Analysis of Foot-and-Mouth Disease Virus Internalization Events in Cultured Cells

Abstract

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the ␣ V subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the ␤G-␤H loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the ␣ V ␤ 3 and ␣ V ␤ 6 integrins, virus adsorbed to the cells at 4°C appears to colocalize almost exclusively with the ␣ V ␤ 6 integrin. Upon shifting the infected cells to 37°C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed livestock caused by Foot-and-mouth disease virus (FMDV), the type species of the genus Aphthovirus of the family Picornaviridae. The virus is a positive-strand RNA virus containing a genome of approximately 8,400 nucleotides (reference 33 and references therein).FMDV initiates infection in cultured cells by binding to any of four members of the ␣ V subgroup of the integrin family of cellular receptors (␣ V ␤ 1 , ␣ V ␤ 3 , ␣ V ␤ 6 , and ␣ V ␤ 8 ) (16,27,36,39,40,60) via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the ␤G-␤H loop of VP1 (6, 30, 46, 52). We have recently shown that the ␣ V ␤ 6 integrin acts as a high-affinity receptor for the virus while ␣ V ␤ 3 interacts with virus with a much lower affinity (28). In addition, viruses of the type A serotype utilize both the ␣ V ␤ 3 and ␣ V ␤ 6 integrins as receptors in cultured cells, while serotype O...

The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1

“…At pH slightly below neutrality, FMDV capsid disassembles into pentameric subunits (9) and there is good correlation between loss of infectivity by in vitro exposure to acidic pH and capsid dissociation (10)(11)(12). The acid sensitivity of FMDV has been related to the mechanism of virus penetration in host cells (13)(14)(15); the current model supports the hypothesis that the acidification of endosomes, where FMDV virions are sorted, triggers viral uncoating and genome release (16)(17)(18). Consistently, blockage of endosome acidification using the lysosomotropic weak base ammonium chloride (NH 4 Cl), which acts as a proton sink within the endosomes (19,20), inhibited FMDV infection by impairing virus uncoating without affecting virus binding or entry (10,13,21).…”

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

“…The surfaceexposed FMDV capsid proteins (all but VP4, which is entirely internal) play a key role in the antigenic properties of the virus (e.g., defining the serotype). In addition, the capsid surface has to interact with various cell surface receptor molecules, such as integrins and heparan sulfate glycosaminoglycans (for some tissue culture-adapted variants) (3), prior to cell entry by clathrin-or caveola-mediated endocytosis (4)(5)(6)(7)(8). Following virus entry, the acidic pH within endosomes triggers capsid disassembly, allowing release of the viral RNA genome into the cytoplasm of the cells where replication occurs.…”

Entry of Feline Calicivirus Is Dependent on Clathrin-Mediated Endocytosis and Acidification in Endosomes

“…Many nonenveloped viruses have been shown to use the clathrin-mediated endocytic route to infect cells; examples include canine parvovirus (41,59), adenovirus (60), and picornaviruses including rhinovirus (15,22), foot-and-mouth disease virus (7,39), and coxsackievirus B3 (11). Recently, two viruses, simian virus 40 and parechovirus 1, have been shown to use caveolae in order to infect cells (3,27), while human echovirus 11 and coxsackievirus B4 have been shown to enter cells in a cholesterol-dependent manner suggesting entry via lipid rafts (51,54).…”