Varicella-zoster Virus: Tegument Proteins in Pathogenesis

水痘带状疱疹病毒（VZV）在初次感染时引起水痘，在感觉神经节中持续存在，并可能从潜伏期重新激活引起带状疱疹。VZV的发病机制取决于其对T细胞、皮肤和感觉神经节的趋向性。VZV疫苗对预防水痘和降低老年人带状疱疹发病率非常有效。然而，第二代重组疫苗可以改善VZV的控制，该疫苗在皮肤中的复制减弱，但对T细胞和神经节的传染性有限。感觉神经节的VZV感染通过潜伏期的再激活确保其在人群中存活，从而导致带状疱疹。我们通过移植人背根神经节（DRG）于严重联合免疫缺陷（SCID）小鼠，建立了一种研究VZV神经发病机制的模型。我们对VZV被膜/调节蛋白的分析将扩展到研究它们在VZV嗜神经性中的功能。我们将重点研究由ORF63编码的重要的即时早期调节蛋白IE63，以及使用在这些基因中具有靶向突变的VZV重组体的ORF66激酶蛋白。SCIDhu DRG模型还提供了独特的机会来研究调节病毒基因启动子的细胞反激活因子如何控制VZV的嗜神经性，并确定持续感染的神经元的扰动可能触发VZV的再激活。IE63和ORF66在VZV感染神经节早期和晚期的作用将在scihu DRG中得到检验。实验将解决四个一般假设：1)初始VZV复制是必需的，或影响神经元中VZV DNA持续拷贝的水平；2) VZ病毒粒子必须高效制造和释放；3)抑制神经细胞凋亡的VZV蛋白促进持久性；4) VZV感染DRG反映了由干扰素（IFN）介导的先天细胞反应的平衡，从而优化了持久性。像所有疱疹病毒一样，VZV基因启动子具有被无处不在的宿主细胞调节蛋白识别的元素。我们的假设是，细胞蛋白在初始感染期间调节关键ORF63启动子的转录，并且需要在感觉神经元中过渡到潜伏期。为了了解持续感染DRG的细胞应激源可能诱导VZV再激活，实验将检查热、化学制剂、组蛋白去乙酰化酶抑制剂、干扰神经生长因子信号和其他已知的增强单纯疱疹病毒再激活的触发因素。在一年级和二年级提出的工作（见具体目标）预计将获得关于人类感觉神经节内分化的外周神经元和卫星细胞中VZV神经发病机制的分子机制的新信息，并确定设计具有遗传变化的第二代VZV疫苗的选择，该疫苗已被证明可以降低SCIDhu DRG模型体内的毒力。

Varicella zoster virus (VZV) causes varicella during primary infection, persists in sensory ganglia and may reactivate from latency to cause zoster. VZV pathogenesis depends upon its tropisms for T cells, skin and sensory ganglia. VZV vaccines to prevent varicella and to reduce zoster morbidity in the elderly are very effective. However, VZV control could be improved with a 2nd generation recombinant vaccine that has attenuated replication in skin, but limited infectivity for T cells and ganglia. VZV infection of sensory ganglia ensures its survival in the human population through reactivations from latency that result in zoster. We have developed a model for studying VZV neuropathogenesis by making xenografts of human dorsal root ganglia (DRG) in mice with severe combined immunodeficiency (SCID). Our analyses of VZV tegument/regulatory proteins will be extended to examine their functions in VZV neurotropism. We will focus on IE63, an important immediate early regulatory protein encoded by ORF63, and the ORF66 kinase protein using VZV recombinants that have targeted mutations in these genes. The SCIDhu DRG model also offers unique opportunities to investigate how cell transactivators that modulate viral gene promoters may control VZV neurotropism and to identify what perturbations of persistently infected neurons may trigger of VZV reactivation. The roles of IE63 and ORF66 at early and late stages of VZV infection of ganglia will be examined in SCIDhu DRG. Experiments will address four general hypotheses: 1) initial VZV replication is required for, or influences the level of persistent VZV DNA copies in neurons; 2) VZ virions must be made and released efficiently; 3) VZV proteins that inhibit neural cell apoptosis facilitate persistence; 4) VZV infection of DRG reflects an equilibrium with innate cellular responses, mediated by interferons (IFN), which optimizes persistence. VZV gene promoters, like those of all herpesviruses, have elements that are recognized by ubiquitous host cell regulatory proteins. Our hypothesis is that cellular proteins regulate transcription from the critical ORF63 promoter during initial infection and are needed for the transition to latency in sensory neurons. Experiments to understand what cellular stressors may induce VZV reactivation from persistently infected DRG will examine heat, chemical agents, histone deacetylase inhibitors, interference with nerve growth factor signaling and other triggers known to enhance herpes simplex virus reactivation. The work proposed for Yr. 1 and Yr. 2 (see Specific Aims) is expected to yield new information about the molecular mechanisms of VZV neuropathogenesis in differentiated peripheral neurons and satellite cells within human sensory ganglia and to identify options for designing a 2nd generation VZV vaccine with genetic changes that have been proved to reduce virulence in the SCIDhu DRG model in vivo.