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，持续存在于感觉神经节，并可能在潜伏期重新激活而导致带状疱疹。VZV的发病机制与其对T细胞、皮肤和感觉神经节的趋向性有关。预防水痘和减少老年人带状疱疹发病率的VZV疫苗非常有效。然而，第二代重组疫苗可以改善VZV的控制，这种疫苗减少了在皮肤中的复制，但限制了对T细胞和神经节的感染力。感觉神经节的VZV感染通过导致带状疱疹的潜伏期重新激活来确保其在人类群体中的生存。我们建立了人背根神经节(DRG)异种移植严重联合免疫缺陷(SCID)小鼠的VZV神经发病机制模型。我们对VZV被膜/调节蛋白的分析将扩展到研究它们在VZV神经趋向性中的功能。我们将关注IE63，一个由ORF63编码的重要的即时早期调节蛋白，以及使用VZV重组体的ORF66激酶蛋白，这些基因具有靶向突变。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的神经趋向性，并确定持续感染的神经元的扰动可能触发VZV的重新激活。IE63和ORF66在神经节VZV感染早期和晚期的作用将在SCIDhu DRG中进行研究。实验将解决四个一般假设：1)初始VZV复制是神经元持续VZV DNA复制所必需的，或影响其水平；2)VZ病毒粒子必须被有效地制造和释放；3)VZV蛋白可以抑制神经细胞的凋亡促进持久性；4)VZV感染DRG反映了由干扰素(干扰素)介导的固有细胞反应的平衡，从而优化了持久性。像所有疱疹病毒一样，VZV基因启动子具有普遍存在的宿主细胞调节蛋白识别的元件。我们的假设是，在最初的感染过程中，细胞蛋白调节关键的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。为了了解哪些细胞应激源可能会导致持续感染的DRG中VZV的重新激活，实验将检查热、化学试剂、组蛋白去乙酰酶抑制剂、对神经生长因子信号的干扰以及其他已知的促进单纯疱疹病毒重新激活的触发因素。这项工作是为您提出的。1和Yr.2(见特定的AIMS)有望提供有关VZV在人类感觉神经节内分化的外周神经元和卫星细胞中神经致病的分子机制的新信息，并确定设计第二代VZV疫苗的选择，该疫苗具有已被证明在体内SCIDhu DRG模型中降低毒力的基因变化。

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.