Koshizuka Tetsuo, Sadaoka Tomohiko, Yoshii Hironori, Yamanishi Koichi, Mori Yasuko
Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Virology. 2008 Aug 1;377(2):289-95. doi: 10.1016/j.virol.2008.04.039.
Varicella-zoster virus (VZV) encodes five genes that do not have herpes simplex virus homologs. One of these genes, VZV open reading frame 1 (ORF1), encodes a membrane protein with a hydrophobic domain at its C-terminus that is predicted to be the transmembrane domain. However, the detailed characterization of ORF1 protein in infected cells has not been reported. Here, we produced mono-specific antibodies against ORF1 protein and characterized the gene products in infected cells. Western blot analyses showed the ORF1 polypeptides had apparent molecular masses of approximately 14-17 kDa. Furthermore, ORF1 was found to be a phosphoprotein by immunoprecipitation assay. In immunofluorescence assays, the VZV ORF1 protein was detected at both the plasma membrane and trans-Golgi network in both VZV-infected and ORF1-transfected cells. Moreover, ORF1 proteins associated with each other to form homodimer, and were incorporated into viral particles. The C-terminal hydrophobic domain was required for the association of ORF1 with the membrane structures, indicating that ORF1 protein is anchored to the membrane thorough its C-terminus, which is a transmembrane domain. Because ORF1 possesses a C-terminal transmembrane domain without an N-terminal signal sequence for its translocation to the ER lumen, ORF1 can be classified as a tail-anchored membrane protein. These results show that the N terminus of ORF1 protein faces the cytoplasm in infected cells and the tegument region in mature virions.
水痘带状疱疹病毒（VZV）编码五个没有单纯疱疹病毒同源物的基因。其中一个基因，VZV开放阅读框1（ORF1），编码一种膜蛋白，其C端有一个疏水区，预计为跨膜结构域。然而，尚未有关于感染细胞中ORF1蛋白详细特征的报道。在此，我们制备了针对ORF1蛋白的单特异性抗体，并对感染细胞中的基因产物进行了表征。蛋白质印迹分析表明，ORF1多肽的表观分子量约为14 - 17 kDa。此外，通过免疫沉淀试验发现ORF1是一种磷蛋白。在免疫荧光试验中，在VZV感染细胞和ORF1转染细胞的质膜和反式高尔基体网络中均检测到VZV ORF1蛋白。此外，ORF1蛋白相互结合形成同二聚体，并被整合到病毒颗粒中。C端疏水区是ORF1与膜结构结合所必需的，这表明ORF1蛋白通过其作为跨膜结构域的C端锚定在膜上。由于ORF1具有C端跨膜结构域但没有用于转运到内质网腔的N端信号序列，因此ORF1可归类为尾锚定膜蛋白。这些结果表明，在感染细胞中ORF1蛋白的N端面向细胞质，在成熟病毒粒子中面向被膜区。
