Vanderheijden Nathalie, Delputte Peter L, Favoreel Herman W, Vandekerckhove Joël, Van Damme Jozef, van Woensel Peter A, Nauwynck Hans J
Laboratory of Virology, Faculty of Veterinary Medicine, Flanders Interuniversity Institute of Biotechnology (VIB), 5830 AA Boxmeer, The Netherlands.
J Virol. 2003 Aug;77(15):8207-15. doi: 10.1128/jvi.77.15.8207-8215.2003.
Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.
猪繁殖与呼吸综合征病毒（PRRSV）对单核细胞/巨噬细胞谱系的细胞具有非常有限的嗜性。它通过受体介导的内吞作用进入细胞。先前已描述了一种能够阻断PRRSV感染猪肺泡巨噬细胞（PAM）并识别一种210 kDa蛋白（p210）的单克隆抗体（MAb）（MAb41D3）（X. Duan、H. Nauwynck、H. Favoreel和M. Pensaert，《病毒学杂志》72:4520 - 4523，1998）。在本研究中，使用MAb41D3通过免疫亲和从PAM中纯化p210蛋白，并在胰蛋白酶消化后进行内部肽段测序。四个p210肽段与巨噬细胞限制性受体小鼠唾液酸粘附素的氨基酸序列同一性在56%至91%之间。利用这些肽段数据，随后确定了完整的p210 cDNA序列（5193 bp）。它与小鼠和人类唾液酸粘附素的氨基酸同一性分别为69%和78%。用克隆的p210 cDNA转染对病毒进入具有抗性的猪（PK - 15）细胞，并接种欧洲或美洲PRRSV毒株。仅在表达重组唾液酸粘附素的PK - 15细胞中检测到内化的病毒颗粒，表明这种糖蛋白介导了两种毒株的摄取。然而，未观察到核衣壳解体，而在感染的Marc - 145细胞中，病毒与内吞泡膜融合后脱壳时会观察到核衣壳解体，这表明融合过程受阻。MAb41D3特异性内化到PAM中证明了猪唾液酸粘附素介导内吞作用的能力。总之，这些结果表明唾液酸粘附素参与了PRRSV在PAM中的进入过程。
