase cleavage, releases a chemiluminescent byproduct (Luminescent ß-Gal Genetic Reporter System II; Clontech). Cells were lysed and enzymatic reactions performed according to the manufacturer's protocol. GFP expression was assayed by immunofluorescence (IF) microscopy and quantitated by flow cytometry.

Electron Microscopy
Noninfected or Ad-infected cells (harvested at 1–2 h after infection) were pelleted, fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and postfixed with osmium tetroxide (1% in 0.1 M cacodylate buffer, pH 7.4). The specimens were dehydrated and embedded in Epon (Epon-812; Fulham, Latham, NY). Sections were stained with 7% uranyl acetate in methanol, and poststained with 2.6% alkaline lead citrate in H2O. Specimens were examined under a Jeol 1200-EX electron microscope, equipped with a MegaView II high resolution transmission electron microscopy camera and a Soft Imaging System of analysis (Eloïse, Roissy, France).

Mycoplasma Detection
Aliquots of 106 cells/ml of culture medium were inoculated to 55-mm agar plate in modified Hayflick medium suplemented with fresh yeast extract and colt serum (40). In positive samples, colonies were visible after incubation at 35°C for 3 wk in anaerobic conditions. Mycoplasma contaminations were eliminated by three cycles of BM-Cyclin 1 + BM-Cyclin 2 treatment (Boehringer Mannheim), according to the manufacturer's protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Restriction of MM-39 and CF-KM4 Cells to Ad Infection
Cell monolayers were taken early after confluence, and their differentiation status was verified by IF microscopy, using anti–cytokeratin-14 and anti–cytokeratin-18 antibodies. All confluent cells were found to be differentiated, as shown by their positive signal for CytK18 and negative for CytK14 (not shown). They were infected by WT Ad2 or Ad5 at the same MOI (10 PFU/cell), and the virus progeny yield was determined by titration in the lysates of cells harvested at 36 h postinfection (pi). Both MM-39 and CF-KM4 cells were found to be infectable by WT Ad5 and Ad2, but produced 5- to 10-fold less virions than HeLa, used as fully permissive reference cells. Cellular synthesis of Ad structural protein was assayed in time-course experiments in aliquots of Ad5-infected cells harvested at various times pi, using SDS-PAGE and immunoblotting analysis with anti-Ad5 antibodies. All the major Ad5 structural proteins were present in lower amounts in MM-39 (4- to 5-fold) and CF-KM4 cells (8- to 10-fold) than in HeLa cells for each corresponding time point, but in the same stoichiometric ratios for the three cell lines (not shown). This suggested that the lower virus progeny yields in MM-39 and CF-KM4 cells did not result from a specific block of synthesis of one single virus structural protein acting as a limiting factor.

Ad-mediated gene transfer was then analyzed in MM-39 and CF-KM4 cells using Ad5Luc3, a replication-competent recombinant Ad5 which harbors the firefly luciferase gene (luc) as a reporter gene (Table 1). A kinetic analysis of luc expression in Ad5Luc3-infected MM-39, CF-KM4, and HeLa cells showed that the expression of the transgene was 6- to 10-fold lower in CF-KM4 cells than in HeLa cells between 18 and 24 h pi (Figure 1). For MM-39 cells, the difference with HeLa cells was less pronounced, but still significant (3- to 4-fold; Figure 1).



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  		Figure 1. Comparison of HeLa (circles), MM-39 (squares), and CF-KM4 (triangles) cell permissivity to Ad5, assayed by Ad5-mediated gene transfer. Cell monolayers were infected at a MOI of 10 PFU/cell of the replication-competent, Ad5Luc3 vector at 37°C for 1 h. Cells were harvested at different times after infection, processed, and cell lysates assayed for luciferase expression. Standard deviation, not visible on the scale, ranged within 1 x 102 to 2 x 104 RLU.

