Construction of a pseudoreceptor that mediates transduction by adenoviruses expressing a ligand in fiber or penton base.

The utility of adenovirus as a vector to deliver therapeutic genes would be greatly enhanced if the virus could be specifically targeted to the tissue of interest. Incorporation of tissue-specific ligands into viral coat proteins which lack their native cell recognition activity could allow selective delivery of transgenes to tissues that bind those ligands, while heterologous tissues normally susceptible to infection would not be modified. This approach requires the identification of appropriate ligands and a strategy for introducing them into the targeted virus. Redirecting virus to the target tissue will also require knocking out those interactions that contribute to the native tropism of the virus, and so an understanding of the molecular basis of the latter is critical. Infection by adenovirus is facilitated by an initial attachment of the virus to its target cell through an interaction of the fiber protein with a cellular receptor. A fiber receptor for adenoviruses of subgroups A, C, D, E, and F has been identified as the CAR (coxsackievirus/adenovirus receptor) protein (2, 23, 26). The importance of the fiber protein for infection is demonstrated by the ability of soluble fiber protein to block virus entry (3, 20) and by the enhanced infectivity of the virus on cells expressing CAR (11, 16). In addition, viruses with chimeric fiber proteins have been shown to acquire the tropism of the virus from which the fiber knob is derived (25). The fiber knob binds to the N-terminal immunoglobulin-like domain of CAR (8). Following attachment via CAR, internalization of the virus is promoted by the interaction of penton base protein with αv integrins (31). This interaction may elicit critical cell signaling events in addition to linking the virus to an endocytic pathway (17, 18). Although the penton base-αv integrin interaction does not mediate direct binding of subgroup C adenoviruses (serotypes 2 and 5 [Ad2 and Ad5]), the infectivity of these viruses is reduced on cells that express little or no αv integrins or when the interaction is blocked by competitors (13, 31). Mutation of the RGD motif within penton base results in a delay of virus replication (1). The lack of contribution of the penton base-αv integrin interaction to initial binding of virus might be a consequence of the 30-fold-lower affinity of this interaction relative to that of fiber for CAR (31) or result from a steric hindrance imposed by the long fiber of subgroup C viruses. The importance of both the fiber- and penton base-mediated interactions in vivo is highlighted by the finding that the failure of adenovirus to efficiently transduce mature airway epithelial cells correlates with absence of both αv integrins and the CAR receptor (21, 36). While the two-step model of adenovirus entry involving fiber-mediated attachment and penton base-mediated internalization is supported by a number of studies and is a useful guide for attempting to alter the native tropism of the virus, variations of this model should be noted. The subgroup D virus Ad9, which has a much shorter fiber (16 nm, versus 37 nm for Ad2), attaches directly to cells via either its fiber or penton base (22). The fiber protein of Ad9 binds to CAR, but infection of many cell types is not inhibited by competing soluble fiber, whereas antibodies to αv integrin or penton base do block binding. In addition, Ad2 has been shown to bind cells in the absence of CAR via an interaction of penton base with β2 integrins (14). An interaction between penton base and αv integrins was still required for Ad2 to enter these cells. These findings demonstrate that penton base can mediate direct binding if it binds to β2 in the context of Ad2 or if it binds to αv in the context of Ad9. While there may be additional components of the native tropism of adenovirus, abolishing the high-affinity binding of fiber to CAR is essential for development of a targeted vector. Since growth of Ad5-based vectors is dependent on fiber binding to the CAR receptor on production cells, a modified virus that no longer bound CAR would require an alternative means of attaching to these cells. We developed an alternative receptor (pseudoreceptor) for adenovirus to mediate CAR-independent transduction. The pseudoreceptor consists of a membrane-anchored single-chain antibody [m-scFv(HA)] which recognizes a linear peptide epitope from the hemagglutinin (HA) protein of influenza virus. This approach might allow a variety of modified viruses to be grown in a single production cell line so long as they express the HA epitope in a context which allows binding to the pseudoreceptor. In addition the HA epitope could serve as a model for a tissue-specific ligand to examine its function following insertion into various sites in different adenovirus coat proteins. In this report we show that the m-scFv(HA) can mediate transduction of adenoviruses carrying the HA epitope within the HI loop of fiber. Moreover, insertion of the HA epitope within penton base allowed the m-scFv(HA) to mediate entry of viruses in the absence of attachment via the fiber receptor. These results suggest that packaging cell lines expressing this type of pseudoreceptor will be useful in producing tissue-specific targeted adenoviral (Ad) vectors in which CAR binding has been ablated.