The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells

Abstract

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAR^Ex8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAR^Ex8 and determine the feasibility of receptor downregulation, the half-life of total and apical localized CAR^Ex8 was determined and correlated with adenovirus transduction. Total and apical CAR^Ex8 has a relatively short half-life of approximately 2 h. The half-life of apical CAR^Ex8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application.

Keywords: coxsackievirus and adenovirus receptor; half-life; human adenovirus; polarized epithelia.

Figure 1

CAR^Ex8 has a half-life of approximately 2 h. MDCK cells that stably expressed FLAG-tagged CAR^Ex8 under the control of a doxycycline-sensitive promoter were seeded in a plastic 12-well dish in FBS tet⁽⁻⁾ media. Cells were induced with 100 ng/mL doxycycline for 24 h. The doxycycline-containing media were then removed and replaced with standard FBS tet⁽⁻⁾ culture media. (A). Cells were lysed at indicated time points before and after removal of Dox and the lysates were analyzed by Western blot (WB) using anti-FLAG or actin-specific primary antibodies. (B). Cells were lysed at indicated time points post Dox removal and lysates were analyzed by WB. (C). CAR^Ex8 bands were normalized to their corresponding actin bands and quantified using ImageJ. (D). MDCK-FLAG-CAR^Ex8 were polarized on semipermeable membranes. At 24 h post Dox treatment, standard FBS tet⁽⁻⁾ culture media were added and surface biotinylation was performed at indicated time points post Dox removal. Cells were then lysed and the lysates (actin) or isolated-biotinylated proteins (CAR^Ex8, FLAG Ab) were analyzed via WB. (E). CAR^Ex8 bands were normalized to their corresponding actin bands and quantified using ImageJ. Error bars represent SEM. (* p < 0.05; ** p < 0.01; One-Way ANOVA; N at least 3).

Figure 2

CAR^Ex8 has a half-life of approximately 2 h. (A) Calu-3 cells were treated with 30 μg/mL cycloheximide. The cells were lysed at the times indicated post cycloheximide addition and analyzed by WB using a CAR^Ex8-specific primary antibody (5678p) or actin (loading control). (B) Quantification of CAR^Ex8 bands normalized to actin. Error bars represent SEM. (* p < 0.05; ** p < 0.01; *** p < 0.001; One-Way ANOVA; N at least 3).

Figure 3

Adenovirus transduction decreases as CAR^Ex8 degrades. MDCK-FLAG-CAR^Ex8 were seeded and polarized in FBS tet⁽⁻⁾ media. The cells were then treated with 100 ng/mL Dox for 24 h. At 24 h post treatment, the doxycycline-containing media were removed and replaced with standard FBS tet⁽⁻⁾ culture media. At 4, 6, 8, 10, 12, and 24 h post dox removal, cells were transduced with AdV-β-Gal. AdV transduction was analyzed by β-galactosidase assay after 24 h. Error bars indicate standard error of the mean. (**** p < 0.0001; One-Way ANOVA; N = 3).