 
Electron Microscopic Observation of Ad5-Infected MM-39 and CF-KM4 Cells
The cellular morphology of MM-39 and CF-KM4 cells were different under electron microscopy (EM; Figures 2a and 2b). A distinguished feature of MM-39 cells was the abundance of pseudopodes emanating from the cell surface, whereas CF-KM4 cells were characterized by a rich intracytoplasmic vesicular network. The intracellular fate of Ad5 in these cells at early times pi was investigated by EM, with quantitation of virions in the different cell compartments. For this quantitative analysis, 293 cells were preferred to HeLa cells as control, permissive cells, as the cytoplasmic area of HeLa cells was too reduced in section and the nuclear envelope too close to plasma membrane to allow for a clear discrimination of compartmentalized virions. Cells were infected with WT Ad5 at high MOI (104 PP/cell) for 1 h at 37°C, harvested, and processed for EM. Ad5 virions were counted as (i) cell plasma membrane-bound virions, (ii) intravesicular-, (iii) intracytoplasmic-, and (iv) nuclear pore complex (NPC)-associated particles. More than a hundred particles were counted in sections from 10 to 20 different cells, and the results expressed as the average number of Ad5 virions per cell recovered in each defined compartment (Figure 3).



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  		Figure 2. EM observation of uninfected MM-39 (a) and uninfected CF-KM4 cells (b), and of Ad5-infected 293 (c), Ad5-infected MM-39 (d, e), and Ad5-infected CF-KM4 cells (f–h). In c–h, cells were infected at a MOI of 104 PP/cell, and harvested after 1 h of infection at 37°C. The different panels show virions in various cell compartments representing early stages of the virus life cycle: attachment to cell surface receptors (c, d, f), endocytosis (c), endosomal escape (e), attachment to the nuclear pore complex (NPC; f, g), and nuclear localization of uncoated virus cores (h). N, nucleus; C, cytoplasm.

 


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  		Figure 3. Quantitative EM analysis of compartmentalized Ad5 virions in 293 (dotted bars), MM-39 (striped bars), and CF-KM4 (solid bars) cells, as examplified in Figure 2. Ad particles were counted in the different compartments of several sections of Ad5-infected cells (at least 20 different cells; total number of 100 particles, at the minimum), as indicated on the x-axis. Results were expressed as the number of particles found per cell in each compartment.

 
As shown in Figures 2c–2h, incoming Ad5 virions were found in all compartments of MM-39 and CF-KM4 cells, e.g., attached to the cell plasma membrane, within endosomes, in the cytosol, and associated with NPC (Figures 2e–2h). However, there were significantly less virions detected in all the cellular compartments of MM-39 and CF-KM4 cells, compared with 293 cells (Figure 2c and Figure 3). Plasma membrane–attached and intravesicular virions were proportionally less (~ 10-fold) than cytoplasmic virions (2- to 3-fold) in MM-39 and CF-KM4 cells (Figure 3). These results suggested that cell attachment and endocytosis of Ad5 were relatively inefficient in both MM-39 and KM4 cells. We next explored Ad–cell surface attachment, endocytosis, and vesicular escape of partially uncoated virions.

Repertoire of Ad5 Receptors on MM-39 and CF-KM4 Cells
Ad binds to the host cell surface via its penton-fiber projections, and involves two types of cellular receptors: the primary receptors, which bind to the distal knob of the fiber; and the secondary receptors, required for endocytosis of the virus into clathrin-coated vesicles. The latter consist of integrins of the {alpha}v subfamily, which recognize the RGD motifs in the penton base (reviewed in Ref. 41). The repertoire of MM-39 and CF-KM4 cell surface molecules serving as potential receptors for Ad was then quantitatively analyzed by flow cytometry, in comparison with control HeLa cells (Table 2). CAR was not detected at the surface of support-detached MM-39 and CF-KM4 cells. This result differed from previous observations on airway epithelial cells, which are polarized and in which CAR is in fact expressed at the plasma membrane, but not exposed at the apical surface (5, 9). HLA class I molecules were present in similar amounts on MM-39 and CF-KM4 cells. Interestingly, {alpha}vß3 integrins, which have been identified as one of the integrin species essential for Ad5 endocytosis (42), were expressed at higher levels on MM-39 and CF-KM4 cells than on HeLa cells (~ 2.5-fold). Likewise, {alpha}M integrins, specific for Ad entry into hematopoietic cells via the penton base RGD motif recognition (43), were found to be highly expressed on both MM-39 and CF-KM4 cells. As for {alpha}vß5 integrins, which have been reported to be mainly involved in membrane permeabilization and probably the vesicular escape of Ad5 virions (44), they were found to be poorly expressed on MM-39 cells (10–15% of the HeLa cell level), but expressed at HeLa-cell levels on CF-KM4 cells (110–120%; Table 2).


