Gilbert L, Toivola J, Lehtomäki E, Donaldson L, Käpylä P, Vuento M, Oker-Blom C
Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, Jyväskylä FIN-40014, Finland.
Biochem Biophys Res Commun. 2004 Jan 23;313(4):878-87. doi: 10.1016/j.bbrc.2003.11.176.
Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1, VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two proteins are produced by differential splicing of a primary transcript of the right ORF of the viral genome. The VP2 protein can be further proteolytically cleaved to form VP3. Previous studies have shown that VP1 and VP3 are unnecessary for capsid formation and consequently, that VP2 alone is sufficient for assembly. We have hypothesized that insertion of the enhanced green fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out without altering assembly. To investigate the possibility to develop fluorescent virus-like particles (fVLPs) from such chimeric VP2 proteins, the corresponding fusion construct was abundantly expressed in insect cells. Confocal imaging indicated that the EGFP-VP2 fusion product was assembled to fluorescent capsid-like complexes. In addition, electron micrographs of purified EGFP-VP2 complexes showed that they displayed a very similar size and appearance when compared to VP2 VLPs. Further, immunolabelling of purified EGFP-VP2 VLPs showed the presence of EGFP within the structure. Fluorescence correlation spectroscopy (FCS) studies confirmed that fVLPs were very similar in size when compared to authentic CPV. Finally, feeding of mammalian cells susceptible to CPV infection with these fVLPs indicated that entry and intracellular trafficking could be observed. In summary, we have developed fluorescent virus-like nanoparticles carrying a heterologous entity that can be utilized as a visualization tool to elucidate events related to a canine parvovirus infection.
犬细小病毒（CPV）是一种小型无包膜单链DNA病毒，由病毒蛋白VP1、VP2和VP3组成，具有T = 1二十面体对称性。VP2嵌套在VP1中，这两种蛋白质是由病毒基因组右开放阅读框初级转录本的差异剪接产生的。VP2蛋白可进一步经蛋白水解切割形成VP3。先前的研究表明，VP1和VP3对于衣壳形成并非必需，因此，仅VP2就足以进行组装。我们推测，在VP2的N端插入增强型绿色荧光蛋白（EGFP）可以在不改变组装的情况下进行。为了研究从这种嵌合VP2蛋白开发荧光病毒样颗粒（fVLP）的可能性，相应的融合构建体在昆虫细胞中大量表达。共聚焦成像表明，EGFP-VP2融合产物组装成了荧光衣壳样复合物。此外，纯化的EGFP-VP2复合物的电子显微镜照片显示，与VP2病毒样颗粒相比，它们的大小和外观非常相似。此外，纯化的EGFP-VP2病毒样颗粒的免疫标记显示结构内存在EGFP。荧光相关光谱（FCS）研究证实，与正宗CPV相比，fVLP的大小非常相似。最后，用这些fVLP感染对CPV敏感的哺乳动物细胞表明，可以观察到病毒的进入和细胞内运输。总之，我们开发了携带异源实体的荧光病毒样纳米颗粒，可作为一种可视化工具来阐明与犬细小病毒感染相关的事件。
