FIG 5.

Complete recovery of CAR function after substitution of the D2 domain by other D2 domains of the IgSF. (A) Schematic illustration of various CAR proteins: left, wild-type hCAR and wild-type chCAR; center, chimeric chCAR/hCAR receptors containing the chCAR-D1 domain fused to the hCAR-D2 domain (chCAR-hCAR) or the hCAR-D1 domain fused to chCAR-D2 (hCAR-chCAR); and right, hCAR-D1 domains fused to the D2 domains of CD80, CD155, or IgSF11. (B) Localization of CAR chimeras expressed in CHO-K1 cells. Cells transfected with CAR chimeras were stained with polyclonal anti-CAR antibodies and examined by immunofluorescence microscopy. Typical localization to cell-cell junctions was observed for wild-type hCAR, the hCAR/chCAR chimeras, and hCAR-IgSF11 (white arrows), whereas the other substitution mutants (hCAR-CD80 and hCAR-CD155) showed little or no localization to the junctions. (C) Binding of with ³⁵S-labeled CVB3 to CHO-K1 cells expressing wild-type CAR or chimeric receptors, measured as described in Materials and Methods. (D) Viral replication in cells expressing chimeric receptors (MOI of 5). Transfected CHO-K1 cells were incubated with CVB3 for 1 h at room temperature, washed twice to remove unbound virus, and then incubated at 37°C. Viral titers were determined by plaque assay at 0 and 24 h. (E) CHO-K1 cells stably expressing hDAF (CHO-hDAF) were transfected with wild-type or chimeric hCAR receptors (or mock transfected). At 48 h after transfection, the cells were exposed to CVB3 at the indicated MOIs, washed, and incubated at 37°C for 24 h. Virus titers were determined by plaque assay. The results are shown as means and the SD for triplicate samples. ns, not significant; *, P < 0.05; **, P < 0.01 (compared to hCAR control value).