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  		TABLE 2 Expression of various surface molecules in MM-39 and CF-KM4 cells*

 
However, because flow cytometry analysis was performed on support-detached cells, a situation totally different from that prevailing in polarized or monolayer cells, the patterns obtained might not reflect the actual level of expression of integrins at the apical pole of the cells. To explore the endocytotic function at the apical surface, monolayers of confluent MM39 and CF-KM4 cells were assayed for their whole capacity of endocytosis of macromolecules at 37°C, using FITC-dextran (FD-70). The intracellular fluorescent signal of endocytosed FD-70, measured by flow cytometry, was found to be consistently higher in both MM39 (3-fold) and CF-KM4 (4-fold) than in HeLa cells, suggesting a significantly higher endocytosis activity. This result was consistent with the overall higher level of cell surface–expressed integrins revealed by flow cytometry.

The number of Ad5 receptors present at the surface of MM-39 and CF-KM4 cells, and their affinity for the virus, were determined from binding experiments using radiolabeled Ad5 (22, 45) and binding data graphically analyzed (36). As shown in Table 3, MM-39 cells expressed a significantly lower number of Ad5 receptors with an apparent affinity similar to that of HeLa cells, whereas CF-KM4 expressed lesser levels of lower-affinity receptors. These results suggested that the early event of Ad attachment to the cell plasma membrane was one of the key mechanisms of the low permissiveness of MM-39 and CF-KM4 cells to Ad5. The biochemical nature of the Ad5 receptors in MM-39 and CF-KM4 cells was investigated in the next experiments.


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  		TABLE 3 Quantitative data for Ad5 receptors at the cell surface

 
Cell Surface Receptors for Ad5 in MM-39 and CF-KM4 Cells
Ad5Luc3-cell binding assays were performed under various conditions: (i) in competition using various cell ligands, and (ii) after modifications or blockage of certain cell surface molecules. Mucin from bovine submaxillary glands was found to have little effect, if any, on Ad attachment to HeLa cells, but some discrete effect on MM-39 and CF-KM4 cells (15–30% inhibition; Figure 4A). Some lectins were found to be efficient in inhibiting the attachment of Ad5 to MM-39 and CF-KM4 cells. Wheat germ agglutinin (WGA), which has ßGlcNAc residues of glycoconjugates as major specificity, and SA (NeuNAc) as minor specificity, showed a significant effect on all three cell lines, 85% inhibition for CF-KM4 cells, 60% for MM-39 cells, and and 40% for HeLa cells. Sambucus nigra agglutinin (SNA), specific for {alpha}NeuNAc-[2->6]-Gal/GalNAc-containing oligosaccharides, had a stronger effect on the binding of Ad5 to CF-KM4 cells (85% inhibition), compared with MM-39 and HeLa cells (60 and 40%, respectively; Figure 4A). Lectin from Maackia amurensis (MAA), specific for {alpha}[2->3]-linked SA-saccharides, had a stimulatory effect on Ad5 attachment to HeLa cells, no detectable effect on MM-39 cells, but a significant inhibitory effect on CF-KM4 cells (40%; Figure 4A).



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  		Figure 4. Effect of various cellular and viral ligands on Ad5-Luc3 attachment to HeLa (dotted bars), MM-39 (striped bars), and CF-KM4 (solid bars) cells. (A) Mucin and lectins. (B) Cell-surface molecule-modifying enzymes (protease and glycosidases) and their corresponding carbohydrate substrates (heparin and sialic acid) as Ad competitor. Incubation of cell monolayers with enzyme or cell ligands was performed at 37°C for 2 h, whereas incubation of Ad5Luc3 with viral ligands was performed at room temperature for 2 h. The different reagents were used at the indicated concentrations. Cells were rinsed and further incubated with Ad5Luc3, or the Ad5Luc3-ligand complex at 0°C for 30 min, a temperature which allows for the cell surface attachment of Ad virions but blocks their endocytosis. Unadsorbed virus was rinsed off, and cells transferred to 37°C for 20 h and assayed for luciferase expression.

 
The difference observed in the effect of SNA and MAA, two lectins with different SA-binding specificities, suggested that the sialyl bonds of SAGP receptors in CF-KM4 cells were mainly of the {alpha}[2->6] type. However, it could not be excluded that the inhibition resulted from a nonspecific interference of the lectin with Ad receptors by steric hindrance, rather than from the specific blockage of a particular type of sugar residues. To distinguish between these possibilities, Ad vector–cell attachment at 0°C and gene transduction at 37°C were tested after treatment of the cells with various glycosidases and in competition with the relevant carbohydrates.

