Expression and Functional Characterization of Bluetongue Virus VP2 Protein: Role in Cell Entry

Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determine various biological functions of VP2. The purified VP2 was shown to have virus hemagglutinin activity and was antigenically indistinguishable from the VP2 of the virion. Whether VP2 is responsible for BTV entry into permissive cells was subsequently assessed by cell surface attachment and internalization studies with an immunofluorescence assay system. The results demonstrated that VP2 alone is responsible for virus entry into mammalian cells. By competition assay, it appeared that both VP2 and the BTV virion attached to the same cell surface molecule(s). The purified VP2 also had a strong affinity for binding to glycophorin A, a sialoglycoprotein component of erythrocytes, indicating that VP2 may be responsible for BTV transmission by the Culicoides vector to vertebrate hosts during blood feeding. Further, by various enzymatic treatments of BTV-permissive L929 cells, preliminary data have been obtained which indicated that the BTV receptor molecule(s) is likely to be a glycoprotein and that either the protein moiety of the glycoprotein or a second protein molecule could also serve as a coreceptor for BTV infection.

本研究针对蓝舌病毒（bluetongue virus, BTV）血清型10的第2基因组片段进行改造，该片段编码病毒衣壳外层蛋白VP2。将其与核糖核酸酶A（RNase A）的S肽段（S-peptide fragment）进行标签融合后，通过重组杆状病毒（recombinant baculovirus）实现表达。随后借助S标签将重组蛋白纯化至均一状态，并对纯化蛋白的寡聚化特性进行鉴定。检测结果显示，该蛋白多数以二聚体形式存在，仅少量以三聚体形式存在。将所得重组蛋白用于VP2的多项生物学功能验证：实验证实纯化的VP2具备病毒血凝素活性，且其抗原性与完整病毒颗粒中的天然VP2无显著差异。为探究VP2是否介导BTV侵入易感细胞，研究人员通过免疫荧光分析法（immunofluorescence assay）开展细胞表面吸附与内化实验。结果表明，仅VP2单一蛋白即可介导病毒进入哺乳动物细胞。竞争结合实验显示，VP2与完整BTV病毒颗粒可结合同一类细胞表面分子。纯化的VP2还可与血型糖蛋白A（glycophorin A）——一种红细胞表面的唾液酸糖蛋白（sialoglycoprotein）——产生强结合活性，这提示VP2可能介导了BTV通过库蠓属（Culicoides）媒介在血液取食过程中传播至脊椎动物宿主的过程。此外，通过对BTV易感的L929细胞开展多种酶处理实验，研究人员获得了初步数据，结果显示BTV的受体分子大概率为糖蛋白（glycoprotein），且该糖蛋白的蛋白组分或另一类蛋白分子可同时作为BTV感染的共受体。