Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers
Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers
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DOI：
10.1006/viro.2000.0625
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发表时间：
2000-11-25
期刊：
影响因子：
3.7
通讯作者：
Harrison, SC
中科院分区：
文献类型：
--
作者：
Dormitzer, PR;Greenberg, HB;Harrison, SC
轮状病毒是导致儿童严重脱水性腹泻的主要原因。VP 7是轮状病毒外壳糖蛋白，是保护性抗体的靶点，负责病毒进入细胞期间的钙依赖性脱壳。我们已经纯化，表征和结晶重组恒河猴轮状病毒VP 7，在昆虫细胞中表达。纯化的一个关键方面是通过EDTA从中和单克隆抗体柱中洗脱VP 7。凝胶过滤色谱和平衡分析超离心证明，在钙的存在下，纯化的VP 7三聚体。三聚体VP 7结晶成六边形板。初步的X-射线分析表明，晶体包装再现的六角组成部分的二十面体晶格的VP 7的三层轮状病毒颗粒。这些数据表明，轮状病毒外壳组装从钙依赖性VP 7三聚体，这些三聚体的解离是EDTA诱导的轮状病毒脱壳和VP 7中和表位的损失的生化基础。(C)北京大学出版社.
Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes. (C) 2000 Academic Press.