Antigenic determinants of varicella virus

描述（由申请方提供）：水痘-带状疱疹病毒（VZV）是水痘和带状疱疹的病因。VZV感染是移植和癌症患者中的复发性问题。带状疱疹和带状疱疹后神经痛也是老年人的主要医疗问题。VZV基因组编码约70个开放阅读框。与其他α疱疹病毒不同，VZV的主要糖蛋白复合物是gE：gI。该项目的长期目标是深入了解VZV gE糖蛋白的作用，VZV gE糖蛋白是一种必需的基因产物，具有细胞表面受体的许多属性。例如，gE在其胞质尾区具有内吞信号和丝氨酸/苏氨酸和酪氨酸磷酸化基序。gE的内吞作用是网格蛋白介导的;随后的gE运输依赖于酪蛋白激酶II磷酸化序列和PACS-1的缔合。用重组生物活性VZV ORF 47蛋白丝氨酸激酶进行分析，发现该病毒激酶具有与酪蛋白激酶II相似的磷酸化共有基序。因此，该假说认为ORF 47蛋白激酶和酪蛋白激酶II将作为团队成员来修饰酸性gE共有位点，并在此过程中确定gE运输途径。具体目标包括：1）表征ORF 47激酶对gE的磷酸化并记录与酪蛋白激酶II磷酸化的差异，2）研究在存在和不存在ORF 47激酶的情况下gE的运输，3）表征gE上的酪氨酸磷酸化位点和酪氨酸磷酸化的细胞位点，和4）研究在磷酸化和运输事件期间gI与gE的相互作用，以及gE：gI复合物与PACS-I的相互作用。磷酸化分析将包括体外激酶测定和2-D磷酸肽凝胶。运输实验将包括野生型和突变gE和gI糖蛋白的转染研究，以及含有突变gE和ORF 47蛋白的重组病毒的感染研究。将通过共聚焦显微镜分析标记病毒和细胞蛋白的内吞和运输数据，然后进行数字图像分析，以获得像素定量。野生型和突变型VZV的初步数据表明，在感染ORF 47突变病毒的细胞中，gE运输被重定向，即gE不前往反式高尔基体。因此，后一个实验提供了强有力的证据，证明病毒特异性磷酸化和gE糖蛋白的定向运输之间存在相互关系。反过来，累积数据表明，VZV gE运输发生在两个磷酸化依赖性途径中：一个不针对TGN的途径可能促进细胞扩散，而另一个针对TGN的途径可能促进糖蛋白掺入病毒体。这些拟议的VZV研究将提供进一步了解多种功能的gE和gE：gI复合物。

DESCRIPTION (provided by applicant): Varicella-zoster virus (VZV) is the cause of chickenpox and herpes zoster. VZV infection is a recurrent problem in both transplant and cancer patients. Herpes zoster and post herpetic neuralgia are also major medical problems in the elderly population. The VZV genome encodes about 70 open reading frames. The predominant VZV glycoprotein complex, in contrast to other alphaherpesviruses, is gE:gI. The long-term goal of this project is an in depth understanding of the role of VZV gE glycoprotein, an essential gene product which has many attributes of a cell surface receptor. For example, gE has endocytosis signals and both serine/threonine and tyrosine phosphorylation motifs in its cytoplasmic tail. The endocytosis of gE is clathrin mediated; subsequent gE trafficking is dependent on a casein kinase II phosphorylation sequence and the association of PACS-1. Analyses with a recombinant biologically active VZV ORF47 protein serine kinase led to the discovery that the viral kinase has a similar phosphorylation consensus motif to that of casein kinase II. Therefore, the hypothesis states that the ORF47 protein kinase and casein kinase II will act as team players to modify the acidic gE consensus site and in the process determine the gE trafficking pathways. The Specific Aims include the following: 1) Characterize the phosphorylation of gE by ORF47 kinase and document differences from casein kinase II phosphorylation, 2) Investigate the trafficking of gE in the presence and absence of ORF47 kinase, 3) Characterize the tyrosine phosphorylation sites on gE and the cellular site of tyrosine phosphorylation, and 4) Investigate the interaction of gI with gE during the phosphorylation and trafficking events, and the interaction of the gE:gl complex with PACS-l. Phosphorylation analyses will include both in vitro kinase assays and 2-D phosphopeptide gels. The trafficking experiments will include both transfection studies with wild type and mutant gE and gI glycoproteins, as well as infection studies with recombinant viruses containing mutated gE and ORF47 proteins. Endocytosis and trafficking data with labeled viral and cellular proteins will be analyzed by confocal microscopy followed by digital image analysis to obtain a pixel quantification. Preliminary data with wild type and mutant VZV suggested that gE trafficking was redirected in cells infected with an ORF47 mutant virus, namely, gE did not travel to the trans Golgi. Thus, this latter experiment provided strong evidence of an inter-relationship between virus-specific phosphorylation and directed trafficking of the gE glycoprotein. In turn, the cumulative data suggest that VZV gE trafficking occurs in two phosphorylation dependent pathways: one pathway not directed to the TGN may facilitate cell spread while the other pathway directed to the TGN may facilitate glycoprotein incorporation into virions. These proposed VZV studies will provide further insight into the multiple functions of gE and the gE:gI complex.