Dormitzer P R, Greenberg H B, Harrison S C
Laboratory of Molecular Medicine, Enders 673, Children's Hospital, 320 Longwood Avenue, Boston, Massachusetts 02115, USA.
Virology. 2000 Nov 25;277(2):420-8. doi: 10.1006/viro.2000.0625.
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.
轮状病毒是导致儿童严重脱水腹泻的主要原因。轮状病毒外衣壳糖蛋白VP7是保护性抗体的靶点，并且在病毒进入细胞过程中负责病毒的钙依赖性脱壳。我们已经对在昆虫细胞中表达的重组恒河猴轮状病毒VP7进行了纯化、表征和结晶。纯化的一个关键环节是通过EDTA从一种中和性单克隆抗体柱上洗脱VP7。凝胶过滤色谱法和平衡分析超速离心表明，在有钙存在的情况下，纯化的VP7会三聚化。三聚体VP7结晶形成六边形板。初步的X射线分析表明，晶体堆积再现了三层轮状病毒颗粒上VP7二十面体晶格的六边形成分。这些数据表明，轮状病毒外衣壳由钙依赖性VP7三聚体组装而成，并且这些三聚体的解离是EDTA诱导轮状病毒脱壳和VP7中和表位丧失的生化基础。
