Varicella-zoster Virus Tegument Proteins in Pathogenesis

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。我们的总体目标是确定调控/被膜基因的要求 对于这些致病阶段中的每一个，使用体外方法和我们的VZV皮肤和T细胞向性的SCIDhu模型， 建立SCIDhu神经细胞模型以研究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， VZV嗜神经性研究的主要系统。作为替代方案，我们将探索SCID动物的VZV接种 植入了神经干细胞开发一个或两个模型将有可能评估 改变VZV在皮肤或T细胞中复制的突变也影响VZV在体内的嗜神经性。调查 调节/被膜蛋白将集中于立即早期（IE）蛋白，IE62、IE63和IE 4。ORF 62编码 VZV和IE 63的主要病毒反式激活因子似乎具有辅助反式激活活性。我们已经证明， IE63的拷贝是必需的，并且已经制备了单拷贝IE63重组体;正在进行生产单拷贝IE63重组体的工作。 ORF 62重组体。这些单拷贝构建体将用于将ORF 62和ORF 63突变引入病毒载体中。 基因组使用亲本Oka（P-Oka）cosmetics。将通过IE62 tlE 63的定位位点选择诱变靶标 相互作用，并从序列基序或通过保守性鉴定ORF 62和63的推定功能区， α疱疹病毒基因。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。我们提出 表征IE 4蛋白中与IE62结合、二聚化、反式激活和核/胞质相关的结构域 本地化这些分析将定义感染性必须完整的IE 4区域。具有以下特征的活重组体 将评估IE62、IE63或IE 4中的靶向突变对VZV在分化的人细胞中复制的影响， 在SCIDhu模型中。为了进一步研究V-Oka减毒，从V-Oka制备嵌合重组体， 将在SCIDhu皮肤和T细胞异种移植物中评估P-Oka cosmetics。最后，我们将利用该模型， 检查P-Oka病毒粒子的结构特征并比较P-Oka和V-Oka病毒粒子。使用这些实验 方法，应该有可能产生缺乏通过感染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设计， “第二代”水痘减毒活疫苗。

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 IE62tlE63 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.