Mechanisms of Interferon Regulatory Factor Dysfunction by Cutaneous Papillomaviruses

项目摘要/摘要 乳头瘤病毒(PV)感染严重影响人类健康。几乎所有病例都是由粘膜疱疹病毒引起的 人类宫颈癌，而皮肤PVs会导致严重的皮肤感染和非黑色素瘤皮肤癌 在因艾滋病毒感染、器官移植或遗传疾病等原因导致免疫缺陷的患者中。 皮肤PVs的治疗是有限的，而且往往无效。因此，迫切需要发展。 针对角质形成细胞的新疗法，角质形成细胞是PV感染的目标细胞。角质形成细胞具有潜在的 以产生抗病毒反应。病毒感染激活干扰素调节因子(IRF)，使其上调 干扰素和干扰素刺激的抗病毒基因。IRFS和IFN也具有肿瘤抑制作用，使它们 作为治疗PV感染的靶点特别有吸引力。然而，粘膜和皮肤静脉曲张都可以 抑制IRF功能和干扰素表达。这种抑制是由粘膜的E6和E7癌基因介导的 PVS。虽然有限的研究表明，粘膜静脉曲张和皮肤静脉曲张的E6和E7功能不同，而且 IRF对角质形成细胞中干扰素反应的调节不同于其他类型的细胞，还有更多 问题多于答案。与人类一样，皮肤PV感染也发生在狗身上，更普遍的是 免疫缺陷的动物。因此，狗提供了一种自然自发的动物模型，用于研究 用于皮肤PV感染和测试新的治疗方法。事实上，我们的初步数据表明， 犬皮肤疱疹病毒E6和E7对干扰素及其刺激的基因表达的干扰作用 角质形成细胞。我们工作的长期目标是开发针对IRFs和干扰素的新疗法 用犬PV模型治疗免疫缺陷患者皮肤PV感染的疗效 感染。我们推测，皮肤疱疹病毒的E6和E7通过抑制IRF而增强病毒感染。 在角质形成细胞中的功能使用皮肤静脉特有的机制。在目标1中，我们将抑制 确定其对皮肤PV感染的影响以及对IFN的结构性和诱导性表达的影响 和角质形成细胞中干扰素刺激的基因。在目标2和目标3中，我们将确定皮肤损伤的机制 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。具体来说， 我们将使用质谱仪鉴定调节IRF表达和/或的E6和E7结合伙伴 功能，然后确定E6和E7对这些结合伙伴的影响机制。使用人类PV和 角质形成细胞将解决人类疾病；使用犬PV和角质形成细胞将促进狗的模型。 这些结果将为开发绕过E6和E7有害的新疗法奠定基础 效果。指导团队包括在角质形成细胞生物学、IRF和干扰素方面拥有丰富专业知识的教师。 分化细胞生物学、光伏生物学、质谱学、蛋白质组学和生物信息学。总而言之， 拟议的研究将解决人类和动物健康中的一个关键问题，利用不同的技能组合 作为一名独立的临床医生科学家，我将成为跨学科指导团队中的一员。

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.