Non-lytic extraction and characterisation of receptors for multiple strains of rotavirus
Non-lytic extraction and characterisation of receptors for multiple strains of rotavirus
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DOI：
10.1007/s007050170093
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发表时间：
2001-01-01
影响因子：
2.7
通讯作者：
Holmes, IH
中科院分区：
文献类型：
--
作者：
Jolly, CL;Beisner, BM;Holmes, IH
为了纯化轮状病毒的细胞受体，我们使用去污剂辛基-β-D-吡喃葡萄糖苷（octyl-glucoside/OG）从MA 104和HT 29细胞中提取牛、猴、猪和人轮状病毒的受体。浓度为0.2%的辛基葡糖苷显著降低了处理细胞对感染的易感性，同时使它们具有代谢活性，结果耗尽的受体能够再生。MA 104和HT 29辛基葡糖苷提取物的高碘酸盐处理显著降低了这些提取物中和轮状病毒感染性的能力，揭示了碳水化合物作为Wa和NCDV的提取受体的组分。用代谢抑制剂衣霉素、脱氧mannojirimycin和BenzylGalNAc处理MA 104细胞表明，在某些轮状病毒株的感染中，N-连接碳水化合物可能比O-连接更重要。此外，通过在耗尽的受体的再生过程中包括放线菌酮，我们发现了猪轮状病毒CRW 8可能使用糖脂基受体，而NCDV和Wa使用糖蛋白的证据。轮状病毒受体的再生特性允许连续几天使用辛基葡糖苷重复收获细胞表面分子，并且这些提取物用于在病毒覆盖蛋白质印迹测定（VOPBA）中可视化病毒结合。使用VOPBAs，我们观察到Wa和NCDV似乎都识别MA 104和HT 29细胞上存在的分子量大致相同的蛋白质。
To characterise the cellular receptors for rotavirus, we used the detergent octyl-beta -D-glucopyranoside (octyl-glucoside/OG) to extract the receptors for bovine, simian, porcine and human rotaviruses from MA 104 and HT29 cells. An octyl-glucoside concentration of 0.2% dramatically reduced the susceptibility of treated cells to infection, while leaving them metabolically active, and as a result the depleted receptors were able to regenerate. Periodate treatment of the MA 104 and HT29 octyl-glucoside extracts significantly decreased the ability of these extracts to neutralise rotavirus infectivity, revealing carbohydrate as component of the extracted receptors for Wa and NCDV. Treatment of MA104 cells with the metabolic inhibitors tunicamycin, deoxymannojirimycin and BenzylGalNAc suggested N-linked carbohydrate may be more important than O-linked in infection by some strains of rotavirus. Furthermore, by including cycloheximide during the regeneration of depleted receptors we found evidence that porcine rotavirus CRW8 may use a glycolipid-based receptor, while NCDV and Wa use a glycoprotein. The regenerating properties of the rotavirus receptors allowed repeated harvesting of cell surface molecules using octyl-glucoside on consecutive days, and these extracts were used to visualise virus binding in a virus overlay protein blot assay (VOPBA). Using VOPBAs, we observed both Wa and NCDV appear to recognise proteins of approximately the same molecular weight present on MA104 and HT29 cells.