monensin and chloroquine-diphosphate

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH(4)Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH(4)Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% +/- 110%, 1,212% +/- 34%, and 1,100% +/- 179%, respectively, in porcine kidney (PK-15) cells; by 128% +/- 7%, 158% +/- 3%, and 142% +/- 11% in swine kidney cells; by 160% +/- 28%, 446% +/- 50%, and 162% +/- 56% in swine testicle (ST) cells; and by 313% +/- 25%, 611% +/- 86%, and 352% +/- 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.

Topics: Ammonium Chloride; Animals; Cell Line; Chloroquine; Circovirus; Endosomes; Epithelial Cells; Ionophores; Lysosomes; Monensin; Serine Endopeptidases; Swine; Virus Attachment

Treatment of porcine kidney (PK-15) cells with either interferon-gamma (IFN-gamma) or endosomal- lysosomal system acidification inhibitors increases replication of porcine circovirus type 2 (PCV2). In the present study, the effect of a combination of these treatments on the number of infected cells and virus yield was tested. The number of PCV2 (Stoon-1010)-infected PK-15 cells increased in cells treated with ammonium chloride (445 +/- 39% increase), IFN-gamma (446 +/- 8%), ammonium chloride + IFN-gamma (1721 +/- 283%), chloroquine diphosphate (1037 +/- 121%), chloroquine diphosphate + IFN-gamma (2199 +/- 255%), monensin (950 +/- 178%) and monensin + IFN-gamma (1948 +/- 60%). Combined IFN-gamma and endosomal-lysosomal system acidification inhibitors increased PCV2 yield by up to 50 times compared to untreated PK-15. This augmented virus replication in PK-15 cells may be helpful in the production of PCV2 vaccines.

Topics: Ammonium Chloride; Animals; Cell Line; Chloroquine; Circovirus; Endosomes; Enzyme Inhibitors; Immunologic Factors; Interferon-gamma; Monensin; Swine