mCAR and human CAR amino acid sequences. The sequence of human CAR (h), the sequence of a murine homolog (clone m1), and the sequence of a murine homolog with an altered C terminus (clone m2) are shown. Predicted hydrophobic leader (determined as described in reference 24) and transmembrane domains are underlined. Potential sites for N-linked glycosylation are marked with an asterisk.

Expression of mCAR on transfected CHO cells. (A) Immunofluorescence analysis. Control CHO cells transfected with the human integrin α2 subunit (CHO-al2) or CHO cells transfected with mCAR cDNA (CHO-mCAR) were incubated first with normal rat serum (dotted line) or with serum from rats immunized with the 46-kDa mouse brain receptor (anti-p46) (solid line) and then with fluorescein isothiocyanate-conjugated goat antibody to rat immunoglobulin. Results with clone m2 are shown; similar results were obtained with clone m1. (B) Immunoblot analysis. Proteins blotted onto nitrocellulose were probed with anti-p46 serum as described in Materials and Methods. Results are shown for extracts of CHO cells transfected with the human integrin α2 subunit and for CHO cells transfected with mCAR clones m1 and m2. Positions of molecular mass markers are shown at the right (in kilodaltons). Specific proteins of 46 to 48 kDa were detected in CHO-mCAR transfectants. A nonspecific band (approximately 60 kDa) was seen in all lanes.

Coxsackie B virus attachment to mCAR on transfected CHO cells. Confluent monolayers of CHO-mCAR or control CHO-al2 cells were incubated with radiolabeled CB3 or CB4 (29,000 cpm) for 4 h at room temperature and then washed and dissolved for scintillation counting. Results with clone m2 are shown; similar results were obtained with clone m1. Some monolayers were preincubated with recombinant adenovirus 5 knob domains (produced in E. coli [0.7 μg]) before exposure to radiolabeled virus. Results for triplicate samples (mean virus bound [counts per minute] + 1 standard deviation [error bar]) are shown.

Coxsackievirus production by transfected CHO cells. CHO-mCAR and CHO-al2 monolayers were exposed to CB3 or CB4 (10 PFU/cell) for 1 h at room temperature and then monolayers were washed and incubated at 37°C for 1 h (0 days), 1 day, or 2 days. Monolayers were frozen and thawed to release virus, and then plaque assays were performed. The figure shows the mean virus titers for triplicate cultures.

Adenovirus interaction with mCAR on transfected CHO cells. (A) Virus attachment. Cell monolayers were incubated with radiolabeled adenovirus 2 (20,000 cpm) for 1 h at room temperature and then washed and dissolved for scintillation counting. Some monolayers were preincubated with adenovirus 2 fiber (5 μg) for 1 h and then washed before exposure to radiolabeled virus. (B) Knob attachment. Monolayers were incubated with ¹²⁵I-labeled adenovirus 5 knob domains (produced in insect cells) for 1 h at room temperature and then washed and dissolved for scintillation counting. Results for triplicate samples (mean virus or knob protein bound [in counts per minute] + 1 standard deviation [error bar]) are shown.

Adenovirus-mediated gene transfer. Duplicate monolayers of CHO-al2, CHO-mCAR, or CHO cells transfected with human CAR cDNA (CHO-hCAR) were exposed to Ad.CMV-βgal for 1 h at room temperature, and then monolayers were washed. After incubation at 37°C for 40 h, β-galactosidase activity was detected by in situ staining with X-Gal. Some monolayers were incubated with 1.5 μg of purified adenovirus 2 fibers (+) before exposure to virus. Results with clone m2 are shown; similar results were obtained with clone m1.

CAR mRNA expression in human and murine tissues. Multiple-tissue Northern blots (Clontech) containing 2 μg of poly(A)⁺ RNA from each of the indicated tissues were probed with human CAR and mCAR cDNA as described in Materials and Methods. Positions of marker RNAs are indicated in kilobases. Hybridization with a human actin probe confirmed the presence of equivalent amounts of RNA in each lane.