A Helper-Independent Adenovirus Vector with E1, E3, and Fiber Deleted: Structure and Infectivity of Fiberless Particles

Adenoviruses (Ads) are nonenveloped DNA viruses with icosahedral symmetry. There are at least 47 known Ad serotypes, many of which are associated with respiratory, gastrointestinal, or ocular disease (23). Ad has served as a model for the study of many biological processes and is in use as a vector for clinical gene therapy (26). Of the known serotypes, the closely related Ad type 2 (Ad2) and Ad5 have been most extensively studied. A multistage pathway by which Ad2 infects epithelial cells has been described elsewhere (21, 50). The outer shell of the Ad capsid contains three major proteins, hexon, penton base, and fiber, along with several minor proteins. Cryoelectron microscopic (cryo-EM) structural studies have revealed the locations of most of these in the viral particle (42, 44) and in some cases have provided clues to their function (18). The majority of the capsid by mass is hexon, which forms the facets of the icosahedral particle (47). Each of the 12 vertices contains a complex of the penton base and fiber proteins (47). A ∼25-Å protrusion at the top of each penton base monomer contains an RGD sequence (43), which interacts with cellular αv integrins to mediate virus internalization and endosome disruption (49, 50). This interaction appears to be conserved across many Ad serotypes (31). In at least some cell types (for example, THP-1 monocytic cells), the penton base can also mediate virus attachment by binding to β2 integrins via its RGD motif (25). The homotrimeric fiber protein forms a prominent spike that protrudes from each vertex of the capsid. Fiber is anchored to the penton base by its N terminus, while its C-terminal domain mediates attachment to cellular receptors (12, 30, 34). The fibers from Ad2 or Ad5 bind to a 46-kDa protein termed CAR (coxsackievirus and adenovirus receptor), expressed on the surface of many cells (4, 45), and the Ad2 fiber protein has also been reported to bind major histocompatibility class I antigens (22). The fiber from Ad3 binds to an as yet unidentified but more widely distributed receptor (14, 41). Since all of these virus serotypes are thought to be internalized via the integrin-penton interaction (31), the fiber-receptor interaction largely determines Ad cell tropism. In addition to its role in targeting Ad infection, the fiber has been proposed to facilitate assembly or to stabilize the viral particles. A number of Ad proteins are synthesized as precursors which are then cleaved to their mature forms by the virally encoded L3 23-kDa protease (2, 3). Studies using fiber mutant viruses have suggested that a defect in the fiber protein might lead to defective proteolytic processing of proteins VI, VII, and VIII and therefore to accumulation of their uncleaved precursors (10, 15, 17). Other defects including abnormal sedimentation on CsCl gradients and incomplete packaging of viral DNA into the mutant particles were reported. These earlier studies led investigators to conclude that defective proteolysis due to lack of the fiber protein leads to a general block in virus maturation. The inability to propagate viral mutants lacking the genes that encode structural proteins has hindered study of their roles in capsid assembly. The aforementioned studies were done with either temperature-sensitive (ts) fiber mutants (10, 15) or deletion mutants that were propagated in the presence of nondefective helper virus (17). Their interpretation may therefore be clouded by leaky expression of the ts protein or by residual helper virus in the preparations. A true null mutant which is completely helper independent would be useful in studying the fiber mutant phenotype. We previously reported the generation of cell lines expressing a functional Ad5 fiber protein, which can complement fiber mutant Ads (48). In the studies reported here, we used these cell lines to generate a helper-independent gene transfer vector with E1, E3, and fiber deleted and have examined the structure and infectivity of Ad5 particles lacking the fiber protein. The fiberless virus, in combination with packaging cell systems that we have previously developed, should be useful in vector retargeting.