Structural analysis of a fiber-pseudotyped adenovirus with ocular tropism suggests differential modes of cell receptor interactions

Abstract

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.

FIG. 1

Cryo-EM particle images and reconstruction of the Ad37f pseudotyped vector. (A and C) Two clear particle images showing density around the capsid corresponding to the short fibers. (B and D) Surface views of the three-dimensional reconstruction shown in the same orientations as determined for the particle images in panels A and C. The arrows in panel A point to strong density from overlapping fiber knobs. The arrows in panel C point to five fibers visible around the capsid in this approximate fivefold view. The fiber is colored green, the penton base is yellow, and the remaining capsid is blue. Bar, 100 Å.

FIG. 2

Vertex region of the Ad37f pseudotyped vector and comparison with Ad2. (A and B) Top and side views of the Ad37f vertex region. (C) Side view of the Ad2 vertex region (7) shown with a low isosurface value to include the weak diffuse density that results from flexibility in the fiber shaft. The color scheme is the same as in Fig. 1. Bar, 100 Å.

FIG. 3

Modeling of the crystal structure of the Ad2 knob and a portion of the shaft into the Ad37f cryo-EM fiber density. (A) Side view. (B) Top view. The AB loop is colored red, and the CD loop is blue. The arrows point to the HI loop that protrudes in the Ad2 crystal structure (29) and fits well within a bulge of the reconstructed Ad37f fiber knob. Bar, 100 Å.

FIG. 4

Pseudotyped Ad with Ad37 fiber uses α_v integrins for internalization. Chang C cells were pretreated with 100 μg of HB5 (anti-CD21) monoclonal antibody, LM609 (anti-α_vβ₃) and P1F6 (anti-α_vβ₅) monoclonal antibodies, 69-6-5 (anti-α_v) antibody, or recombinant Ad5 penton base (PB) per ml in Dulbecco's modified Eagle medium with 10% fetal bovine serum for 1 h at 37°C before infection with Ad5.GFP.ΔF/37F. Cell fluorescence from GFP transgene expression was measured by flow cytometry. Error bars indicate standard deviations from three experiments. The error for the penton base experiment was too small to show on the graph.

FIG. 5

Interaction of Ad37 and Ad2 with their cell receptors. (A) The geometry of Ad37 with short, rigid fibers (green) prevents the side of the fiber knob from contacting the appropriate surface of CAR (black). (B) If the side of the fiber knob on Ad37 were to bind CAR, there would be steric hindrance between the viral capsid and the host cell membrane (orange). The clash is indicated by transparent density. (C) Ad37 is shown binding a 50-kDa receptor that is preferentially expressed on conjunctival cells (33). Modeling indicates that concurrent binding of the fiber with the 50-kDa receptor (purple) and of the penton base (yellow) with α_v integrin (red) should be possible. (D) The Ad types with long, bent fibers, such as Ad2 and Ad5, contact CAR with the side of the fiber knob.