The crystal structure of a coxsackievirus B3-RD variant and a refined 9-angstrom cryo-electron microscopy reconstruction of the virus complexed with decay-accelerating factor (DAF) provide a new footprint of DAF on the virus surface

Abstract

The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.

Fig 1

(A) Ribbon diagram of the CVB3-RD crystal structure, with the VP1 to VP4 proteins in blue, green, red, and yellow, respectively, and the pocket factor surface rendered in orange. The RMSD of the CVB3-RD structure relative to that reported under PDB accession number 1COV is <1 Å for all four structural proteins (0.36, 0.39, 0.32, and 0.41 Å for VP1 to VP4, respectively). The two most variable regions of the puff and the knob had RMSDs of 0.36 and 0.27, respectively. This near equality upon superimposition quantifies that the two structures are nearly identical. (B and C) Representative region of the electron density map with the structure of CVB3-RD rendered at 1.0 σ to show the quality of the map (B) and a close-up with CVB3-M (PDB accession number 1COV) (green backbone) superimposed on the structure reported under PDB accession number 4GB3 (gold backbone) to show the sites of two differences in sequence between CVB3-RD and CVB3-M (C).

Fig 2

Cryo-EM structure of the complex of CVB3 strain RD with DAF SCR1 to SCR4. (A) Radially colored, surface-rendered cryo-EM reconstruction of the map density, displayed at a threshold value of 0.6. The strong DAF density lies over the virus surface as a bent cylinder crossing in the northeast-to-southwest direction and bridging the canyon. (B) View of a central section of the map, perpendicular to the icosahedral 2-fold axis. Arrows point to 2-, 3-, and 5-fold axes of icosahedral symmetry. (C) Plot of the three-dimensional reconstruction for calculation at an FSC value of 0.5 in the 9.0-Å-resolution map. (D) Radial density graph describing the spherical average distribution of the density, in which yellow corresponds to the RNA core, green corresponds to the virus capsid, and blue corresponds to the bound DAF density.

Fig 3

Determination of the handedness of the cryo-EM reconstruction. (A) An 8-Å-resolution map calculated from the crystal structure (white) was used to measure the correlation (CC) of both possible hands of the cryo-EM maps (42). The calculated map is placed within the new 9-Å cryo-EM map, depicted in blue mesh, at 1 with the handedness previously reported (18), which gave a CC of 0.14. The X-ray-calculated map placed into the cryo-EM reconstruction of the alternate hand is shown in green mesh at 1, producing a CC of 0.6. Not only does the poor correlation of the blue map indicate the wrong hand assignment, the map calculated from the crystal structure also protrudes significantly from the blue density. In contrast, the correct-handed green map fits better with the calculated map of CVB3-RD. (B) Close-up view of the 5-fold vertex showing the disagreement and agreement in directionality for the incorrect hand and the correct hand, in blue and green, respectively.

Fig 4

(A) Refined fit of DAF (yellow ribbon) into the difference density, displayed at 1.0 σ. (B) Capsid-DAF interaction rendered to show capsid surface topology. Five protomers of the virus capsid were colored by radius from the center of the virus, with the key shown at the right. DAF is shown as a highlighted yellow ribbon that binds to a depression in the virus capsid via SCR2 and crosses the virus canyon to interact beneath the puff via SCR3. Residues known to be necessary for DAF binding, residues 2138 and 3234, are highlighted in magenta (41). The C_α-to-C_α distance between residues 2138 and 3234 is 17.6 Å. (C) Each DAF bridges two protomers of the virus capsid, delineated in blue and green. The residues that define the region of DAF that binds convertases are highlighted in red on the surface-rendered DAF molecule (yellow).

Fig 5

(A) Two protomers of CVB3-RD, E7, and E12 with one molecule of DAF bound are surface rendered to compare the DAF contact residues of three closely related DAF-binding viruses. (B) For each virus, the DAF footprint is shown in yellow, orange, and red, colored according to the interaction with SCR2, SCR3, or SCR4 (the key is at the left). (C) The DAF molecule rotated 180° relative to the virus surface in an “open book” configuration to show virus-binding residues, colored according to the viral protein with which they interact: blue, VP1; green, VP2; red, VP3. In CVB3 and E7, DAF interacts with two adjacent protomers. Thus, protomers are differentiated by dark shades of blue, green, and red and light tints of the same colors.

Fig 6

The virus surface represented as a quilt of amino acids, shown as a projection, with the icosahedral asymmetric unit indicated by the triangular boundary. Virus residues representing two adjacent protomers are shown in light blue and green, with DAF contacts shown in yellow, orange, and red, corresponding to the SCR (SCR2, -3, and -4, respectively) that is predicted to interact with the virus surface. Rather than showing sequence-equivalent residues plotted previously (18, 45), this figure shows the CVB3-RD–DAF contacts on the CVB3-RD virus roadmap, the E7-DAF contacts on the E7 roadmap (PDB accession number 2X5I) (45), and the E12-DAF contacts on the E11 roadmap (accession number 1H8T) (43, 57).

Fig 6

The virus surface represented as a quilt of amino acids, shown as a projection, with the icosahedral asymmetric unit indicated by the triangular boundary. Virus residues representing two adjacent protomers are shown in light blue and green, with DAF contacts shown in yellow, orange, and red, corresponding to the SCR (SCR2, -3, and -4, respectively) that is predicted to interact with the virus surface. Rather than showing sequence-equivalent residues plotted previously (18, 45), this figure shows the CVB3-RD–DAF contacts on the CVB3-RD virus roadmap, the E7-DAF contacts on the E7 roadmap (PDB accession number 2X5I) (45), and the E12-DAF contacts on the E11 roadmap (accession number 1H8T) (43, 57).

Fig 7

CVB3-RD and echovirus sequence-equivalent residues from a clustaw alignment shown to compare DAF interactions (43, 45). The puff region of the virus consists of VP2 residues 129 to 180, outlined by a gray box. The four residues defined in the DAF footprint that are common to DAF binding in all three viruses are boxed. The two residues essential for DAF binding to CVB3-RD are circled (41). The domains of DAF SCR1 to SCR4 were assigned to residues 1 to 62, 63 to 127, 128 to 189, and 190 to 253, respectively, from data reported under Uniprot accession number P08174-2 (DAF 1) (PDB accession number 1OJV) (32). There are no DAF residues in common that interact with all three viruses.