Interaction of decay-accelerating factor with coxsackievirus B3

Abstract

Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor (CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of echoviruses, suggesting independent evolutionary processes.

FIG. 1.

Diagrammatic view of a picornavirus (top left). A thick black line outlines a protomer, an assembly intermediate. Blue, green, and red correspond to VP1, VP2, and VP3, respectively. One icosahedral asymmetric unit is enlarged (center) to show the location of the canyon and the entrance (white disc) to the hydrophobic binding pocket (white dashed lines) containing the lipid pocket factor. On the right is shown a side view of the pocket, showing the entrance to the pocket from the canyon.

FIG. 2.

Surface-rendered cryoEM reconstruction of CVB3-RD complexed with full-length DAF molecules. Density further than a 160-Å radius from the center of the virus is shown in gray. An asymmetric unit is outlined in black. Density corresponding to a full-length DAF molecule with a His tag lies across the asymmetric unit stretching from a threefold axes of symmetry, across and partially blocking the canyon, to the neighboring protomer, rising towards a fivefold vertex. The figure was produced using the program DINO (DINO: Visualizing Structural Biology [2002], http://www.dino3d.org).

FIG. 3.

DAF fitted into the difference density map. The SCR domains are labeled, and the C_α backbone of DAF is shown in red. The figure was produced using the program O (17).

FIG. 4.

The surface of CVB3 around a fivefold vertex, with VP1, VP,2, and VP3 in blue, green, and red, respectively. One DAF and one CAR receptor are shown as C_α backbones in yellow and cyan, respectively, where they would be bound to the viral surface. The DAF contact area is shown in white in each of the five protomers. CAR is seen to clash with SCR3 of DAF if both receptors were to bind at the same time to the same icosahedral asymmetric unit of the virus. The stereo figure was produced using Chimera (34, 46).

FIG. 5.

Echovirus surface residues predicted to interact with DAF, given as equivalent CVB3 residues based on multiple-sequence alignment (CLUSTALW) (57). Virus contact residues are color coded to identify specific DAF SCR interactions, with SCR2 shown in orange, SCR3 in purple, and SCR4 in green. Residues marked with ′ are located in symmetry-related proteins. EV7 and DAF contacts are according to fit (a) in Table 2 in reference . EV12 and DAF contacts are from Fig. 6D and E in reference .

FIG. 6.

The viral surface is shown as a stereographic projection where the polar angles θ and ϕ represent latitude and longitude, respectively (65). The virus surface is represented as a quilt of amino acids (44). The icosahedral asymmetric unit of the virus is indicated by the triangular boundary. The footprint of DAF for echoviruses was plotted using the equivalent CVB3 residues based on multiple-sequence alignment (CLUSTALW) (57). The three viruses have sequence identities of 61% for VP1, 70% for VP2, and 67% for VP3. DAF contact residues for EV7 (14) and EV12 (4) are green and blue, respectively. The overlap between DAF footprints for the two echoviruses is shown in cyan. The DAF and CAR (14) footprints on CVB3 are outlined in black and red, respectively.

FIG. 7.

A surface rendering of the full-length fitted DAF structure, in stereo. (Left) The CVB3 footprint onto DAF (blue) compared to the EV12 footprint onto DAF (green) shows no overlap. (Right) The DAF area predicted to interact with convertases (from reference 20) (magenta) shows no overlap with the echovirus footprint on DAF and a slight overlap of five residues with the CVB3-RD footprint.

FIG. 8.

Interaction of wild-type CVB3 virus (light blue) or mutant CVB3-RD virus (green) with HeLa cells (dark blue) or RD cells (red). HeLa cells express both DAF (yellow) and CAR (lavender) receptors, whereas the RD cells express DAF and possibly an unidentified receptor (blue). The wild-type virus normally contains a pocket factor (pink), which can be displaced by binding of a receptor into the canyon, shown as a U-shaped depression at the top of the virus.