Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2008
Publication Date: N/A
Citation: 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 receptor on the cell surface. We have shown that FMDV can utilize a number of cell surface molecules as receptors. One of these receptors is part of a large family of membrane proteins called integrins. A second receptor utilized by the virus is a molecule called heparan sulfate (HS). Field viruses virulent to animals enter the cells utilizing integrins, whereas viruses grown in tissue culture exhibit reduced virulence over time, resulting in the utilization of HS as a receptor. Viral entry after receptor binding has been well described for integrin-binding viruses but entry mechanism for HS binding viruses remained unknown. In this study we have examined the steps immediately after the virus binds to the HS receptor and found that the virus enters into an invagination in the cell membrane called a caveolae. From there, the pit breaks off from the cell membrane and forms a separate structure in the cell called a caveosome. The virus then moves into another structure, called an endosome, and due to the acidic nature in the interior of this vesicle, it breaks open, releasing the viral nucleic acid to begin replication. These findings can now be expanded to examine the role of drugs that inhibit these processes as possible anti-viral agents, and also to perform structural studies which will be useful in antiviral drug design.
Technical Abstract: Foot-and-mouth disease virus (FMDV) utilizes different cell surface macromolecules to facilitate infection of cultured cells. Virus which is virulent for susceptible animals infects cells via four members of the alpha V subclass of cellular integrins. In contrast, tissue culture adaptation of some FMDV serotypes results in the loss of viral virulence in the animal accompanied by the loss of ability to use integrins as receptors. These avirulent viral variants acquire positively charged amino acids on surface-exposed structural proteins resulting in the utilization of cell surface heparan sulfate (HS) molecules as receptors. We have recently shown that FMDV serotypes utilizing integrin receptors enter cells via a clathrin-mediated mechanism into early endosomes. Acidification within the endosome results in a breakdown of the viral capsid releasing the RNA, which enters the cytoplasm by a still undefined mechanism. Since there is evidence that HS internalizes bound ligands via a caveolae-mediated mechanism, it was of interest to analyze the entry of FMDV by cell-surface HS. Using a genetically engineered variant of type O1Campos (O1C3056R) which can utilize both integrins and HS as receptors, and a second variant (O1C3056R-KGE), which can only utilize HS as a receptor, we followed viral entry using confocal microscopy. After binding virus to cells at 4 deg C, followed by shifting the temperature to 37 deg C, type O1C3056R-KGE colocalized with caveolin-1 while O1C3056R colocalized with both clathrin and caveolin-1. Compounds which either disrupt or inhibit the formation of lipid rafts inhibit the replication of O1C3056R-KGE. Furthermore, a caveolin-1 knockdown by RNA interference also considerably reduced the efficiency of O1C3056R-KGE infection. These results indicate that HS-binding FMDV, enters the cells via the caveolae-mediated endocytosis pathway and caveolae can associate and traffic with endosomes. In addition, these results further suggest that the route of FMDV entry into cells is a function solely of the viral receptor.