Broad spectrum Vibrio cholerae neuraminidase showed a stimulatory effect on Ad5 binding to MM-39 cells (as well as to HeLa cells; Figure 4B), an observation already reported for human bronchial epithelial cells (11). In contrast, neuraminidase had a strong negative effect on Ad5 binding to CF-KM4 cells (~ 90% inhibition at 400 mU/ml; Figure 4B). Two neuraminidase preparations with restricted sialyl bond specificity were also used, {alpha}[2->3]-specific neuraminidase, and a neuraminidase preparation with dual {alpha}[2->3] and {alpha}[2->6] specificity. A 50–60% inhibition of gene transduction was observed with the {alpha}[2->3]+{alpha}[2->6]-specific enzyme at 300 mU/ml, whereas no effect was obtained with the single {alpha}[2->3] specificity (not shown). This result and the data with SNA and MAA suggested that the sialyl bonds of the SAGP recognized by Ad5 at the surface of CF-KM4 cells were mostly (but not exclusively) of the {alpha}[2->6] type.

Free SA efficiently competed with Ad5 receptors on CF-KM4 cells (Figure 4B) in a dose-dependent manner. The 50%-inhibition concentration was 0.8 mg/105 cells (IC50 = 0.8–1.0) for CF-KM4 cells, and two times higher (IC50 = 1.75–2.0) for MM-39 cells. Treatment of the cells with the broad spectrum protease subtilisin severely reduced the Ad5 binding to both HeLa and CF-KM4 cells, but showed an enhancing activity on Ad5 binding to MM-39 cells (Figure 4B). The sensitivity to both neuraminidase and protease of the Ad5 receptors on CF-KM4 cells implied that the SA residues did not belong to gangliosides, but to the sugar moiety of membrane SAGP.

Heparinase treatment of MM-39 cells (at 1 U/ml) reduced the Ad binding to background levels (5–6%; Figure 4B), whereas it showed little effect, if any, on Ad binding to CF-KM4 cells. Likewise, preincubation of heparin with Ad5Luc3 resulted in a strong inhibitory effect on MM-39 cells, but had no effect on CF-KM4 cells (Figure 4B). In control HeLa cells, heparinase reduced Ad–cell binding to 30% of the binding level to untreated cells, a result already observed (14, 15). The fact that Ad binding to MM-39 cells was sensitive to heparinase and heparin, but resistant to both subtilisin and neuraminidase treatment, suggested that their Ad receptors consisted of acidic HS-GAG.

Ad5 Binding to MM-39 and CF-KM4 Cell Receptors Is Mediated by the Fiber Knob Domain
We next sought to determine which viral protein(s) was involved in the attachment step. Three major capsid proteins (hexon, penton, and fiber) and the fiber knob domain were tested in competition assays for Ad5Luc3-cell attachment at 0°C. They were used in large excess over the content of the corresponding protein in the input virions (103- to 104-fold). No significant competition effect was observed with penton base or hexon protein for Ad binding to MM-39 and CF-KM4 cells (Figure 5A), suggesting that the mechanism of Ad5 attachment to MM-39 and CF-KM4 cells did not involve penton base or hexon capsomers.



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  		Figure 5. Competition of viral (A) or nonviral (B) components for Ad5-Luc3 attachment to HeLa (dotted bars), MM-39 (striped bars), and CF-KM4 (solid bars) cells. (A) Cell monolayers were preincubated with Ad capsid components for 1 h at 0°C and at 103- to 104-fold excess over the theoretical numbers of cell receptors. Unadsorbed material removed by rinsing and Ad5Luc3 vector added for 30 min at 0°C. Unadsorbed virus was rinsed off, and cells transferred to 37°C for 20 h, and assayed for luciferase expression. (B) Ad5Luc3 was mixed with a large excess of anti-fiber knob mAb 1D6.14, and incubated at room temperature for 2 h, then cooled to 0°C before addition to cell monolayers. Cells were incubated at 0°C for a further 30 min. Unadsorbed virus was rinsed off, and cells transferred to 37°C for 20 h and assayed for luciferase expression.

 
Attachment of Ad5Luc3 to control HeLa cells was significantly inhibited in the presence of Ad5 fiber or fiber knob protein: a 70–75% inhibition was observed at a concentration of competing fiber equivalent to a 1,000-fold excess over the fiber content of the virus. However, no effect on MM-39 and CF-KM4 cells was observed at this concentration with Ad5 fiber or fiber knob protein (Figure 5A). The nonsaturable nature of Ad5 receptors by fiber or fiber knob suggested that isolated fiber proteins bound to receptors different from the ones used by the virions, or rather that the accessibility and reactivity of Ad5 receptors were significantly higher for free fiber (or fiber knob) molecules than for fibers carried by the virions.

