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.

Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin.

Heparan sulphate and sialoadhesin were previously identified on porcine macrophages as receptors for porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the exact role and cooperation of heparan sulphate and sialoadhesin during PRRSV attachment and internalization was analysed. It was observed that both heparan sulphate and sialoadhesin mediate PRRSV attachment and that only these two receptors are involved in attachment. Analysis of attachment kinetics of PRRSV to macrophages revealed that early attachment is mediated mainly via an interaction with heparan sulphate, followed by a gradual increase in interaction with sialoadhesin. By using wild-type CHO and CHO deficient in heparan sulphate expression, it was shown that heparan sulphate alone is sufficient to mediate PRRSV attachment, but not entry, and that heparan sulphate is not necessary for sialoadhesin to function as a PRRSV internalization receptor, but enhances the interaction of the virus with sialoadhesin.