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细胞趋向性的SCIDhu模型来确定每个致病阶段的调控/修饰基因需求，开发SCIDhu神经细胞模型来研究VZV神经趋向性，并进一步表征减毒疫苗Oka （V-Oka）病毒。我们将使用在SCID小鼠眼睛前房内建立的人类神经植入物作为VZV嗜神经性研究的主要系统。作为一种替代方法，我们将探索在SCID动物中植入神经干细胞接种VZV。开发一种或两种模型将有可能评估改变VZV在皮肤或T细胞中的复制的突变是否也会影响VZV在体内的嗜神经性。调控/包覆蛋白的研究将集中在即时早期（IE）蛋白，IE62， IE63和IE4。ORF62编码VZV的主要病毒反激活子，而IE63似乎具有辅助反激活活性。我们已经证明了IE63的一个拷贝是必不可少的，并制作了一个单一的IE63重组拷贝；目前正在进行产生单拷贝ORF62重组的工作。这些单拷贝构建体将使用亲本Oka (P-Oka) cosmids将ORF62和ORF63突变引入病毒基因组。将通过绘制IE62/lE63相互作用位点，从序列基序中确定orf62和orf63的假定功能区，或通过α疱疹病毒基因的保守性来选择诱变靶点。结构域将被定义为细胞培养中复制所必需的或可有可无的。我们建议表征IE4蛋白中与IE62结合、二聚化、反激活和核/细胞质定位相关的结构域。这些分析将定义IE4区域，这些区域必须是完整的传染性。在SCIDhu模型中，将评估具有IE62、IE63或IE4靶向突变的活的重组体对VZV在体内分化的人细胞中的复制的影响。为了进一步研究V-Oka的衰减，将在SCIDhu皮肤和T细胞异种移植中评估由V-Oka和P-Oka cosmids制成的嵌合重组。最后，我们将利用该模型来检验P-Oka病毒粒子的结构特征，并比较P-Oka和V-Oka病毒粒子。利用这些实验方法，应该有可能创造出缺乏通过感染T细胞传播能力的VZV重组，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。更好地了解水痘病毒在皮肤、T细胞和神经细胞中产生毒力所需的遗传机制，将指导“第二代”减毒水痘活疫苗的设计。

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.