Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells
Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells
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DOI：
10.1128/jvi.01229-07
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发表时间：
2008-02-01
影响因子：
5.4
通讯作者：
Nauwynck, Hans J.
中科院分区：
文献类型：
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作者：
Misinzo, Gerald;Delputte, Peter L.;Nauwynck, Hans J.
最近，Misinzo等人（G. Misinzo，P. Meidou，M. Bublot，J. Mast，H. M. Weingartl和H. J. Nauwynck，J. Gen. Virol. 86：2057-2068，2005）报道了抑制内体-溶酶体系统酸化减少了猪圆环病毒2（PCV 2）对单核细胞3D 4/31细胞的感染。本研究检查了抑制上皮细胞中内体溶酶体系统酸化的效果，因为上皮细胞在体内支持PCV 2感染并用于体外培养PCV 2。氯化铵（NH 4Cl），氯喹二磷酸盐（CQ），莫能菌素被用来抑制内体-溶酶体系统酸化。NH 4Cl、CQ或莫能菌素增加PCV 2（Stoon-1010）感染在猪肾（PK-15）细胞中分别降低726% +/-110%、1，212%+/-34%和1，100%+/-179%;在猪肾细胞中分别降低128% +/-7%、158% +/-3%和142% +/- 11%;在猪睾丸（ST）细胞中分别为160% +/-28%、446% +/-50%和162% +/- 56%;在原代肾上皮细胞中分别为313% +/-25%、611% +/-86%和352% +/- 44%。类似地，在用内体-溶酶体系统酸化抑制剂处理的PK-15细胞中，观察到其他六种PCV 2菌株的PCV 2感染增加。使用CQ在PK-15细胞中进一步研究了PCV 2感染增加背后的机制。PK-15细胞的PCV 2感染仅当在PCV 2感染期间早期添加CQ时增加。CQ不影响PCV 2病毒样颗粒（VLP）与PK-15细胞的附着，但增加了内化PCV 2 VLP的分解。在未处理的PK-15细胞中，内化的PCV 2 VLP定位在内体-溶酶体系统内。未处理的3D 4/31和PK-15细胞以及CQ处理的PK-15细胞的PCV 2感染被丝氨酸蛋白酶抑制剂[4-（2-氨基乙基）苯亚磺酰氟盐酸盐]阻断，但不被乙酰基蛋白酶（胃酶抑素A）、半胱氨酸蛋白酶（E-64）和金属蛋白酶（磷酰胺）抑制剂阻断。这些结果表明，丝氨酸蛋白酶介导的PCV 2解体增强猪上皮细胞，但抑制单核细胞后，抑制内体-溶酶体系统酸化。
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 endosomelysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH4Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH4Cl, 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) benzenesulfimyl 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.