Downloaded from www.microbiologyresearch.org by IP: 54.161.171.221 On: Tue, 06 Sep 2016 19:41:23 Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31 G. Misinzo, 1 P. Meerts, 1 M. Bublot, 2 J. Mast, 3 H. M. Weingartl 4 and H. J. Nauwynck 1 Correspondence H. J. Nauwynck hans.nauwynck@Ugent.be 1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2 Merial, Biological Research, Lyon, France 3 Veterinary and Agrochemical Research Centre, Brussels, Belgium 4 Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada Received 29 September 2004 Accepted 24 March 2005 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. INTRODUCTION Porcine circoviruses (PCVs) are small, non-enveloped viruses with a single-stranded, closed-circular DNA (Todd et al., 2000), classified in the genus Circovirus (Pringle, 1999) of the family Circoviridae. The first porcine circovirus (PCV1) was detected as a contaminant of the continuous porcine kidney (PK-15) cell line (Tischer et al., 1974) and was found to be non-pathogenic for pigs (Tischer et al., 1986). In 1997, an agent morphologically similar to but genetically and antigenically distinct from PCV1 was iso- lated from pigs with post-weaning multisystemic wasting syndrome (PMWS) (Ellis et al., 1998; Morozov et al., 1998). This virus was designated PCV2. The PCV2 genome con- tains two major open reading frames (ORFs). ORF1 encodes the replicase protein (Rep) involved in rolling-circle PCV2 DNA replication, whilst ORF2 encodes the PCV2 capsid protein (Hamel et al., 1998; Mankertz et al., 1998; Nawagitgul et al., 2000). The PCV2 virion has an icosahedral T=1 structure containing 60 capsid protein molecules arranged in 12 pentamer-clustered units (Crowther et al., 2003). PCV2 has been associated with different diseases and syndromes, two of which have been reproduced experimentally: PMWS (Allan et al., 1999) and reproductive problems (Sanchez et al., 2001). Recently, PCV2 target cells have been shown to be cardio- myocytes, hepatocytes and macrophages (mw) during fetal life and mainly monocytes/mw in early post-natal life (Sanchez et al., 2003). In experimentally inoculated pigs, the 0008-0652 G 2005 SGM Printed in Great Britain 2057 Journal of General Virology (2005), 86, 2057–2068 DOI 10.1099/vir.0.80652-0