Structure-function studies of adenovirus type 5 fiber protein and its receptor CAR: Implications for adenovirus cell entry mechanism and gene delivery

The physiological function of coxsackievirus and adenovirus receptor (CAR) is not known. Using a yeast two-hybrid screen we found that filamin-a (ABP-280) interacts with human CAR through CAR's D1 and D2 domains. There is a reverse correlation between filamin levels and surface CAR levels. In a filamin deficient cell line M2, CAR is predominantly expressed on the cell surface; whereas in A7 cells in which filamin levels have been restored, CAR becomes predominantly localized intra-cellularly. Over-expression of either CAR-binding domain of filamin or filamin-binding domain of CAR can alter the subcellular expression of CAR. We propose that filamin regulates CAR's subcellular distribution. We also speculate that CAR-filamin interaction might have physiological significance in adenoviral entry and coxsackieviral myocarditis.; Any strategy to design a targeted adenoviral vector for cell-specific gene delivery must be based on the structure of the Ad virion and the biology of Ad infection. We used the cutting-edge technique, site-directed spin labeling and EPR, to study the structure of Ad5 fiber protein in its biological context during the Ad infection. We found that knob domain of fiber may display different structure in solution from that in crystal; CAR receptor binding to the knob might induce fiber conformational changes which hypothetically alter the assembly of Ad5 capsid. In addition, our experiments show that Ad5 fiber knob domain may undergo secondary structural change in response to low pH during the endosomal compartment. These studies attract interests of adenoviral gene therapy and help understand cell entry events of Ad virion.