| The coxsackievirus and adenovirus receptor (CAR) is, as the name indicates, a cellular receptor for two diverse classes of virus. CAR is a crucial receptor for coxsackievirus B, and is an attachment receptor for most adenoviruses. CAR is a 46 kD immunoglobulin-superfamily protein that is expressed in most organs in both man and mouse. The natural function of the protein is still an enigma. The goal of this thesis project was to create tools that would allow further investigation of CAR's natural function as well as exploring its role as a viral receptor.; A system to produce recombinant CAR by insect cell secretion was developed. Purified soluble CAR protein was used to generate new anti-CAR antibodies both by rat vaccination and through screening of a recombinant human antibody library. Several new monoclonal antibodies that recognized cell surface CAR were isolated.; A yeast two-hybrid screen was performed using the cytoplasmic tail of CAR to probe a human cDNA library for potential interacting proteins. One particularly interesting candidate was a novel protein that contained several PDZ protein interaction domains.; A CAR knockout mouse was created and revealed that the CAR-/- genotype was lethal to the fetus at about 10.5 days post conception. The visible phenotype was one of extensive hemorrhaging and cellular autolysis. The manifestation of the moribund phenotype in the CAR-/- fetuses corresponded to the time of first expression of CAR in normal mice. In order to circumvent fetal lethality, a mouse that will enable the conditional knockout of CAR using the Cre-loxP recombination system was created.; Studies were also conducted in mice that overexpressed human CAR, either with near ubiquity or only in lymphocytes. When these mice were infected with coxsackievirus B, there was no effect on organ viral titers. However, a novel transgenic mouse that expressed hCAR on erythrocytes using a GATA1 promoter displayed a significant reduction in viremia and viral replication in several organs.; These novel protein reagents, antibodies, and transgenic mice are expected to be powerful tools for future studies of CAR. Further examination of the CAR knockouts will hopefully help elucidate CAR's natural function. |
