Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles

Abstract

The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.

FIG. 1.

Formation of receptor-decorated liposomes. Coomassie-stained SDS-PAGE of liposome flotation gradient fractions. Lanes 1 to 10 represent fractions, from top to bottom, of flotation gradients in which liposomes banded at fraction 3. M, molecular weight markers. (A) Flotation after binding of 4 μg of PVR onto 100 μg of liposomes. (B) Flotation after mixing 20 μg of PVR with 100 μg of liposomes: some unbound PVR does not float and remains in fractions 8 to 10, illustrating saturation of binding under these conditions. Images were captured with a gel documentation system (Bio-Rad).

FIG. 2.

Receptor-mediated binding of virus to liposomes. Location of radiolabeled virus in liposome flotation gradients, from top (fraction 1) to bottom (fraction 10), after incubation at room temperature for 5 min with either receptor-decorated (closed circles) or control (open circles) liposomes. Liposomes banded at fraction 2.

FIG. 3.

Receptor-decorated liposomes induce conversion of virus to cell entry-intermediate particles at physiological temperature. Sucrose density gradient analysis of radiolabeled poliovirus after incubation with receptor-decorated (closed circles) or control (open circles) liposomes for 5 min at room temperature (A) or 37°C (B). Arrows indicate the positions of 160S, 135S, and 80S particles after sedimentation through parallel gradients.

FIG. 4.

Temperature dependence of virus conversion by receptor-decorated liposomes Sucrose density gradient analysis of radiolabeled poliovirus after incubation for 32 min with receptor-decorated liposomes at room temperature (closed circles) and 31°C (open squares), 33°C (open triangles), 35°C (crosses), 37°C (open circles), and 39°C (barred crosses).

FIG. 5.

Time course of conversion of virus bound to receptor-decorated liposomes. Sucrose density gradient analysis of radiolabeled virus particles before incubation (T = 0; closed circles) and after 0.5 (open circles), 2 (squares), 8 (crosses), and 32 (triangles) min of incubation at 37°C. Rapid temperature elevation was achieved by a sample dilution of 1:20 into prewarmed buffer; reactions were stopped by freezing in liquid nitrogen.

FIG. 6.

Receptor density controls efficiency of virus conversion. The graph shows the relative proportions of native virus and converted particles after incubation with receptor-decorated liposomes for 40 min at 37°C. The density of receptor on liposomes was varied by altering the percentage of NTA-lipid during preparation of liposomes as indicated. Values represent the sum of counts assigned to peaks in sucrose density gradient profiles representing native or converted particles. RT, room temperature.

FIG. 7.

(A) Converted virus particles remain membrane associated after removal of receptor. The proportion of radiolabeled virus particles (in counts per minute) remaining liposome associated (dark bars) or released (light bars) after temperature-dependent conversion of particles (37°C) and/or removal of receptor by imidazole is shown. Bars represent the sum of counts in top or bottom regions of flotation gradients. Liposome-associated counts (dark bars) in panel 4 represent converted particles anchored to the membrane in the absence of receptor. (B) Converted virus particles are released from liposomes by V8 protease digestion. The proportion of particles (in counts per minute) remaining liposome associated (dark bars) or released (light bars) from liposomes after V8 protease digestion of samples harvested from the flotations shown in panel A is shown. The particles released (light bars) in panels 3 and 4 have been freed from liposomes by protease cleavage of the VP1 membrane anchor. ND, not done, due to the low level of liposome-associated counts in sample 2, as shown in panel A.

FIG. 8.

Virus particles released from liposomes by V8 protease are 135S. Sucrose density gradient profiles showing sedimentation of radiolabeled particles spontaneously released from liposomes at 37°C (closed squares) or released by a combination of incubation at 37°C, followed by V8 protease digestion (open squares), are shown. 160S material in control reactions (remaining profiles, as indicated) represents excess virus not bound to liposomes.

FIG. 9.

VP4 is membrane associated after conversion of virus by receptor-decorated liposomes. SDS-PAGE autoradiograph of the liposome fraction (left) and nonfloating fraction (right) of a flotation gradient following incubation of receptor-decorated liposomes with radiolabeled virus at 37°C for 1 h. Autoradiographs were scanned (Epson 4870).