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Strengthening epidermal defenses for the prevention of HPV infection and replication

加强表皮防御，预防 HPV 感染和复制

基本信息

批准号：
10524745
负责人：
Susanne I Wells
金额：
$ 31.55万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-04 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10524745
关键词：
3-Dimensional Acceleration Adhesions Anemia Attenuated Basal Cell Bioinformatics Biological Models Bone marrow failure Bulla Categories Cell Adhesion Cell Differentiation process Cells Chemopreventive Agent Childhood Complex DNA Interstrand Cross-Link Repair DNA Repair Data Defect Dependence Desmosomes Disease Electron Microscopy Elements Endowment Enzymes Epidermis Fanconi Anemia pathway Fanconi&apos s Anemia Ganglioside Biosynthesis Pathway Gangliosides Genes Genetic Genetic Transcription Genome Genomic Instability Goals Guanosine Triphosphate Phosphohydrolases Hematopoietic stem cells Host Defense Human Human Papillomavirus Human papilloma virus infection Human papillomavirus 16 Immunofluorescence Immunologic Impairment In Situ Hybridization Infection Inherited Intercellular Junctions Investigation Laboratories Large Keratinocyte Life Cycle Stages Link Lipids Malignant Neoplasms Maps Mass Spectrum Analysis Membrane Microdomains Modeling Molecular Mucous Membrane Mutation Names Pathway interactions Patients Penetration Phenotype Predisposition Prevention Production Productivity Public Health Publications Publishing Reporting Research Resistance Risk Role Route Signal Transduction Skin Squamous cell carcinoma Supporting Cell Surface System Testing Tissues Undifferentiated Viral Viral Genome Virus Virus Diseases Virus Replication Work antiviral drug development cancer prevention cancer therapy cell motility cell type clinically relevant crosslink epidemiology study experimental study genome integrity induced pluripotent stem cell inhibitor innovation keratinocyte keratinocyte differentiation knock-down lipid metabolism loss of function loss of function mutation migration patient population permissiveness prevent response restoration single-cell RNA sequencing stem stem cells therapeutic target transcriptome transcriptomics

项目摘要

ABSTRACT Human papillomavirus (HPV) infection is a global threat to public health, but infection and replication remain poorly understood, thus hindering the development of antivirals for cancer prevention and treatment. The goals of this proposal are two-fold. First, we define the genetic role of the Fanconi anemia pathway in suppressing epidermal susceptibility to HPV infection and replication. Second, we will test clinically relevant inhibitors of ganglioside biosynthesis and signaling (Aim 1), and define new targets (Aim 2), to prevent or attenuate such susceptibility. The HPV life cycle takes place in human epidermis, and is intricately linked to the integrity of this stratified tissue and the differentiation of keratinocytes. There are two basic categories of keratinocytes – epidermal stem and progenitor cells (ESPCs) located in the basal cell layer, and differentiated progeny located in more superficial layers. For the viral life cycle to begin, HPV must infect ESPCs. Access to these basal cells requires a temporary breakdown in epidermal integrity. Infected ESPCs then migrate to the surface, differentiating en route. Viral genome amplification is triggered in a poorly characterized subset of terminally differentiated cells, followed by encapsidation and release of infectious progeny. Our recent epidemiological studies of the inherited genome instability disorder Fanconi anemia (FA) demonstrated that FA patients have a significantly increased risk of HPV positivity, suggesting that FA pathway loss of function may increase susceptibility to HPV infection and/or proclivity for amplification. Our published and preliminary data indicate that FA pathway deficiency stimulates the HPV life cycle at two critical stages: initial infection and late amplification. In the absence of HPV, FA pathway deficiency diminished keratinocyte adhesion, and accelerated skin blistering – suggesting structural impairment of the host tissue, which could facilitate HPV infection. This will be tested in patient-derived FA-inducible and conventional systems using electron microscopy, molecular investigation of mechanisms focused on lipid metabolism, and studies of HPV infectivity. Available ganglioside biosynthesis and Rac1 inhibitors will be tested for their ability to prevent initial HPV infection via restoration of epidermal integrity. In the presence of HPV, FA pathway deficiency triggered excessive and ectopic viral genome amplification – suggesting that the intact FA pathway suppresses HPV replication and progeny production. This hypothesis will be tested by generating an HPV+ replication system conditional for FA, and by single-cell RNA sequencing that will identify transcriptomic distinctions between HPV-replicating and -nonreplicating cells, in the presence and absence of a functional FA pathway (Aim 2). Candidate regulators will be validated and mapped in 3D epidermis, and putative effectors targeted to attenuate HPV replication in the FA hyper-permissive (and normal) human host. Together, we take the first required step towards discovering new targets and chemopreventive agents that endow human epidermis with maximal integrity and resistance to HPV infection and amplification.

摘要人乳头瘤病毒(HPV)感染是对公共卫生的全球威胁，但感染和复制仍然存在了解甚少，从而阻碍了用于癌症预防和治疗的抗病毒药物的开发。目标这项提议有两个方面。首先，我们定义了Fanconi贫血途径在抑制表皮对HPV感染和复制的易感性。第二，我们将测试临床相关的抑制物神经节苷脂的生物合成和信号传递(目标1)，并定义新的靶点(目标2)，以防止或减弱这种情况敏感度。人类乳头状瘤病毒的生命周期发生在人的表皮中，并与其完整性密切相关。复层组织与角质形成细胞的分化。角质形成细胞有两种基本类型：表皮干细胞和祖细胞(ESPC)位于基底层，分化的后代位于在更浅的层面上。要开始病毒生命周期，HPV必须感染ESPC。通向这些基底细胞需要暂时破坏表皮的完整性。然后，受感染的ESPC迁移到地表，在途中进行差异化。病毒基因组扩增在特征不佳的终末期亚群中被触发分化细胞，然后包埋和释放感染性后代。我们最新的流行病学研究对遗传性基因组不稳定性疾病范可尼贫血(FA)的研究表明，FA患者有一种显著增加HPV阳性的风险，提示FA途径功能丧失可能增加对HPV感染的易感性和/或扩增倾向。我们公布的和初步的数据表明 FA途径缺陷在两个关键阶段刺激HPV的生命周期：初始感染和晚期放大。在没有HPV的情况下，FA途径缺陷会降低角质形成细胞的粘附性，并且皮肤起泡加速-表明宿主组织结构受损，这可能促进HPV 感染。这将在患者衍生的FA诱导系统和传统系统中进行测试，使用电子显微镜，以脂代谢为重点的机制的分子研究，以及HPV感染性的研究。现有的神经节苷脂生物合成和rac1抑制剂将被测试其预防初始HPV的能力。通过恢复表皮完整性而感染。在HPV存在的情况下，FA途径缺陷被触发病毒基因组过度和异位扩增--表明完整的FA途径抑制HPV 复制和后代生产。这一假设将通过生成HPV+复制系统来验证有条件的FA，并通过单细胞RNA测序，将识别转录差异 HPV复制和非复制细胞，在存在和不存在功能性FA途径的情况下(目标2)。候选调节剂将在3D表皮中得到验证和映射，并针对假定的效应器减弱HPV在FA过度许可(和正常)人类宿主中的复制。我们一起夺得第一名需要一步来发现新的靶点和化学预防药物，赋予人类表皮最大的完整性和对HPV感染和扩增的抵抗力。