Conformational changes in the capsid of a calicivirus upon interaction with its functional receptor

Abstract

Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37 degrees C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-A structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37 degrees C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

FIG. 1.

Neutralization of FCV-5 by sfJAM-A. FCV-5 (∼1 × 10⁵ PFU) was incubated with various concentrations of soluble GST-fJAM-A (A) or cleaved fJAM-A at 37°C for 30 min (B). The virus samples were then plaque titrated on CRFK cell monolayers. Each data point represents the average of two replicates from a single representative experiment.

FIG. 2.

X-ray structure of FCV (FCV-5) capsid. (A) Two sample regions in the calculated electron density map with modeled amino acid residues 197 to 207 (S domain) and 586 to 596 (P domain), respectively. (B) X-ray structure of FCV-5 viewed along the icosahedral 2-fold axis. Location of a set of A/B and C/C dimers and icosahedral 5-fold and 3-fold axes are shown. (C) Ribbon representation of the B subunit structure. The NTA (green), S domain (blue), and P1 (red) and P2 (yellow) subdomains are indicated.

FIG. 3.

Ribbon representation of the P2 subdomain in FCV-5 (A) and SMSV-4 (B). The β-strands are labeled from A′ to F′ in each case. (C) The superposition of the P domain of FCV-5 (red) and SMSV-4 (blue) shows that the six β-strands in the P2 subdomains are similarly spatially arranged into a compact barrel, despite the low sequence similarity. Arrows indicate the loops connecting β-strands C and D, and E and F, of the FCV-5 P2 subdomain. (D) The FCV-5 A/B dimer (top) as viewed along the line joining the icosahedral 3- and 5-fold axes and the C/C dimer (bottom) as viewed along the line joining the adjacent icosahedral 3-fold axes demonstrate the bent and flat conformations, respectively, assumed by the subunits. In both cases, the dimeric 2-fold axis is vertical. (E to G) Positions of antigenic sites and conserved residues on the P2 subdomain of the C/C dimer of FCV-5. (E) Neutralizing epitopes mapped on P2 (gray). Antigenic sites (Ags1 to -4; colored pink) represent B-cell linear epitopes identified by Radford et al. (43). MAb escape mutations identified by Tohya et al. are colored red (58). (F) Degree of conservation of surface-exposed residues on the P2 subdomain as predicted by Consurf 3.0. The solid yellow line indicates dimer interface; the dashed yellow line indicates the axis of conservation. (G) Locations of N-terminal and C-terminal HVRs, region C and central conserved portion of region E mapped on surface of P2 subdomain.

FIG. 4.

Positions of mutations found in srr mutants in the structure of FCV-5. (A) The residues mutated in the srr mutants are illustrated on a schematic of the FCV capsid protein. The locations of the different structural domains in the primary capsid sequence are indicated. (B) Locations of the mutations in the FCV-5 atomic resolution structure.

FIG. 5.

Neutralization of srr mutants by sfJAM-A. A subpanel of eight srr mutants and FCV-5 (1 × 10⁵ PFU) was incubated in the presence or absence of soluble GST-fJAM-A (55 μM) at 37°C for 30 min. The infectivity of each sample was assayed by plaque titration. The change in log titer was calculated by subtracting the titer of samples incubated with receptor from that of samples incubated without receptor. The mean change in log₁₀ titer ± standard deviation (SD) of three replicates of a representative experiment is shown.

FIG. 6.

Binding of srr mutants to CHO cells expressing fJAM-A. (A) Nonpermissive CHO-S cells were transfected with a DNA construct encoding fJAM-A. At 24 h posttransfection, FCV (MOI = 5) was adsorbed to the cells on ice for 30 min. After being washed with cold PBS to remove unbound virus, the cells were fixed and bound virus and cell surface fJAM-A were detected with mouse anti-FCV MAb and rabbit anti-fJAM-A antibodies, followed by Alexa 488-conjugated goat anti-mouse IgG and Alexa 647-conjugated goat anti-rabbit IgG. Virus binding and receptor expression were analyzed by flow cytometry. Flow cytometry dot plots for four representative samples are shown. (B) Virus binding was assayed by determining the percentage of fJAM-A-positive cells that bound virus. The means of the results for three independent experiments (two samples of 1 × 10⁴ cells per experiment) ± SE are shown.

FIG. 7.

Growth of srr mutants. CRFK monolayers were infected with FCV-5 and the indicated srr mutants under single-cycle (MOI = 5) (A) or multiple-cycle (MOI = 0.01) (B) conditions. For clarity, the data from only four mutants have been included in each graph. The change in virus titer was determined by plaque assay. The mean log₁₀ titer (log₁₀ titer at each time point − log₁₀ titer at T = 0) for each time point is shown. The error bars shown at 4 and 8 h pi for the multiple-cycle growth represent the standard deviation of four replicates; for other data points, error bars have been omitted from the figure for clarity. The asterisks indicate significant differences between viral isolates as determined by ANOVA at these time points.

FIG. 8.

Binding of srr mutants to immobilized fJAM-A in the absence (A) or presence (B) of increasing concentrations of sfJAM-A. ELISA plates were coated with 100 ng of sfJAM-A ectodomain. Various concentrations of purified FCV-5 and G329D, V516I, and K572E srr mutants were incubated with the immobilized protein for 3 h on ice. To investigate the effect of sfJAM-A on the binding of virus to immobilized receptor, 750 ng of each virus and various concentrations of soluble receptor were incubated together with plate-bound fJAM-A for 3 h on ice. Bound FCV was detected with rabbit anti-FCV serum, followed by HRP-conjugated goat anti-rabbit IgG. Colorimetric HRP substrate was added, and the amount of bound FCV-5 was quantified by absorbance at 595 nm. The means of three and four plates, respectively (two replicates per plate), ± SE are shown. ANOVA was performed on three concentrations of soluble receptor (0.04, 0.33, and 2.7 μM) to determine statistical differences; significant differences are indicated by asterisks.

FIG. 9.

Changes in bis-ANS binding upon mixing of FCV with sfJAM-A. At time zero, bis-ANS was added to the indicated purified virions, soluble JAM-A (human [A] or feline [B and C]), or virions preincubated with JAM-A. Fluorescence was recorded every 2 s continuously for 10 min at excitation and emission wavelengths of 395 and 495 nm, respectively. (D to G) Stabilized fluorescence intensities measured during the last minute for each sample were averaged. Broken lines indicate the additive average fluorescence intensity measured for both native virus and receptor. The means ± SE (n = 3) are shown.

FIG. 10.

Ability of sfJAM-A to neutralize select FCV isolates. A panel of six FCV field isolates (FCV-5, Kaos, Deuce, FCV-127, FCV-131, and FCV-796) and two tissue culture-adapted strains (F9 and Urbana) (1 × 10⁵ PFU) was incubated in the presence or absence of soluble GST-fJAM-A (45 μM) at 37°C for 30 min. The remaining infectivity in each sample was assayed by plaque titration. The change in log titer was calculated by subtracting the titer of samples incubated with receptor from the titer of samples incubated without receptor. The mean change in log₁₀ titer ± SD (n = 4) is shown.