Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic
wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (
clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2
infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47+/-5.0 % of cells. Internalization was continuous, with 70.5+/-9.7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with
clathrin. PCV2
infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including
cytochalasin D (75.5+/-7.0 % reduction) and
latrunculin B (71.0+/-3.0 % reduction), and (ii)
clathrin-mediated endocytosis, including
potassium depletion combined with hypotonic shock (50.2+/-6.3 % reduction), hypertonic medium (56.4+/-5.7 % reduction), cytosol acidification (59.1+/-7.1 % reduction) and
amantadine (52.6+/-6.7 % reduction). Inhibiting macropinocytosis with
amiloride and caveolae-dependent endocytosis with
nystatin did not decrease PCV2
infection significantly. PCV2
infection was reduced by the lysosomotropic weak bases
ammonium chloride (47.0+/-7.9 % reduction) and
chloroquine diphosphate (49.0+/-5.6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via
clathrin-mediated endocytosis and requires an acidic environment for
infection.