Adenovirus transduction: More complicated than receptor expression

Abstract

The abundance and accessibility of a primary virus receptor are critical factors that impact the susceptibility of a host cell to virus infection. The Coxsackievirus and adenovirus receptor (CAR) has two transmembrane isoforms that occur due to alternative splicing and differ in localization and function in polarized epithelia. To determine the relevance of isoform-specific expression across cell types, the abundance and localization of both isoforms were determined in ten common cell lines, and correlated with susceptibility to adenovirus transduction relative to polarized primary human airway epithelia. Data show that the gene and protein expression for each isoform of CAR varies significantly between cell lines and polarization, as indicated by high transepithelial resistance, is inversely related to adenovirus transduction. In summary, the variability of polarity and isoform-specific expression among model cells are critical parameters that must be considered when evaluating the clinical relevance of potential adenovirus-mediated gene therapy and anti-adenovirus strategies.

Keywords: Adenovirus; Apical and basolateral; Coxsackievirus and adenovirus receptor (CAR); Epithelia; Non-polarized; Polarized; Tight junction.

Figure 1

Transepithelial electrical resistance (TER) increases over time in cell lines able to polarize into epithelia. No increase in the TER above background level (grey dotted line) was observed in cell lines unable to polarize on semipermeable membranes (CHO-K1, COS-7, HeLa, HEK-293, 293T and A549). By contrast, a rapid increase in TER over time was observed for most epithelial cell lines (MDCK, Caco-2, Calu-3, NuLi-1, and primary human airway epithelia (HAE)).

Figure 2

CAR^Ex7 transcript and protein levels are more abundant than CAR^Ex8 transcript and protein levels. A) CAR^Ex7 or B) CAR^Ex8 gene expression was quantified by qPCR with exon specific primers after cDNA synthesis from total RNA obtained from non-polarized and polarized cell types grown on semi-permeable membranes. Gene expression shown is relative to HAE (see also Table 1). A comparison to CHO-K1 cells is shown in Supplementary Table 1. C–F) Lysate from each cell type was subjected to SDS–PAGE and analyzed by Western blotting. Total CAR expression was detected in C) the cell lines that do not polarize or D) polarized cells with a CAR-specific polyclonal antibody (rabbit anti-CAR-1605p) that recognizes the C-terminus of both CAR isoforms. CAR^Ex8 specific protein expression was detected in E) the cell lines that do not polarize or F) polarized cells by a CAR^Ex8-specific antibody (rabbit anti-CAR^Ex8-5678p). As expected, no CAR^Ex7 or CAR^Ex8 protein was detectable in CHO-K1 cells.

Figure 3

CAR^Ex7 localizes at basolateral junctions while CAR^Ex8 localizes within the subapical and apical compartments and epithelial polarization decreases susceptibility to adenovirus transduction. A) and B) Immunocytochemistry for total CAR staining (rabbit anti-CAR-1605p) in green and nuclei in blue (DAPI). A) In non-polarized cells (A549 and COS-7), CAR^Ex7 staining and localization is discontinuous due to lack of formation of tight junction, B) whereas CAR^Ex7 localization in polarized cells (MDCK, Caco-2, Calu-3, and HAE) presents as a “chicken wire” pattern due to the formation of tight junctions. C) and D) CAR^Ex8 localization (rabbit anti-CAR^Ex8-5678p) is shown in green and nuclei in blue (DAPI). C) CAR^Ex8 localization is similar to CAR^Ex7 in non-polarized cells. D) However, CAR^Ex8 localization in well differentiated and polarized epithelial cells is distinct and is found in the cytoplasm consistent with the subapical compartment. (60× oil immersion confocal microscopy, white bar equals 10 µm. E) All cell types were seeded onto semi-permeable membranes and maintained at the air-liquid interface prior to infection from the “apical” surface with recombinant adenovirus carrying the gene for beta-galactosidase (AdV5-Beta-Gal) and Beta-galactosidase activity evaluated 24 h post-transduction.