To test whether the fiber knob domain was involved in the cell attachment of Ad5 virions, Ad5Luc3 vector was preincubated at room temperature with 1D6.14, a well-characterized ligand of the knob domain. 1D6.14 is an anti-Ad5 fiber knob mAb with a virus-neutralizing activity (27, 32). We have mapped its epitope to peptide sequence PEYWNFRNGDLTEGTAY within residues 475–491 in Ad5 fiber (Hong and Boulanger, unpublished data), a region involved in CAR recognition (46–48). As shown in Figure 5B, preincubation of virions with 1D6.14 decreased the attachment of Ad5Luc3 vector to HeLa, MM-39, and CF-KM4 cells, an effect which was not observed with anti-hexon mAb, or other irrelevant mAb used within the same concentration range. This suggested that the attachment of Ad5 to MM-39 and CF-KM4 cells was mediated by the knob domain of its fibers.

RGD-Integrins and Uptake of Ad5 Virions by MM-39 and CF-KM4 Cells
The role of penton base and its RGD motifs was evaluated using recombinant WT penton base capsomer, its R340E mutant (carrying an Arg-to-Glu substitution in the RGD motif at position 340 [20]), and the RGD-containing oligopeptide GRGDS (45) in endocytosis competition assays with Ad5 virions preattached to cells at 0°C. As expected from previous studies, GRGDS, WT penton base, but not its R340E mutant, were found to compete with Ad5 virions for endocytosis in HeLa cells: a 50% inhibition was obtained with 30–50 µg penton base per 5 x 104 cells, i.e., a 9 x 103 to 15 x 103 excess over the theoretical number of penton base receptors (42), and a 70% inhibition with GRGDS at 5 mM. No effect was observed with control, irrelevant peptides used within the same concentration range (data not shown). In contrast to HeLa cells, GRGDS and penton base had no detectable effect on the endocytosis of Ad5 by MM-39 and CF-KM4 cells (not shown). This suggested that after binding to HS-GAG and SAGP receptors at the surface of MM-39 and CF-KM4 cell, Ad5 followed an entry route different from the penton base– and RGD-integrin–dependent endocytotic pathway observed in HeLa cells. However, as shown below using modified Ad5 vectors, RGD-dependent integrins could substitute for HS-GAG and SAGP receptors and lead to efficient gene transduction.

Ad-Mediated Endosomolysis in MM-39 and CF-KM4 Cells
Ad5 virions co-endocytosed with toxins enhanced their cytotoxic effect due to their simultaneous vesicular release into the cytosol (37, 38, 49). Ad-mediated endosomolysis was then assayed by the degree of augmentation of cell protein synthesis inhibition by the toxin in the presence of the virus. We used increasing quantities of ricin agglutinin (RcA) or transferrin-conjugated RcA (Tf-RcA) (38) with a constant virus input of 500 PP/cell. No Ad-induced early cytopathic effect was detected at this MOI (38). As shown in Figure 6, co-endocytosis of Ad5 virions with RcA resulted in a net augmentation of its cytotoxicity for HeLa cells, with 90% inhibition of cell protein synthesis at 5 µg RcA per 105 cells. A less pronounced cytotoxic effect was observed in CF-KM4 and MM-39 cells (60 and 25% inhibition, respectively). Similar results were obtained with Tf-RcA (not shown). The data were consistent with our EM observations and analysis of Ad5 receptors (Figure 2, Figure 3, and Table 2), suggesting that: (i) less virions were attached to and endocytosed by MM-39 and CF-KM4 cells, compared with HeLa cells; or/and that (ii) Ad5 entered MM-39 and CF-KM4 cells via a pathway different from that followed by RcA, as mentioned above.



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  		Figure 6. Ad5-mediated augmentation of cytotoxicity of co-endocytosed ricin agglutinin (RcA) and Ad virions, as assayed by the inhibition of cell protein synthesis. Cells were incubated with increasing amounts of RcA, in the presence of Ad virions at a constant MOI of 5 x 102 PP/cell, an input which did not provoke any detectable cytopathic effect. Results were expressed as the percentage of protein synthesis in control cells treated with the same RcA concentrations in the absence of Ad5. The curves represent the means of four separate experiments, and the standard deviations were within 15% of the mean values. Diamonds, HeLa; squares, MM-39; triangles, CF-KM4.

