Interaction of poliovirus with its receptor affords a high level of infectivity to the virion in poliovirus infections mediated by the Fc receptor

Abstract

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcgamma receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.

FIG. 1

Expression of mouse FcRI on LmFcRI cells. LmFcRI cells (A and C) or LpCI-neo cells (B and D) were analyzed by rosette assay (A and B) or indirect immunofluorescence (C and D) to measure the expression of mouse FcRI on LmFcRI cells, as described in Materials and Methods.

FIG. 2

PV1 infection of LmFcRI cells mediated by anti-PV1 MAb. LmFcRI cells or LpCI-neo cells (4.0 × 10⁴ cells) were incubated in the presence of PV1 (9.6 × 10⁹ virions, 1.3 × 10⁻¹⁰ M), anti-PV1 MAb 7m012 (1.3 × 10⁻⁸ M), or both, as described in Materials and Methods. Indirect immunofluorescence was performed at 8 h p.i.

FIG. 3

Purified recombinant PVR-IgG2a. Purified recombinant PVR-IgG2a was analyzed by polyacrylamide gel electrophoresis followed by silver staining (A) or Western blotting (B) to detect the protein, as described in Materials and Methods. Purified recombinant PVR-IgG2a (C) and, as a control, mouse IgG2a (D) were also analyzed by gel filtration. For Western blot analysis (B), PVR-IgG2a was examined under reducing (left lane) and nonreducing (right lane) conditions. Positions of molecular mass markers (in kilodaltons) are indicated by arrows on the left of panels A and B and at the top of panel C. BD, blue dextran 2000; Th, thyroglobulin (669 kDa); Fe, ferritin (440 kDa); Ca, catalase (232 kDa); Al, aldolase (158 kDa); Ab, albumin (67 kDa); Ov, ovalbumin (43 kDa); Ch, chymotrypsinogen A (25 kDa).

FIG. 4

Time course of PV1 infection mediated by anti-PV1 MAbs or PVR-IgG2a. LmFcRI cells (4.0 × 10⁴) were challenged with PV1 (9.6 × 10⁹ virions, 1.3 × 10⁻¹⁰ M) in the presence of anti-PV1 MAb (1.3 × 10⁻⁸ M for 7m012 [open circles] and 7m039 [closed circles] and 1.3 × 10⁻⁹ M for Mah45i [open squares] and Mah49e [closed squares]) or PVR-IgG2a (6.5 × 10⁻⁹ M) (open triangles), and the cells were fixed at the times indicated, followed by indirect immunofluorescence as described in Materials and Methods. The data represent means from three independent experiments, and error bars indicate standard deviations.

FIG. 5

Infectivity of PV1 mediated by the anti-PV1 MAbs or PVR-IgG2a. LmFcRI cells (4.0 × 10⁴) were challenged with PV1 in the presence of anti-PV1 MAbs (7m012 [open circles], 7m039 [closed circles], Mah45i [open squares], and Mah49e [closed squares]) or PVR-IgG2a (open triangles) at the indicated concentrations. The concentrations of PV1 used were 1.3 × 10⁻¹⁰ M (9.6 × 10⁹ virions), 1.3 × 10⁻¹¹ M (9.6 × 10⁸ virions), and 1.3 × 10⁻¹² M (9.6 × 10⁷ virions) (A, B, and C, respectively). At 8 h p.i., indirect immunofluorescence was performed to detect viral antigens. The data represent means from three independent experiments, and error bars indicate standard deviations.

FIG. 6

PV1 binding to protein G or cells mediated by anti-PV1 MAbs or PVR-IgG2a. [³⁵S]methionine-labeled PV1 and the anti-PV1 MAbs or PVR-IgG2a were incubated with a protein G gel (A) or cells (LmFcRI cells [open bars] or LpCI-neo cells [closed bars]) (B), and radioactivities associated with the protein G gel or cells were measured as described in Materials and Methods. Symbols in panel A are the same as in Fig. 5. The data represent means from three independent experiments, and error bars indicate standard deviations.

FIG. 7

Virion conformational alteration induced by anti-PV1 MAbs or PVR-IgG2a. (A) The viral conformational change was examined at the optimal concentration of MAbs or PVR-IgG2a for mediation of PV1 infection, as shown in Fig. 5A: 1.3 × 10⁻⁸ M for 7m012 and 7m039, 1.3 × 10⁻⁹ M for Mah45i and Mah49e, and 6.5 × 10⁻⁹ M for PVR-IgG2a. Arrows indicate positions of intact particles (160S), altered particles (80S and 135S), and released VP4 (top fraction). (B) Each of the peak fractions in panel A was incubated in the presence of 1% SDS at room temperature for 20 min and then subjected to sucrose density gradient centrifugation.

FIG. 8

Direct infection of LmFcRI cells with 135S particles. PV1 (9.6 × 10⁹ virions, 1.3 × 10⁻¹⁰ M) was preincubated with or without PVR-IgG2a (1.3 × 10⁻⁸ M), and the infectivities for LmFcRI cells were examined in the presence of 1.3 × 10⁻⁸ M PVR-IgG2a. The data represent means from two independent experiments, and error bars indicate standard deviations.