Varicella-zoster Virus Tegument Proteins in Pathogenesis

水痘-带状疱疹病毒(VZV)感染及其传播的三个关键事件是病毒血症， 皮肤复制和神经潜伏期。我们的总体目标是定义监管/被盖基因要求 使用体外方法和我们的VZV皮肤和T细胞趋向性的SCIDhu模型，对于这些致病阶段的每一个阶段， 建立SCIDhu神经细胞模型研究VZV的神经嗜性并进一步研究OKA减毒活疫苗的特性 (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神经趋向性研究的主要系统。作为替代方案，我们将探索对SCID动物接种VZV 植入了神经干细胞。一个或两个模型的开发将使评估 改变VZV在皮肤或T细胞中复制的突变也会影响VZV在体内的神经趋向性。调查 调节/被膜蛋白将集中在即刻早期(IE)蛋白，IE62、IE63和IE4。ORF62编码 VZV和IE63的主要病毒反式激活剂似乎具有辅助反式激活活性。我们已经证明了这一点 IE63的复制是必不可少的，并已使IE63的单个副本得到重组；生成单个副本的工作正在进行中 ORF62重组。这些单拷贝结构将用于将ORF62和ORF63突变引入病毒 使用亲本Oka(P-Oka)宇宙体的基因组。通过对IE62tlE63的定位来选择突变靶点 从序列基序或保守序列中鉴定ORF62和63的相互作用和功能区域 甲型疱疹病毒基因。在细胞培养中，结构域将被定义为复制所必需的或必不可少的。我们建议 研究IE4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IE4区域，这些区域必须是完整的，才能具有传染性。有活力的重组体 将评估IE62、IE63或IE4中的靶向突变对分化的人类细胞中VZV复制的影响 在SCIDhu模型中的活体。为了进一步研究V-Oka的衰减，由V-Oka制成的嵌合重组体 P-Oka宇宙体将在SCIDhu皮肤和T细胞异种移植中进行评估。最后，我们将利用该模型来 研究P-Oka病毒粒子的结构特征，并比较P-Oka病毒粒子和V-Oka病毒粒子。使用这些实验 方法，应该有可能创造VZV重组体，缺乏通过感染T细胞进行传播的能力， 或者在神经细胞中建立持续感染，同时保持在皮肤中复制的能力。更好地理解 在皮肤中VZV毒力所需的遗传机制中，T细胞和神经细胞将指导设计 “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.