 
Gene Transduction of MM-39 and KM4 Cells by Capsid-Modified Ad5 Vectors
As both types of surface molecules responsible for the initial attachment of Ad5 to MM-39 and CF-KM4 cells were acidic in nature and carried negative charges at neutral pH, we then genetically modified Ad5 vectors so as to introduce extra basic charges via a cluster of seven lysine residues (abbreviated K7) fused to the C-terminus of the fiber knob domain (Table 1). The resulting virions would theoretically bind to MM-39 cells and CF-KM4 cells with a higher affinity resulting from a higher number of complementary charges, and overcome the possible limiting step occurring at the Ad–cell attachment level (Figures 1–3 and Figure 6). A similar stategy had been successfully used to retarget Ad vectors to heparan-containing receptors (16). Three Ad5LacZ vectors were then tested for lacZ gene transduction of HeLa, MM-39, and CF-KM4 cells. They were abbreviated Ad5LacZ-FiWT (carrying WT fibers), Ad5LacZ-FiK7 (carrying a CAR-binding fiber knob with a K7 tail), and Ad5LacZ-Fi408K7 (K7-fused, CAR-binding defective fiber knob), respectively. Ad5LacZ-Fi408K7 was derived from Ad5LacZ-FiK7 by an additional mutation in the knob domain, a Ser-to-Glu substitution at position 408 (Table 1). This substitution has been found to be detrimental to the binding of Ad5 to CAR (46, 47), as it involves one of the major contact sites between the knob and CAR (48, 50, 51).

For MM39 cells, the transduction efficiency was drastically increased over the Ad5LacZ-FiWT level: a 12-fold augmentation was observed with Ad5LacZ-FiK7, and a factor of 18-fold with Ad5LacZ-Fi408K7. In CF-KM4 cells however, only a slight increase (2.5-fold) was detected with the double mutant fiber Ad5LacZ-Fi408K7, and no difference with Ad5LacZ-FiK7 (Figure 7A). This suggested that S408E mutation could have modified some function(s) of the fiber knob other than the primary event of cell receptor-binding (52).



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  		Figure 7. Efficiency of gene transduction (A, C) and cell binding (B, D) by Ad5 vectors carrying genetically-modified fibers and expressing LacZ (A, B), or GFP (C, D). (A) Control, Ad5LacZ-FiWT carried WT fibers, whereas Ad5LacZ-FiK7 and Ad5LacZ-Fi408K7 carried a stretch of seven lysine residues (K7) in fusion to the C-terminus of the knob domain, and Ad5LacZ-Fi408K7 contained an additional fiber knob mutation (S408E). Results were normalized to the level of ß-galactosidase expres sion in Ad5LacZ-FiWT-infected cells, which was attributed the 1.0 value. (C) Control, Ad5GFP-R7-knob carried the natural knob domain at the extremity of a seven-repeat fiber shaft (R7), whereas Ad5GFP-R7-RGD had its deleted knob domain replaced by a RGD motif. Results were normalized to the level of GFP expression in Ad5GFP-R7-knob–infected cells, which was attributed the 1.0 value. (B and D) Cy3-labeled vectors Ad5LacZ-FiWT, Ad5LacZ-FiK7, Ad5LacZ-Fi408K7, Ad5GFP-R7-knob, and Ad5GFP-R7RGD were incubated with monolayers of confluent cells for attachment at 0°C, and the intensity of plasma membrane-bound fluorescent signal was quantitated in IF microscopy. The cell binding index (no. of fluorescent cells x mean cellular fluorescence intensity) was determined for each vector, and data (average of twenty determinations ± SD), were normalized to the binding index of Ad5GFP-R7-knob (arbitrary value of 1.0).

 
As MM-39 and KM4 cells were found to express significantly more {alpha}Vß3 and {alpha}M integrins at their surface than HeLa cells (Table 2), we next determined whether RGD-dependent integrin molecules could function as attachment receptors for Ad5 in these two cells. We therefore assayed the gene transfer efficiency of a knob-deleted Ad5 vector carrying a RGD motif at the extremity of each fiber monomer (Ad5GFP-R7-RGD; Table 1). The results showed that Ad5GFP-R7-RGD was significantly more efficient in transducing GFP into MM-39 and KM4 cells (20- and 50-fold, respectively) than the parental vector (Ad5GFP-R7-knob) carrying its natural knob domain (Figure 7C). This indicated that the block to Ad5 entry in MM-39 and KM4 cells could be overcome by redirecting the virus to integrins, which in this case acted as both primary (attachment) and secondary (endocytosis) receptors.

