|O'Donnell, Vivian - UNIV. OF CONNECTICUT|
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 7, 2005
Publication Date: July 1, 2005
Citation: O'Donnell, V.K., Larocco, M.A., Duque, H., Baxt, B. 2005. Analysis of foot-and-mouth disease virus internalization events in cultured cells. Journal of Virology 79: 8506-8518. 2005 Interpretive Summary: Foot-and-mouth disease is a highly contagious foreign animal disease of livestock, which results in large economic losses to countries where it occurs. As part of an overall FMD control strategy we are examining host and viral targets which may be useful in the development of antiviral compounds to be used in conjunction with vaccination. The virus initiates infection in cells by first binding to a specific molecule on the cell surface, called a receptor. We have shown that the specific receptor used by FMDV is part of a large family of membrane proteins called integrins. After binding to the integrin the virus enters the cell in a series of coordinated steps that leads to the beginning of the viral replication cycle in the cell. Interference with both virus-receptor interaction and viral entry into the cell are a number of antiviral strategies we are investigating. To this end we have examined the steps which occur immediately after the virus binds to the cell. We have utilized specialized microscopy instruments to follow the virus from the cell surface into the cell interior. We have found that the virus enters into an invagination in the cell membrane called a coated pit. From there, the pit breaks off from the cell membrane and forms a separate structure in the cell called a coated vesicle. The virus then breaks apart, because the interior of the vesicle is acidic, and the viral nucleic acid is released from the vesicle to begin replication. We have also analyzed the cell proteins involved in these processes. These findings now be expanded to examine the role of drugs which inhibit these processes as possible anti-viral agents, and also to perform structural studies which will be useful in antiviral drug design.
Technical Abstract: It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the alphaV subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the betaG-betaH (G-H) loop of VP1. While there have been extensive studies on virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell are 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 cells expressing both the alphaVbeta3 and alphaVbeta6 integrins, virus adsorbed to the cells at 4C appears to colocalize almost exclusively with the alphaVbeta6 integrin, while in cells expressing the alphaVbeta3 integrin, only the type A virus can be observed colocalizing with the receptor. Upon shifting the infected cells to 37C, 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 caveolae-mediated endocytosis. Virus remained associated with the integrin until about 1 hr 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 were 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.