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 receptordecorated 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.

Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

“…Picornaviruses such as poliovirus also release a myristoylated peptide, VP4, during cell entry, and there is some evidence that VP4, like 1N, is the critical agent of membrane perforation (54). The structural parallels are also suggestive.…”

Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus–receptor–membrane complex at 30 Å resolution

“…Liposomes were prepared using a mixture of phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, cholesterol and phosphatidic acid in a molar ratio of 1:1:1.5:0.3 with 10% DOGS-NTA and 1% rhodamine-PE (Tuthill et al, 2006). The lipids were dried, re-hydrated and extruded through 100 nm pores to produce unilaminar vesicles 100-150 nm in diameter.…”

“…In this model membrane system, liposomes containing low levels of lipids with NTA head-groups are used to capture C-terminally His-tagged ectodomains of Pvr. These receptor-decorated liposomes have been shown to bind virus, to induce the transition to form 135S particles, and to result in the insertion of VP4 and the Nterminus of VP1 into membranes (Tuthill et al, 2006). Structural studies of the virus in complex with a membrane-anchored receptor (Bubeck et al, 2005b) demonstrate that poliovirus approaches the membrane along its five-fold axis and binds five copies of Pvr.…”

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism

“…The acidic environment of the endosomes triggers the uncoating of some picornaviruses, including minor group rhinoviruses (15,16). In contrast, major group rhinoviruses, which use receptors from the Ig superfamily, are stable at low pH and their genome release requires receptor binding (17,18). Regardless of the trigger mechanism, enterovirus genome release is preceded by the formation of an uncoating intermediate called the altered (A) particle that has an expanded capsid with pores, N termini of VP1 subunits exposed at the Significance Here, we present a structural analysis of the genome delivery of slow bee paralysis virus (SBPV) that can cause lethal infections of honeybees and bumblebees.…”