A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

Group B coxsackieviruses (CVBs) must cross the epithelium as they initiate infection, but the mechanism by which this occurs remains uncertain. The coxsackievirus and adenovirus receptor (CAR) is a component of the tight junction and is inaccessible to virus approaching from the apical surface. Many CVBs also interact with the GPI-anchored protein decay-accelerating factor (DAF). Here, we report that virus attachment to DAF on the apical cell surface activates Abl kinase, triggering Rac-dependent actin rearrangements that permit virus movement to the tight junction. Within the junction, interaction with CAR promotes conformational changes in the virus capsid that are essential for virus entry and release of viral RNA. Interaction with DAF also activates Fyn kinase, an event that is required for the phosphorylation of caveolin and transport of virus into the cell within caveolar vesicles. CVBs thus exploit DAF-mediated signaling pathways to surmount the epithelial barrier.

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.G roup B coxsackieviruses and a number of adenovirus serotypes initiate infection by binding to the coxsackievirus and adenovirus receptor (CAR) (1-3). CAR is a 46-kDa integral membrane protein with a typical transmembrane region, a long cytoplasmic domain, and an extracellular region composed of two Ig-like domains (1, 2). Both adenovirus (4) and coxsackievirus (5) interact with the N-terminal domain. Homologs of human CAR have been characterized in mice (2, 6), rats, pigs, dogs (7), and zebrafish (8). The murine and human proteins are very similar (91% amino acid identity within the extracellular domain, 77% within the transmembrane domain, and 95% identity within the cytoplasmic domain). Variant isoforms of CAR, which differ only at the C terminus and which most likely result from alternative splicing, have been identified in mice (6), humans, and rats (7). Despite evidence of its evolutionary conservation in mammals and nonmammalian vertebrates, the function of CAR is not known.Tight junctions are continuous circumferential intercellular contacts at the apical poles of lateral cell membranes, appearing in electron micrographs as a series of discrete contacts between the plasma membranes of adjacent cells (9). Tight junctions form a barrier that regulates the paracellular transit of water, solutes, and immune cells across an epithelium (10), and are essential for establishing cell polarity by separating the apical and basolateral domains of polarized epithelial cells (11). ZO-1, the first tight junction protein identified (12, 13), is an intracellular peripheral membrane scaffolding protein important for tight junction structure and assembly. In the present study, we found that in polarized epithelial cells CAR is expressed at the tight junction, where it associates with ZO-1 and functions in the barrier to the movement of macromolecules and ions. Materials and MethodsCell Culture. T-84, CALU-3, and 16HBE14o-cells were grown in 10% CO 2 in a 1:1 mixture of DMEM and Ham's F-12 medium with 10% FCS. To establish polarized monolayers, 5 ϫ 10 5 cells per well were plated on 12-mm diameter polyester filters with a ...