Interaction of poliovirus with its purified receptor and conformational alteration in the virion

Abstract

Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 +/- (0.86) x 10(-8) M at 4 degrees C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37 degrees C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.

FIG. 1

Schematic structures of recombinant PVRs expressed in a baculovirus system. The number of amino acids in each region is shown for hPVRα and recombinant PVRs. Possible N-linked glycosylation sites are indicated by triangles. The box represents a transmembrane domain.

FIG. 2

Purity of recombinant PVRs expressed by a baculovirus system. Recombinant PVRs were expressed in a baculovirus system and purified by immunoaffinity column chromatography as described in Materials and Methods. (A) Western blot analysis of purified PVR142, PVR163, and PVR241. (B) Silver staining of purified PVR241 and PVR330. (C) Autoradiography of [³⁵S]methionine-labeled PVR241 and PVR330. (Positions of molecular mass markers are indicated on the left of panels A to C.) PVR241 (D) and PVR330 (E) were analyzed by a gel filtration. BD, Blue Dextran 2000; Fe, ferritin (440 kDa); Ca, catalase (232 kDa); Al, aldolase (158 kDa); Ab, albumin (67 kDa); Ov, ovalbumin (43 kDa), and Ch, chymotrypsinogen A (25 kDa).

FIG. 3

Recognition of purified PVR241 and PVR330 by MAb p286. ³⁵S-labeled PVR241 (A) or PVR330 (B) was incubated with MAb p286 (closed symbols) or without MAb p286 (open symbols) in PBS containing 1% BSA at 4°C for 24 h. The final concentrations of PVRs and MAb p286 were 6.06 × 10⁻⁸ and 5.60 × 10⁻⁷ M, respectively. The mixtures were analyzed by chromatography on Sephacryl S-300 superfine (Pharmacia). The chromatography was carried out in PBS at 4°C. Radioactivities in aliquots of fractions were measured in a liquid scintillation counter.

FIG. 4

Number of PVR330 molecules bound per virion. Poliovirions (approximately 1.33 × 10⁻¹⁴ mol per well) bound to the well by means of MAb 7m012 were reacted with ³⁵S-labeled PVR330 in various concentrations as described in Materials and Methods. Numbers of bound PVR330 molecules were calculated by using the specific activity of the labeled compound.

FIG. 5

K_d value for recombinant PVR330 and its binding site on poliovirions. Poliovirions were fixed to the bottom of the well by means of MAb 7m012. Poliovirion concentrations of 1.31 × 10⁻¹³, 6.67 × 10⁻¹⁴, and 2.60 × 10⁻¹⁴ mol bound per well are indicated by closed circles, open squares, and open circles, respectively. Poliovirions in the well were incubated with various concentrations of PVR330 (67,000 cpm; adjusted to a final concentration of 2.29 × 10⁻⁸ to 21.2 × 10⁻⁸ M by the addition of unlabeled PVR) as described in Materials and Methods. Two independent experiments were performed.

FIG. 6

Effect of concentration of PVR330 on rate of conformational change of poliovirions. [³⁵S]methionine- and [³⁵S]cysteine-labeled poliovirions were incubated with PVR330 at 37°C, and the conformational changes were analyzed at the indicated times by using sucrose density gradient centrifugation as described in Materials and Methods. Concentrations of 7.8 × 10⁸ virions (1.8 × 10⁵ cpm) per μl (A and D) and 3.9 × 10⁸ virions (1.2 × 10⁵ cpm) per μl (B and C) were mixed with 1.92 × 10⁻⁸ M (A), 3.37 × 10⁻⁸ M (B), 10.1 × 10⁻⁸ M (C), and no (D) PVR330. Fractions are numbered from the top to bottom of gradients. Positions of 80S and 160S particles are indicated by arrows.

FIG. 7

Materials in fractions of sucrose density gradients. (A) [³⁵S]methionine- and [³⁵S]cysteine-labeled poliovirus (1.4 × 10⁵ cpm) at a concentration of 1.01 × 10⁸ virions per ml was mixed with 4.13 × 10⁻⁸ M PVR, incubated at 37°C for indicated times, and analyzed under the same conditions described in the legend for Fig. 6. Positions of VP4, 80S particles, and 160S particles are indicated by arrows. (B) Autoradiography of top fractions (fractions 1, 2, and 3) after separation by polyacrylamide gel electrophoresis. In this fraction, radioactivities of VP1, VP2, VP3, and VP4 were 0.99, 1.77, 1.73, and 95.5% of the total radioactivity, respectively. (C) Materials in the top fraction (lane 1) and fraction 11 (lane 2) and 160S intact virion particles (lane 3) were analyzed by Western blot analysis with a mixture (1:1) of rabbit hyperimmune serum against poliovirus type 1 and its capsid protein VP4.

FIG. 8

Gel filtration of mixtures of PVR330 and virion-related particles. PVR330 was incubated at 4°C with 160S intact poliovirions (▪) or their conversion products (135S) (□) and analyzed by gel filtration. ▵, PVR330 alone.

FIG. 9

Neutralizing activity of culture fluids containing PVR330. Culture fluid (134 μl [bar 1], 121 μl [bar 2], or 107 μl [bar 3]) containing PVR330 was mixed with 1.5 × 10⁶ PFU of PV1(M) in PBS containing 1% BSA and incubated at 4°C for 3 h and then at 37°C for 1 h. Plaque assay was performed as described in Materials and Methods. Similar experiments with 134 μl of the culture fluid were carried out after the treatment of the fluid at pH 3.0 in a glycine-HCl buffer at 4°C for 1 (bar 4), 3 (bar 5), and 5 (bar 6) min, neutralization, and dialysis against PBS. Experiments were performed in triplicate, and error bars indicate standard deviations.