Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31

Abstract

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.

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

“…For PCV2 attachment and internalization studies, PCV2 VLP were used as previously described (44). PCV2 VLP were purified in a cesium chloride gradient from Spodoptera frugiperda 9 (Sf9) insect cells infected with recombinant baculoviruses harboring PCV2 ORF2, the gene encoding the PCV2 capsid.…”

“…PCV2 VLP were purified in a cesium chloride gradient from Spodoptera frugiperda 9 (Sf9) insect cells infected with recombinant baculoviruses harboring PCV2 ORF2, the gene encoding the PCV2 capsid. PCV2 VLP used in this study have been characterized using PCV2-specific monoclonal antibodies and porcine polyclonal antibodies from PCV2-infected pigs and by fluorescence and electron microscopy (44).…”

“…Analysis of PCV2 VLP binding to 3D4/31 cells using fluorescence confocal microscopy has been used before (44). To investigate the effect of GAG on binding of PCV2 VLP to 3D4/31 cells, PCV2 VLP were incubated with 2,500 g/ml of heparin, CS-A, or CS-B at 37°C for 90 min.…”

“…Besides, a variety of cell types including hepatocytes, enterocytes, renal and alveolar epithelial cells, vascular endothelial cells, pancreatic acinar and ductular cells, lymphocytes, smooth muscle cells, and fibroblasts have also been shown to contain PCV2 antigens and/or nucleic acid (12). The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells (44). PCV2 has been shown to bind to all 3D4/31 cells and to internalize via clathrin-mediated endocytosis in a very low proportion of cells (44).…”

“…The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells (44). PCV2 has been shown to bind to all 3D4/31 cells and to internalize via clathrin-mediated endocytosis in a very low proportion of cells (44).…”