The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry

Abstract

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.

FIG. 1

Binding of empty capsids and mature virions to HeLa cells. Various amounts of labeled particles (empty capsids or mature virus) were added to a constant number of HeLa cells in suspension. After incubation, the cells were pelleted and the radioactivity copelleting with the cells was measured. (A) Saturation binding curve. (B) Scatchard plot of the data in panel A. As a rough approximation, for each of the binding curves two lines have been fitted by the linear least-squares method. The solid and broken lines are the best-fit lines for the empty capsid and mature virus, respectively. The heavy lines represent a high-affinity mode of binding, whereas the light lines represent a low-affinity mode of binding.

FIG. 2

Competition for cell surface virus receptors by radioactively labeled particles (empty capsids or mature virions) and unlabeled mature virions. Competition for cell-surface poliovirus receptor was assayed by determining the amount of labeled particle attaching to HeLa cells as the concentration of competing unlabeled virus was increased. The effect of increasing amounts of unlabeled virus is presented by plotting the amount of label copelleting with the cells (normalized to the value obtained with no unlabeled virus added) versus the amount of competing unlabeled virus added.

FIG. 3

V8 protease sensitivity of heated and unheated empty capsids, native virus, and 80S particles. Native virus, 80S particles, unheated empty capsids, and heated empty capsids were treated with V8 protease. The capsid proteins were separated by SDS-polyacrylamide gel electrophoresis. Fragments containing the antigenic sequence of interest were visualized specifically by immunoblotting and staining with the procedures outlined in the text. As controls, particles not treated with V8 protease were analyzed in parallel. (A) Western blot analysis with polyclonal antibodies binding the pep1 region of VP1 (residues 24 to 40). (B) Western blot analysis with polyclonal antibodies binding the pep9 region of VP1 (residues 270 to 287). PV, poliovirus; EC, empty capsid.

FIG. 4

Immunoprecipitation titrations of heated and unheated empty capsids. Titrations were with the following antibodies (Ab). (A) Polyclonal antibodies binding the pep0 region of VP1 (residues 7 to 24). (B) Polyclonal antibodies binding the pep1 region of VP1 (residues 24 to 40). (C) Monoclonal antibody raised against pep1. (D) Polyclonal antibodies binding VP4. In each panel, the percent immunoprecipitated versus the log of the dilution is plotted. Symbols: , empty capsids; ▵, heated empty capsids; ◊, pentamers; , mature virus; ⧫, 135S; ▴, 80S. The dashed line in panel D represents the background level for pentamer immunoprecipitation determined as described in the text. The reactivity of the undiluted anti-pep1 and anti-pep0 polyclonal antibodies toward unheated empty capsids may be due to instability of the empty capsids in serum concentration. The high background in control experiments using pentamers is due to adventitious binding of pentamers to the protein A-Sepharose beads. Mock experiments in which no anti-VP4 antibody was added resulted in precipitation of the pentamers approximately equal to the background level observed in panel D.

FIG. 5

View of the poliovirus protomer. The view is from inside the particle looking out. The N-terminal arm of VP1 (residues 6 to 10 and 20 to 69) and all of VP4 are represented as blue and green ribbons, respectively. Residues 1 to 5 and 11 to 19 are disordered and therefore are not shown. The remaining portions of the protomer are represented by the surfaces, with blue, yellow, and red corresponding to VP1, VP2, and VP3, respectively. The sphere indicates the position of the VP0 scissile bond in the empty capsid. The quasi-threefold axis relating VP1, VP2, and VP3 in the protomer is located near the VP0 scissile bond and is roughly perpendicular to the page.