Lee J, Yoo D, Redmond M J, Attah-Poku S K, van den Hurk J V, Babiuk L A
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon.
Can J Vet Res. 1998 Jan;62(1):56-62.
Rotavirus VP8*, the N-terminal trypsin cleavage product of VP4, has been shown to bind to MA-104 cells and human O type erythrocytes. To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E. coli and radiolabelled with 35S-methionine. The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV. To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8*. rVP8* showed HA which could be inhibited by antiserum to BRV. This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction. To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used. First, a peptide spanning aa 230-247 was synthesized and antisera was raised against the peptide to see whether it could inhibit HA of rVP8*. Second, a truncated form of VP8* (tVP8*: aa 1-229) was expressed to examine its hemagglutinating activity. Third, the dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA. To characterize the interaction between the HA domain and sialic acid receptors, erythrocytes were treated with sialidases of different specificities. Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.
轮状病毒VP8是VP4的N端胰蛋白酶裂解产物，已被证明可与MA-104细胞和人O型红细胞结合。为了检测细菌表达的VP8是否与MA-104细胞的细胞成分结合，VP8*（氨基酸1-247）在大肠杆菌中表达并用35S-甲硫氨酸进行放射性标记。用抗牛轮状病毒C486（BRV）抗血清对放射性标记的rVP8进行免疫沉淀。发现rVP8与MA-104细胞结合，并且其结合被BRV竞争。为了研究VP8与红细胞受体之间的相互作用，使用可溶性rVP8进行血凝（HA）和血凝抑制（HI）试验。rVP8表现出血凝活性，该活性可被抗BRV抗血清抑制。这种相互作用也被神经节苷脂抑制，表明存在唾液酸依赖性相互作用。为了研究VP8的C端区域对HA的贡献，采用了多种方法。首先，合成了一段跨越氨基酸230-247的肽段，并制备了针对该肽段的抗血清，以观察其是否能抑制rVP8的血凝活性。其次，表达了VP8的截短形式（tVP8*：氨基酸1-229）以检测其血凝活性。第三，在使用天然SDS-PAGE进行电泳后，通过蛋白质印迹法比较rVP8和tVP8的二聚化情况。结果表明，针对氨基酸230-247的抗体通过阻止VP8的二聚化来抑制血凝，而这反过来又使该分子能够引起血凝。为了表征HA结构域与唾液酸受体之间的相互作用，用不同特异性的唾液酸酶处理红细胞。解脲棒杆菌、产气荚膜梭菌和α2-8连接特异性神经氨酸酶破坏了红细胞唾液酸与rVP8相互作用的能力，表明牛轮状病毒C486结合需要α2-8连接，但唾液酸的乙酰化并非必需。