The mechanism of improvement of gene transfer by fiber-modified, retargeted vectors Ad5LacZ-FiK7, Ad5LacZ-Fi408K7, and Ad5GFP-R7-RGD could take place at the attachment level, or/and involve further steps in the vector–cell interaction. Cell attachment of Cy3-conjugated Ad5 vectors at 0°C on confluent cell monolayers was then studied by quantitative IF microscopy (Figures 7B and 7D). No correlation was observed between the Ad5LacZ-FiK7– and Ad5LacZ-Fi408K7–mediated gene transfer to MM-39 cells and their respective cell binding capacity, which was similar to that of the WT-fiber vector (Figure 7, compare A and B). For the Ad5GFP-RGD vector however, the binding to MM-39 cells increased by 15-fold, compared with the fiber knob-carrying vector (Figure 7D), a factor consistent with the level of increase in gene transduction efficiency (Figure 7C). The increase in the Ad5GFP-RGD binding to CF-KM4 cells was only 5-fold, and no difference was observed with HeLa cells (Figure 7D). Compared with the 50-fold enhancement of gene transduction of CF-KM4 cells by Ad5GFP-RGD, this implied that the higher level of CF-KM4 cell surface binding by this vector was not the only factor responsible for its augmentation of gene transduction. Taken together, these results suggested that modifications of Ad fiber aimed at retargeting Ad vectors to alternative cell surface receptors could also influence further steps in Ad–cell interaction such as endocytosis and cell entry, and hence the overall efficiency of gene transfer.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, two serous cell lines from submucosal tracheal glands, MM-39 and CF-KM4 cells, were investigated for their permissiveness to Ad2 and 5, and their susceptibility to gene transduction by Ad5 vectors in vitro. MM-39 cells were isolated from a young non-CF adult, and CF-KM4 cells were obtained from a patient with CF homozygous for the {Delta}F508 mutation (12, 13). Both cell lines have been shown to retain their original features of native gland serous cells. Upon pharmacologic stimulations, an increase in secretory leukocyte proteinase inhibitor (SLPI) was observed with MM-39 cells (53), whereas the CF-specific secretory functions were found to be defective in CF-KM4 cells (13). MM-39 cells were thus meant to serve as the control cells for CF-KM4. In addition, the airway submucosal glands have been shown to be prominent sites of human CFTR expression in subjects carrying the WT alleles of the cftr gene (54).

We found that the initial steps of Ad5 attachment to surface receptors and cell entry were mainly responsible for the low degree of permissiveness of both MM-39 and CF-KM4 cells to Ad5 infection, and for their relatively low efficacy of gene transduction using Ad vectors. As the high-affinity CAR receptors were not expressed in the two cell lines, Ad5 utilized alternative receptors. Cell surface molecules acting as primary receptors for Ad5 were probed for, and we identified two types of cellular receptors for Ad5, HS-GAG in MM-39 cells, and terminal SA of the oligosaccaride moiety of SAGP in CF-KM4 cells, respectively (Figure 4). Both types of acidic surface molecules have already been identified as primary receptors for Ad, SA for Ad37 (17) and HS-GAG for Ad5 (14–16). Experiments using SA-binding lectins and neuraminidases with various specificities suggested that the majority of SA residues acting as Ad5 receptors in CF-KM4 cells were linked via {alpha}-[2->6] bonds to the oligosaccharide chains. This was different from Ad37, which has been shown to preferentially utilize {alpha}-[2->3]-linked SA (17). The binding to these molecules occurred via the knob domain of the Ad5 fiber, as suggested by the pattern of inhibition observed with anti-knob mAb 1D6.14 (Figure 5B).

Considering the acidic nature of the cell surface molecules involved in Ad5 recognition, we then tested whether Ad5 vectors carrying extra basic charges (K7) on their fiber knobs would bind better to acidic receptors of MM-39 and CF-KM4 cells, and transduce reporter genes with a better efficiency than the Ad5LacZ-FiWT vector carrying WT fibers. As expected, K7-containing vectors were found to transduce MM-39 cells with a higher efficiency (12- to 18-fold) than Ad5LacZ-FiWT. With CF-KM4 cells, however, the effect of the fiber modification on transducing efficiency was negligible (Figure 7A). This confirmed that the Ad5 receptors and the mechanisms of Ad5 binding were different in MM-39 and CF-KM4 cells.

