CAR mediates virus uptake

Tight junctions are important in the natural defense against invasion by microbial patho-gens. In order to initiate infection, many pathogens must breach the epithelial barrier to gain access to the body.

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Unsuccessful efforts to use viral vectors for therapeutic gene delivery to airway epithe-lium have led to the recognition that intact epitheepithe-lium is highly resistant to adenovirus infection and adenovirus mediated gene transfer. Efficient gene delivery to epithelium in vivo, and to epithelial monolayers in culture, depends on disruptions of intercellular junc-tions, at least in part because the virus is unable to attach to the epithelial surface recep-tors when junctions are intact. The importance of receptor accessibility is demonstrated by the increased efficiency of infections noted in epithelial cultures engineered to express CAR on the apical surface (Davis et al., 2004; Pickles et al., 2000; Walters et al., 2001).

Nonetheless, many viruses are known to infect by crossing the airway or intestinal epithe-lium.

At least three viruses are now known to initiate infection by attaching to receptors within the tight junction: both coxsackieviruses and adenoviruses bind to CAR, reoviruses attach to JAM (Barton et al., 2001). Because CAR and JAM are normally sequestered within junctions, and thus not readily available to viruses at the apical cell surface, it is not clear how interaction with CAR and JAM can promote virus infection of epithelial surfaces.

Viruses may attach to CAR and JAM when intercellular contacts are opened, the receptor molecules may sometimes be exposed on the apical membrane, or alternative receptors may be required for infection of intact epithelium. It is also possible that viruses cross the epithelium by alternative mechanisms, such as transport through intestinal M-cells, which do not require infection of the epithelial cells themselves (Clark and Jepson, 2003).

The crystal structure of the reovirus σ1 protein (the attachment protein responsible for interaction with JAM) reveals striking similarity between σ1 and the adenovirus fiber knob (Chappell et al., 2002). The structures of the JAM N-terminal domain and of CAR D1 (including the surfaces involved in receptor dimerization and virus attachment) are also strikingly similar. Because viruses bind with high affinity to these receptors, it is conceivable that virus attachment proteins could disrupt the low affinity dimers

responsi-21 ble for junction formation as described in 1.1.2 (although this would require that attach-ment proteins gain physical access to the sequestered receptors).

It is reported that adenovirus fibers applied to the basal surface of a polarized epithelial monolayer can disrupt intercellular junctions (Walters et al., 2002). Based on this obser-vation, a model has been proposed in which excess production of adenovirus fiber knob leads to the disruption of cellular junctions, thus contributing to virus spread into the air-way lumen. It is not yet known whether the junctional rearrangements induced by fiber knob result directly from interference with CAR dimerization, or whether fiber knob trig-gers signals that lead to junctional reorganization, which occurs only after 24 hours. In-flammatory cytokines have marked effects on the integrity of airway tight junctions, and it is possible that adenovirus or soluble fiber may disrupt cell-cell junctions by triggering a cytokine response (Coyne et al., 2002).

Figure 3: Model of adenovirus and coxsackivirus escape from airway epithelia. CAR does not only mediate cell-cell junction via its extracellular Ig domain homodimerization, but also uptakes adenovirus and cox-sackievirus. CAR (green) is localized on the basolateral membrane below the level of the tight junction seal and on basal cells. Once infection is established, adenovirus (blue) and fiber protein (black) are released basolaterally. Fiber increases paracellular permeability by competing CAR-mediated cell-cell adhesion.

This allows adenovirus escape to the apical surface.. Independent from or together with its co-receptor DAF (decay accelerating factor/CD55) (yellow), CAR mediates coxsackievirus binding and uptake and escape from the infected cells. Figure modified from (Walters et al., 2002), according to the results from (Meier et al., 2005) and (Chung et al., 2005).

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Like adenoviruses, coxsackieviruses also appear limited in their capacity to infect pola-rized epithelium when the CAR receptor is engaged in intercellular contacts. The CVB susceptibility of cells in vitro is clearly related to the measurable presence of CAR (Sha-fren et al., 1997), so it has been presumed that the tissue tropism of CVB in vivo is related to differential expression of the receptor among cell types. The exceptions, however, sug-gest that the situation is more complicated in vivo. Some tissues, such as liver, with readi-ly measurable CAR are not associated with significant CVB pathology (Wessereadi-ly et al., 2001), and cytoplasmic host proteins may inhibit the ability of CVB to replicate in some cells (Cheung et al., 2005). In contrast, CVBs have been documented in cells of some organs that have not been reported to express CAR at readily detectable levels (Anderson et al., 1996; Mena et al., 1999). However, some coxsackievirus isolates bind to an addi-tional receptor, the complement regulatory protein decay acceleration factor (DAF/CD55) (Shafren et al., 1995). Because of its glycosyl-phosphatidylinositol-linked membrane anchor, DAF is sorted to the apical surface of polarized cells, and is thus likely to be ac-cessible to pathogens in the airway or intestinal lumen. DAF-binding CVB isolates, un-like those that interact only with CAR, are capable of infecting polarized epithelial mono-layers; thus, the capacity to bind to DAF may provide these viruses with a mechanism by which to cross the epithelium despite the inaccessibility of CAR (Shieh and Bergelson, 2002).