Miyazaki Naoyuki, Taylor David W, Hansman Grant S, Murata Kazuyoshi
National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan.
National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA.
J Virol. 2015 Dec 23;90(5):2664-75. doi: 10.1128/JVI.02916-15.
The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes.
We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.
所有杯状病毒的衣壳蛋白（VP1）形成一个二十面体颗粒，具有两个主要结构域，即外壳（S）结构域和突出（P）结构域，它们通过一个柔性铰链区相连。S结构域形成围绕核酸的支架，而P结构域形成与受体相互作用的同源二聚体。P结构域进一步细分为两个亚结构域，称为P1和P2。P2亚结构域可能是插入P1亚结构域中的；因此，P结构域被分为P1-1、P2和P1-2亚结构域。为了研究衣壳抗原性，在两种萨波病毒GI.1和GI.5基因型之间交换了N端（N-term）/S/P1-1和P2/P1-2。嵌合VP1构建体在昆虫细胞中表达，并显示能自组装成形态上与亲本病毒样颗粒（VLP）相似的病毒样颗粒。有趣的是，与亲本VLP相比，嵌合VLP对异源抗血清具有更高水平的交叉反应性。为了从结构角度更好地理解抗原性，我们确定了一种嵌合VLP的中等分辨率（8.5埃）冷冻电子显微镜（cryo-EM）结构，并建立了VP1同源模型。冷冻电子显微镜结构显示，P结构域二聚体比S结构域略高（约5埃）。VP1同源模型使我们能够预测S结构域（67-229）以及P1-1（229-280）、P2（281-447）和P1-2（448-567）亚结构域。我们的结果表明，升高的P二聚体可能暴露免疫反应性的S/P1-1亚结构域表位。因此，与嵌合VLP更高水平的交叉反应性是由GI.1和GI.5表位的组合导致的。
我们构建了萨波病毒嵌合VP1构建体，并在昆虫细胞中产生了嵌合VLP。我们发现，与亲本VLP相比，两种嵌合VLP对异源VLP抗血清均具有更高水平的交叉反应性。解析了一种嵌合VLP（横手/ Mc114）的冷冻电子显微镜结构，分辨率达到8.5埃。VP1的同源模型首次表明了推定的S和P（P1-1、P2和P1-2）结构域。横手/ Mc114的整体结构包含其他杯状病毒共有的特征。我们表明，P2亚结构域主要参与同源二聚体界面，而P1亚结构域之间的大间隙相互作用较少。
