Expression and functional characterization of bluetongue virus VP2 protein: Role in cell entry
Expression and functional characterization of bluetongue virus VP2 protein: Role in cell entry
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DOI：
10.1128/jvi.73.12.9832-9842.1999
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发表时间：
1999-12-01
影响因子：
5.4
通讯作者：
Roy, P
中科院分区：
文献类型：
--
作者：
Hassan, SS;Roy, P
用RNase A的S肽片段标记蓝舌病病毒（BTV）血清10型VP2基因片段2，并在重组杆状病毒中表达。重组蛋白用S标签纯化至均一，并对纯化蛋白的寡聚性质进行了测定。所获得的数据表明，大部分蛋白质形成二聚体，并且在较小程度上形成一些三聚体。利用重组蛋白对VP2的多种生物学功能进行了鉴定。纯化的VP2显示出具有病毒血凝素活性，并且与病毒粒子的VP2在抗原性上无法区分。随后通过免疫荧光测定系统的细胞表面附着和内化研究来评估VP2是否负责BTV进入允许细胞。结果表明，VP2是BTV病毒进入哺乳动物细胞的唯一途径。通过竞争试验，VP2和BTV病毒粒子似乎都附着在同一个细胞表面分子上。纯化的VP2对红细胞的唾液糖蛋白成分血型糖蛋白A也有很强的亲和力，表明VP2可能是吸血过程中库蠓介体向脊椎动物宿主传播BTV的原因。此外，通过对BTV允许的L929细胞进行各种酶处理，已经获得了初步数据，这些数据表明BTV受体分子可能是一种糖蛋白，并且该糖蛋白的蛋白部分或第二种蛋白分子也可以作为BTV感染的辅助受体。
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 ex-tent, 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.