Varicella-zoster Virus Tegument Proteins in Pathogenesis

描述（由申请人提供）：水痘带状疱疹病毒（VZV）感染及其传播的发病机制中的三个关键事件是病毒血症、皮肤复制和神经潜伏期。我们的总体目标是使用体外方法和我们的VZV皮肤和T细胞嗜性的SCIDhu模型来定义这些致病阶段中的每一个的调节/被膜基因要求，以开发SCIDhu神经细胞模型来研究VZV嗜神经性并进一步表征减毒疫苗Oka（V-Oka）病毒。我们将使用在SCID小鼠眼前房内建立的人类神经植入物作为VZV向神经性研究的主要系统。作为替代方案，我们将探索用神经元干细胞移植的SCID动物的VZV接种。一种或两种模型的开发将使评估改变皮肤或T细胞中VZV复制的突变是否也影响体内VZV嗜神经性成为可能。调节/被膜蛋白的研究将集中在立即早期（IE）蛋白，IE62，IE63和IE 4。ORF 62编码VZV的主要病毒反式激活因子，IE63似乎具有辅助反式激活活性。我们已经证明，一个拷贝的IE63是必不可少的，并已作出了单拷贝IE63重组体，工作正在进行中，以产生一个单拷贝的ORF 62重组体。这些单拷贝构建体将用于使用亲本Oka（P-Oka）cosplay将ORF 62和ORF 63突变引入病毒基因组中。通过定位IE62/IE63相互作用的位点并从序列基序中鉴定ORF 62和63的推定功能区或通过α疱疹病毒基因中的保守性来选择诱变靶标。结构域将被定义为细胞培养中复制所必需的或不可缺少的。我们建议表征IE 4蛋白中与IE62结合、二聚化、反式激活和核/胞质定位相关的结构域。这些分析将定义感染性必须完整的IE 4区域。将在SCIDhu模型中评价在IE62、IE63或IE 4中具有靶向突变的活重组体对体内分化的人细胞中VZV复制的影响。为了进一步研究V-Oka减毒，将在SCIDhu皮肤和T细胞异种移植物中评价由V-Oka和P-Oka cosmetry制备的嵌合重组体。最后，我们将利用该模型来研究P-Oka病毒体的结构特征，并比较P-Oka和V-Oka病毒体。使用这些实验方法，应该可以创建缺乏通过感染T细胞传播的能力的VZV重组体，或者在神经细胞中建立持续感染，同时保留在皮肤中复制的能力。更好地理解VZV在皮肤、T细胞和神经细胞中的毒力所需的遗传机制将指导“第二代”水痘减毒活疫苗的设计。

DESCRIPTION (provided by applicant): The three critical events in the pathogenesis of varicella-zoster virus (VZV) infection and its transmission are viremia, cutaneous replication and neural latency. Our overall objectives are to define regulatory/tegument gene requirements for each of these pathogenic phases using in vitro methods and our SCIDhu model of VZV skin and T cell tropism, to develop a SCIDhu neural cell model to study VZV neurotropism and to further characterize the attenuated vaccine Oka (V-Oka) virus. We will use human neural implants established within the anterior chamber of the SCID mouse eye as the primary system for VZV neurotropism studies. As an alternative, we will explore VZV inoculation of SCID animals engrafted with neuronal stem cells. Development of one or both models will make it possible to assess whether mutations altering VZV replication in skin or T cells also affect VZV neurotropism in vivo. Investigations of regulatory/tegument proteins will focus on the immediate early (IE) proteins, IE62, IE63 and IE4. ORF62 encodes the major viral transactivator of VZV and IE63 appears to have accessory transactivating activity. We have shown that one copy of IE63 is essential and have made a single copy IE63 recombinant; work is in progress to generate a single copy ORF62 recombinant. These single copy constructs will be used to introduce ORF62 and ORF63 mutations into the viral genome using parent Oka (P-Oka) cosmids. Mutagenesis targets will be selected by mapping sites of IE62/lE63 interaction and identifying putative functional regions of ORFs 62 and 63 from sequence motifs or by conservation in alphaherpesvirus genes. Domains will be defined as essential or dispensable for replication in cell culture. We propose to characterize domains in IE4 protein related to IE62 binding, dimerization, transactivation, and nuclear/cytoplasmic localization. These analyses will define IE4 regions that must be intact for infectivity. Viable recombinants that have targeted mutations in IE62, IE63 or IE4 will be evaluated for effects on VZV replication in differentiated human cells in vivo in the SCIDhu model. In order to further investigate V-Oka attenuation, chimeric recombinants made from V-Oka and P-Oka cosmids will be evaluated in the SCIDhu skin and T cell xenografts. Finally, we will exploit the model to examine structural characteristics of P-Oka virions and to compare P-Oka and V-Oka virions. Using these experimental approaches, it should be possible to create VZV recombinants that lack the capacity to disseminate by infecting T cells, or to establish persistent infection in neural cells, while retaining the capacity to replicate in skin. A better understanding of the genetic mechanisms that are required for VZV virulence in skin, T cells and neural cells will guide the design of 'second generation' live attenuated varicella vaccines.