Basolateral Localization of Fiber Receptors Limits Adenovirus Infection from the Apical Surface of Airway Epithelia

Abstract

Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I ␣-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency.The mechanism of infection by type 2 and type 5 adenovirus has been extensively studied. However, most of the knowledge on adenoviral infection has been obtained from studies done on immortalized cell lines. The first steps in adenovirus infection are thought to involve primarily two proteins in the capsid, fiber and penton base (1-3). The fiber protein is important for binding to a high affinity fiber receptor. In a human oral epidermoid carcinoma cell line (KB cells), A549 cells, and HeLa cells, this receptor is thought to be present in the range of 3,000 -10,000 receptors/cell (4 -6). NIH 3T3 cells, which are resistant to adenovirus infection, have less than 100 receptors/ cell (7). After binding to the fiber receptor, penton base interaction with ␣ V ␤ 3 and ␣ V ␤ 5 integrins facilitates internalization via receptor-mediated endocytosis (2,8,9). The acidic pH the virus encounters in the endosome may trigger a conformational change that releases the virus into the cytoplasm (10 -12) and allows the adenovirus capsid to travel to the nucleus (2, 13). Then viral proteins and DNA bind to the nuclear pore complex, capsid disassembly continues, and DNA enters the nucleus accompanied by DNA-associated protein 7 (1, 2, 14). These studies have concluded that a high affinity fiber receptor is required for binding and infection and that an ␣ V ␤ integrin acts as a co-receptor.We and others (15-22) have found infection of ciliated airway epithelia by adenovirus to be inefficient. In an in vitro model of human airway epithelia, we found that unlike infections of HeLa cells adenovirus infection of ciliated airway epithelia was quite limited and that which did occur w...

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli–Sertoli and Sertoli–germ cell interface

“…After only 4 hours of treatment, we saw a significant decline in the level of occludin and JAM-A, in comparison to the time interval of 2 to 3 days before we detected a decline in CAR level. In some types of cells, such as human airway epithelia, CAR was located within the adherens junctions at the basolateral side of the columnar cell layer, rendering the cells resistant to viral infection from the apical surface [47]. Here, it is not certain whether CAR also resides near the basal side of the Sertoli cell layers as it does in human airway epithelia.…”

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…Several studies have determined that disruption of calciumdependent intercellular junctions by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 23,25,26,28 Previous evidence demonstrated that polarized apical membranes of normal human bronchial epithelial (NHBE) airway cells are resistant to lipofection. NHBE cells form multicellular islands similar to that of long-term primary rat hepatocyte cultures maintained in DMSO.…”

“…24 Several studies have determined that disruption of calcium-dependent paracellular junction complexes by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 14,23,[25][26][27][28] Transient depletion of extracellular calcium may overcome the inefficiency of baculovirus-mediated gene transfer by disassociating paracellular epithelial junction complexes and exposing the basolateral surface. We hypothesized that in normal medium, which contains calcium, the baculovirus binds and delivers its genome only to peripheral cells and baculovirus attachment, and entry to internal cells is prevented because paracellular junctions mask the baculovirus binding motif of these internal hepatocytes.…”

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…Receptor expression also differs with regards to the location on the cell membrane. For instance, the receptor for adenovirus serotype 5 (the coxsackie and adenovirus receptor CAR) is expressed apically in mice (and thus easily accessible to topical vector delivery), but basolaterally in humans [48,49]. This disparity in receptor distribution likely underpins the failure of rAd in human trials to deliver on the early successes in murine studies.…”