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. 2001 Aug;75(16):7703-11.
doi: 10.1128/JVI.75.16.7703-7711.2001.

Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia

R W Walters  1 , W van't Hof, S M Yi, M K Schroth, J Zabner, R G Crystal, M J Welsh
Affiliations

Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia

R W Walters et al. J Virol. 2001 Aug.

Abstract

In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.

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Figures

FIG. 1
FIG. 1
Effect of CAR expression on adenovirus-mediated gene transfer to NIH 3T3 cells. Cells were infected with varying MOIs of Ad-CaPi coprecipitates encoding wt-CAR (◊). Tailless-CAR (○), GPI-CAR (▵), or CFTR (□) as a control. One day later cells were infected with varying MOIs of Ad2/GFP. Data are the percentage of GFP-positive cells for cells infected with the Ad/CAR vectors at MOIs of 50 (A), 20 (B), 6 (C), and 1 (D).
FIG. 2
FIG. 2
Expression of adenovirus-encoded CAR constructs in primary cultures of human airway epithelia. Differentiated human airway epithelia were mock infected (Naive), infected with Ad2/GFP (Control), or infected with adenovirus vectors encoding wt-CAR, Tailless-CAR, or GPI-CAR. Two days after infection, cellular lysates were assessed for expression of Flag-tagged CAR proteins by Western blot analysis using anti-Flag M2-HRP monoclonal antibody. Arrows indicate the observed migration profiles of full-length CAR, Tailless-CAR, and GPI-CAR; migration of the three bands was consistent with the predicted molecular masses. The band at approximately 70 kDa was nonspecific as it was also observed in naive and mock-infected cells.
FIG. 3
FIG. 3
Cell surface distribution of modified CAR proteins expressed in human airway epithelia. (A) Apical localization of CAR molecules was evaluated in airway epithelia with immunocytochemistry. GFP-positive cells are shown in green, and apical Flag antibody binding is shown in red. Polarized surface distribution of CAR molecules was quantitated with a radioimmunoassay on the apical surface (B), or the basolateral surface (C). Data are means + standard errors of the means (error bars) (n = 6). ∗, P < 0.01 compared to control.
FIG. 3
FIG. 3
Cell surface distribution of modified CAR proteins expressed in human airway epithelia. (A) Apical localization of CAR molecules was evaluated in airway epithelia with immunocytochemistry. GFP-positive cells are shown in green, and apical Flag antibody binding is shown in red. Polarized surface distribution of CAR molecules was quantitated with a radioimmunoassay on the apical surface (B), or the basolateral surface (C). Data are means + standard errors of the means (error bars) (n = 6). ∗, P < 0.01 compared to control.
FIG. 4
FIG. 4
Effect of CAR expression on adenovirus binding to the apical surface of CAR-expressing human airway epithelia. Data are en face projections of Cy3-labeled adenovirus (red) bound to the apical surface of control and CAR-expressing airway epithelia. DAPI-stained nuclei are blue.
FIG. 5
FIG. 5
Effect of CAR expression on adenovirus-mediated gene transfer to airway epithelia. Two days after CAR gene transfer, airway epithelia were infected with Ad2/GFP from the apical surface (A), Ad2/βGal from the apical surface (B), Ad2/βGal from the apical surface following PI-PLC treatment (C), or Ad2/βGal from the basolateral surface (D) (each at an MOI of 10) for 30 min. Epithelia were studied 48 h later. β-Gal data are means + standard errors of the means (error bars) (n = 6). ∗, P < 0.01 compared to control. Lu, light units.
FIG. 6
FIG. 6
Effect of neuraminidase or glycosidase treatment on adenovirus-mediated gene transfer to CAR expressing airway epithelia. (A) Two days after CAR gene transfer, airway epithelia were pretreated with neuraminidase (Neur), O-glycosidase (O-gly), or N-glycosidase (N-gly) and stained with FITC-labeled WGA. Data are en face projections of WGA binding to the apical membrane of airway epithelia. WGA binding (in relative intensity units) decreased following treatment with neuraminidase (14.5 ± 1.9), O-glycosidase (21.0 ± 1.4), and N-glycosidase (20.3 ± 1.2) compared to control (29.5 ± 1.0) (n = 9 for each). (B) Treated epithelia were also assayed for gene transfer with Ad2/βGal. Fluorescence and β-Gal data are means + standard errors of the means (error bars) (n = 6).
FIG. 6
FIG. 6
Effect of neuraminidase or glycosidase treatment on adenovirus-mediated gene transfer to CAR expressing airway epithelia. (A) Two days after CAR gene transfer, airway epithelia were pretreated with neuraminidase (Neur), O-glycosidase (O-gly), or N-glycosidase (N-gly) and stained with FITC-labeled WGA. Data are en face projections of WGA binding to the apical membrane of airway epithelia. WGA binding (in relative intensity units) decreased following treatment with neuraminidase (14.5 ± 1.9), O-glycosidase (21.0 ± 1.4), and N-glycosidase (20.3 ± 1.2) compared to control (29.5 ± 1.0) (n = 9 for each). (B) Treated epithelia were also assayed for gene transfer with Ad2/βGal. Fluorescence and β-Gal data are means + standard errors of the means (error bars) (n = 6).
FIG. 7
FIG. 7
Effect of CAR expression on adenovirus-mediated CFTR expression by CF airway epithelia. Two days after CAR gene transfer, CF airway epithelia were infected with Ad2/CFTR-16 (MOI, 10) for 30 min from the apical surface. Forty-eight hours later the epithelia were studied in Ussing chambers. Data are changes in current (means + standard errors of the means [error bars]) after the addition of bumetanide to cyclic AMP-stimulated, amiloride-treated epithelia (ΔIBum) (n = 6). ∗, P < 0.01 compared to control.
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