Porcine circovirus 2 infection of epithelial cells is clathrin-, caveolae- and dynamin-independent, actin and Rho-GTPase-mediated, and enhanced by cholesterol depletion

Epithelial cells are the major in vivo target cells for porcine circovirus type 2 (PCV2). Although these cells are used for most studies of PCV2 gene expression and, little is known on PCV2 entry, attachment and internalization, in epithelial cells. PCV2 attachment to epithelial cells occurred rapidly and in a time-dependent manner. In contrast to attachment, internalization was slow. Immunofluorescent stainings revealed that during internalization, PCV2 co-localized with clathrin, but not caveolin. Blocking clathrin-mediated endocytosis increased instead of decreased the number of PCV2-infected cells by threefold, suggesting that it does not represent the main internalization pathway leading to a full replication. Further analysis with different inhibitors revealed that also macropinocytosis, dynamin-dependent internalization and membrane cholesterol play no role in PCV2 entry that leads to infection. Inhibition of small GTPases with Clostridium difficile toxin B reduced the number of PCV2-infected PK-15, SK and STs to 63 ± 25%, 47 ± 21% and 14 ± 6%, respectively. Finally, inhibiting actin polymerization also blocked PCV2 infection, showing the need for actin during PCV2 infection. Together, these data indicate that a dynamin- and cholesterol-independent, but actin- and small GTPase-dependent pathway, allows PCV2 internalization in epithelial cells that leads to infection and that clathrin-mediated PCV2 internalization in epithelial cells is not followed by a full replication.