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Adenovirus (Ad) entry into cells is initiated by the binding of the fiber knob to a cell surface receptor. The coxsackie- and adenovirus receptor (CAR) functions as the attachment receptor for many, but not all, Ad serotypes. Ad type 37 (Ad37), a subgroup D virus that causes keratoconjunctivitis in humans, does not infect cells via CAR despite demonstrated binding of the Ad37 knob to CAR. We have pseudotyped a fiber deletion Ad5 vector with the Ad37 fiber (Ad37f), and this vector retains the ocular tropism of Ad37. Here we present a cryo-electron microscopy reconstruction of Ad37f that shows the entire Ad37 fiber, including the shaft and knob domains. We have previously proposed that Ad37 may not utilize CAR for cell entry because of the geometric constraints imposed by a rigid fiber (E. Wu, J. Fernandez, S. K. Fleck, D. Von Seggern, S. Huang, and G. R. Nemerow, Virology 279:78–89, 2001). Consistent with this hypothesis, our structural results show that the Ad37 fiber is straight and rigid. Modeling of the interaction between Ad37f and host cell receptors indicates that fiber flexibility or rigidity, as well as length, can affect receptor usage and cellular tropism.

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.

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.