Mechanisms of Interferon Regulatory Factor Dysfunction by Cutaneous Papillomaviruses

项目概要/摘要 乳头瘤病毒（PV）感染严重影响人类健康。几乎所有的病例都是由粘液肺静脉引起的 人类宫颈癌，而皮肤PV导致严重的皮肤感染和非黑色素瘤皮肤癌 在由例如HIV感染、器官移植或遗传疾病引起的免疫缺陷患者中。 皮肤肺静脉的治疗是有限的，往往无效。因此，迫切需要发展 针对PV感染的靶细胞角质形成细胞的新疗法。角质细胞有潜力 来产生抗病毒反应病毒感染激活干扰素调节因子（IRF）， 干扰素（IFN）和IFN刺激的抗病毒基因。IRF和IFN也是肿瘤抑制剂，使它们 作为PV感染的治疗靶点特别有吸引力。然而，粘膜和皮肤PV都可以 抑制IRF功能和IFN表达。这种抑制是由粘膜上皮细胞的E6和E7癌基因介导的。 肺静脉虽然有限的研究表明，粘膜和皮肤PV在E6和E7功能方面存在差异， IRF对角质形成细胞中IFN应答的调节与其他细胞类型不同， 问题多于答案与人类一样，皮肤PV感染也发生在犬中， 免疫缺陷动物因此，狗提供了一种自然自发的动物模型，用于研究 皮肤PV感染和测试新的治疗方法。事实上，我们的初步数据表明， 犬皮肤PV E6和E7对犬IFN和IFN刺激基因表达的不同干扰 角质形成细胞我们工作的长期目标是开发针对IRFs和IFN的新疗法 使用PV犬模型治疗免疫缺陷患者皮肤PV感染的反应 感染我们假设E6和E7通过抑制IRF而增强病毒感染 使用皮肤PV特有的机制在角质形成细胞中起作用。在目标1中，我们将抑制 IRF，以确定其对皮肤PV感染以及IFN的组成型和诱导型表达的影响 和干扰素刺激的基因。在目标2和3中，我们将确定皮肤 PV E6和E7介导的干扰素和干扰素刺激的角质形成细胞基因表达的破坏。具体地说， 我们将使用质谱法来鉴定调节IRF表达和/或 功能，然后确定E6和E7对这些结合伴侣的作用机制。使用人类PV和 使用狗PV和角质形成细胞将解决人类疾病;使用狗PV和角质形成细胞将推进狗模型。 结果将为开发新的治疗方法奠定基础，这些方法将绕过E6和E7的有害作用。 方面的影响.指导团队包括在角质形成细胞生物学，IRF和IFN方面具有广泛专业知识的教师 分化细胞生物学、PV生物学、质谱蛋白质组学和生物信息学。总之， 拟议的研究将解决人类和动物健康的一个关键问题，利用各种技能， 跨学科指导团队的成员，并增强我作为独立临床医生科学家的成熟度。

Project Summary/ Abstract Papillomavirus (PV) infections significantly impact human health. Mucosal PVs cause virtually all cases of human cervical cancer, while cutaneous PVs cause severe skin infections and non-melanoma skin cancers in patients with immunodeficiencies resulting from e.g. HIV infection, organ transplants, or genetic disorders. Treatments for cutaneous PVs are limited and often ineffective. Thus, there is a critical need for development of novel therapeutics directed at keratinocytes, the target cell of PV infections. Keratinocytes have the potential to mount an antiviral response. Viral infection activates interferon regulator factors (IRFs), which upregulate interferons (IFN) and IFN-stimulated antiviral genes. IRFs and IFNs are also tumor-suppressing, making them particularly attractive as therapeutic targets for PV infections. However, both mucosal and cutaneous PVs can repress IRF function and IFN expression. This repression is mediated by the E6 and E7 oncogenes of mucosal PVs. While limited studies suggest that mucosal and cutaneous PVs differ in their E6 and E7 functions and that IRF regulation of the IFN response in keratinocytes differs from that in other cell types, there are many more questions than answers. Like humans, cutaneous PV infections occur in dogs and are more prevalent in immunodeficient animals. Thus, the dog offers a natural spontaneous animal model for investigation of cutaneous PV infections and for testing novel therapeutics. Indeed, our preliminary data demonstrate that canine cutaneous PV E6 and E7 differentially disrupt IFN and IFN-stimulated gene expression in canine keratinocytes. The long-range goal of our work is to develop novel therapeutics targeting IRFs and the IFN response for treatment of cutaneous PV infections in immunodeficient patients, using a canine model of PV infection. We hypothesize here that E6 and E7 from cutaneous PVs enhance viral infection by inhibiting IRF function in keratinocytes using mechanisms unique to cutaneous PVs. In Aim 1, we will inhibit expression of IRFs to determine their impact on cutaneous PV infection and on constitutive and inducible expression of IFNs and IFN-stimulated genes in keratinocytes. In Aims 2 and 3, we will determine the mechanisms for cutaneous PV E6- and E7-mediated disruption of IFNs and IFN-stimulated gene expression in keratinocytes. Specifically, we will use mass spectrometry to identify E6 and E7 binding partners that modulate IRF expression and/or function, and then identify mechanisms for E6 and E7 effects on these binding partners. Using human PV and keratinocytes will address the human disease; using canine PV and keratinocytes will advance the dog model. Results will lay the foundation for development of novel therapeutics that would by-pass E6 and E7 deleterious effects. The mentoring team includes faculty with extensive expertise in keratinocyte biology, IRF and IFN biology in differentiated cells, PV biology, and mass spectrometry proteomics and bioinformatics. In sum, the proposed research will address a critical issue in human and animal health, capitalize on the diverse skill sets of the interdisciplinary mentoring team, and enhance my maturation as an independent clinician scientist.