Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol

We have characterized the entry leading to productive infection of a type C FMDV in two cell lines widely used for virus growth, BHK-21 and IBRS-2. Inhibition of clathrin-mediated endocytosis by sucrose treatment decreased both cell entry and virus multiplication. Evidence of a direct requirement of clathrin for productive viral entry was obtained using BHK21-tTA/anti-CHC cells, which showed a significant reduction of viral entry and infection when the synthesis and functionality of clathrin heavy chain was inhibited (Tet- cells). This was also observed for vesicular stomatitis virus (VSV) productive entry. The effect of NH(4)Cl and concanamycin A on FMDV entry and infection was consistent with the requirement of acidic compartments for decapsidation and virus replication. As expected from its higher stability at acidic pH, this requirement was higher for VSV. Since BHK-21 and IBRS-2 cells expressed caveolin-1, we explored the effect on productive virus entry of drugs that interfere with caveolae-mediated endocytosis. Treatment with nystatin did not reduce entry and infection of FMDV or VSV, while cholesterol depletion with MbetaCD significantly inhibited both steps of the FMDV cycle, indicating that plasma membrane cholesterol is required for virus productive entry.