Varicella-zoster Virus: Tegument Proteins in Pathogenesis

水痘带状疱疹病毒（VZV）在原发感染期间引起水痘，持续存在于感觉神经节中，并可能从潜伏期重新激活以引起带状疱疹。 VZV 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 疫苗对于预防水痘和减少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水痘带状疱疹病毒的控制，该疫苗减弱了皮肤中的复制，但限制了 T 细胞和神经节的感染性。感觉神经节的水痘带状疱疹病毒感染通过导致带状疱疹的潜伏期重新激活来确保其在人群中的生存。我们通过在严重联合免疫缺陷 (SCID) 小鼠中制作人背根神经节 (DRG) 异种移植物，开发了一种研究 VZV 神经发病机制的模型。我们对 VZV 皮膜/调节蛋白的分析将扩展到检查它们在 VZV 趋神经性中的功能。我们将重点关注 IE63，这是一种由 ORF63 编码的重要立即早期调节蛋白，以及使用 VZV 重组体的 ORF66 激酶蛋白，这些重组体在这些基因中具有靶向突变。 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式激活因子如何控制 VZV 向神经性，并确定持续感染的神经元的哪些扰动可能触发 VZV 重新激活。 SCIDhu DRG 将检查 IE63 和 ORF66 在神经节 VZV 感染早期和晚期的作用。实验将解决四个一般假设：1）初始 VZV 复制是神经元中持续 VZV DNA 拷贝水平所必需的，或者影响其水平； 2) VZ病毒体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) DRG 的 VZV 感染反映了干扰素 (IFN) 介导的先天细胞反应的平衡，从而优化了持久性。 VZV 基因启动子与所有疱疹病毒的启动子一样，具有被普遍存在的宿主细胞调节蛋白识别的元件。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，并且是感觉神经元向潜伏期过渡所必需的。旨在了解哪些细胞应激源可能诱导持续感染的 DRG 中 VZV 重新激活的实验将检查热、化学制剂、组蛋白脱乙酰酶抑制剂、神经生长因子信号传导干扰以及其他已知可增强单纯疱疹病毒重新激活的触发因素。为 Yr 提出的工作。 1 岁2（参见具体目标）预计将产生有关人类感觉神经节内分化的周围神经元和卫星细胞中 VZV 神经发病机制的新信息，并确定设计第二代 VZV 疫苗的选择，该疫苗具有已被证明可降低 SCIDhu DRG 模型体内毒力的遗传变化。

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.