Analysis of Filovirus Entry into Vero E6 Cells, Using Inhibitors of Endocytosis, Endosomal Acidification, Structural Integrity, and Cathepsin (B and L) Activity

Ebola and Marburg viruses are believed to enter host cells by receptor-mediated endocytosis. The process has been studied through the use of inhibitors that affect host cell properties and recombinant pseudotyping systems in which filovirus structural glycoproteins mediate entry of foreign virus particles. The aim of the present study was to determine the effects of such treatments on the entry of wild-type filoviruses. Vero E6 cells were exposed to various inhibitors before, during, and after infection with filoviruses. Infected cultures were harvested early (18-24 h) and late (72 h) after infection, and effects of treatment on entry were measured by fluorescent antibody staining of cells or by antigen capture immunoassays, respectively. These prelimary results suggest that filoviruses enter host cells through receptor-mediated endocytosis via clathrin-coated pits and caveolae, that actin filaments and microtubules are important in the entry process, and that proteolytic digestion of glycoprotein 1 by endosomal proteases facilitates entry. These observations obtained using wild-type viruses confirm the results of studies utilizing recombinant systems and offer additional insights into filovirus entry.