The cellular uptake of HS-GAG–bound or SAGP-bound Ad5 in MM-39 or CF-KM4 cells was apparently less efficient than the endocytotic pathway observed in HeLa or 293 cells. This implied that the HS-GAG and SAGP molecules recognized as primary receptors by the fiber knob domain of Ad5 at the surface of MM-39 and KM4 cells cooperate poorly with integrins for endocytosis, or, alternatively, that the entry pathway of Ad5 in MM-39 and CF-KM4 cells is independent of the RGD-integrin endocytotic pathway, as suggested by our experimental data. The block in Ad5 entry in MM-39 and KM4 cells could be overcome by redirecting the virus to integrin receptors via RGD peptide ligands displayed at the extremity of the fiber shaft (Figure 7B). Thus, integrins could act as functional receptors for both attachment and entry of Ad5-RGD vectors in MM-39 and CF-KM4 cells.

However, except for RGD-liganded Ad and MM-39 cells, we found a poor correlation between quantitative cell binding data of the RGD-liganded vector and its efficiency of gene transfer to CF-KM4 cells (Figures 7C and 7D), and no correlation for the K7-modified vectors and MM-39 cells (Figures 7A and 7B). These results suggested that factors and steps other than cell attachment (e.g., endocytosis, endosomal release, intracellular transport) could be positively influenced by fiber modifications aimed at retargeting the virus to alternative receptors. They also suggested that molecules that anchor virus or vector to the cell surface have to cooperate with the endocytosis machinery to lead to virus entry and/or efficient vector-mediated gene transfer.

Our results confirmed that the early steps of virus–cell interactions are critical for the overall efficacy of Ad-mediated gene transfer to airway cells. However, it has to be kept in mind that our model suffered from some limitations: (i) data from a single cell line from a particular patient with CF, compared with those from a single cell line from a non-CF individual, could be hardly generalized to other patients with CF, even though the mutation involved ({Delta}F508) in CF-KM4 cells was one of the most frequently found, and/or be directly correlated with CFTR function; (ii) although both MM-39 and CF-KM4 cells were studied as confluent cell monolayers attached to collagen-coated plates to mimic the airway epithelium, the airway epithelial cells are polarized and their local environmental conditions are different from cultured cells. Nevertheless, our study shows the feasibility of retargeting Ad vectors to surface molecules of tracheal gland cells which were more efficient than their natural receptors for Ad-mediated gene transduction. It also provides some information on the usage of alternative cellular uptake pathways by Ad5. We will now refine our targeting strategy by selecting peptide ligands with a high specificity and affinity for accessible surface molecules of CF-deficient airway cells, which could act as alternative receptors for Ad vectors, and lead to efficient cellular entry and gene delivery.


   Acknowledgments
 
This work was financially supported by the French Cystic Fibrosis association Vaincre la Mucoviscidose (VLM). F.G. was the recipient of a VLM fellowship. The authors thank Simone Peyrol (Centre d'Imagerie de la Faculté de Médecine Laennec) for her help in EM analyses and Jean-Claude Cortay for his valuable advice in FPLC chromatography. They are deeply grateful to Jeff Engler (University of Alabama at Birmingham; UAB), David Curiel (UAB), Silvio Hemmi (Zurich University) and Jeff Bergelson (Children's Hospital of Philadelphia) for their gift of monoclonal antibodies, and to Frank Graham (McMaster University, Toronto) for his Ad5-luciferase vector. The authors thank the Vector Core of the University Hospital of Nantes (Dr. Ph. Moullier) supported by the Association Française contre les Myopathies (AFM) for the production of titered stocks of Ad5LacZ-FiK7 and Ad5LacZ-Fi408K7 vectors. They also thank the Fondation pour la Recherche Médicale (FRM) for its contribution to the financing of our DAKO flow cytometer, and to the Direction Générale des Hospices Civils de Lyon for the financing of our MegaView II transmission electron microscopy camera and automatic MT-X ultramicrotome (RMC EM Products Group, Ventana Medical Systems, Inc. Tucson, AZ).

Received in original form February 13, 2002

Received in final form June 24, 2002


   References
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 Introduction
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 Results
 Discussion
 References
 

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