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

• 批准号: 10304915
• 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
• 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/10304915
• 关键词: 3-Dimensional; Adhesions; Anemia; Attenuated; Basal Cell; Bioinformatics; Biological Models; Bone marrow failure; Bulla; Categories; Cell Adhesion; Cell Differentiation process; Cells; Centers for Disease Control and Prevention (U.S.); Chemopreventive Agent; Childhood; Complex; DNA Interstrand Crosslinking; DNA Repair; Data; Defect; Dependence; Desmosomes; Disease; Electron Microscopy; Elements; Enzymes; Epidermis; Fanconi Anemia pathway; Fanconi' 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; Phenotype; Predisposition; Prevention; Production; Public Health; Publications; Publishing; Reporting; Research; Resistance; Risk; Role; Route; Signal Transduction; Skin; Squamous cell carcinoma; Supporting Cell; Surface; System; Testing; Tissue Differentiation; 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; epidermal stem cell; experimental study; genome integrity; induced pluripotent stem cell; inhibitor; innovation; keratinocyte; keratinocyte differentiation; knock-down; lipid metabolism; loss of function; loss of function mutation; metabolic abnormality assessment; patient population; prevent; response; restoration; single-cell RNA sequencing; stem cells; targeted agent; therapeutic target; transcriptome; transcriptomics

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）感染是一个全球性的公共卫生威胁，但感染和复制仍然存在， 人们对此知之甚少，从而阻碍了用于癌症预防和治疗的抗病毒药物的开发。的目标 这项建议有两个方面。首先，我们定义了范可尼贫血通路在抑制贫血中的遗传作用。 表皮对HPV感染和复制的易感性。其次，我们将测试临床相关的抑制剂， 神经节苷脂生物合成和信号传导（Aim 1），并定义新的靶标（Aim 2），以预防或减弱这种 易感性HPV的生命周期发生在人类表皮，并与此完整性错综复杂地联系在一起。 复层组织和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（ESPCs）位于基底细胞层，分化的后代位于 在更浅的层中。对于病毒生命周期的开始，HPV必须感染ESPCs。进入这些基底细胞 需要表皮完整性的暂时破坏受感染的ESPCs然后迁移到表面， 在途中鉴别病毒基因组扩增在一个特征不佳的终末亚群中被触发， 分化的细胞，随后是感染性子代的增殖和释放。我们最近的流行病学 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患者具有 HPV阳性的风险显著增加，表明FA途径功能丧失可能增加 HPV感染易感性和/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生命周期：初始感染和晚期感染。 放大在没有HPV的情况下，FA途径缺陷会减少角质形成细胞的粘附，并且 加速皮肤起泡-表明宿主组织的结构损伤，这可能有助于HPV 感染这将在患者源性FA诱导和常规系统中使用电子探针进行测试。 显微镜，脂质代谢机制的分子研究，以及HPV感染性的研究。 将测试可用的神经节苷脂生物合成和Rac 1抑制剂预防初始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通路缺陷引发了 过度和异位病毒基因组扩增-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+复制系统进行检验 条件FA，并通过单细胞RNA测序，将确定转录组之间的区别 HPV复制和非复制细胞，在存在和不存在功能性FA途径（目的2）。 候选调节因子将在3D表皮中进行验证和映射，并且推定的效应物靶向于 减弱FA超容许（和正常）人类宿主中的HPV复制。我们一起， 这是发现新靶点和化学预防剂的必要步骤， 最大的完整性和对HPV感染和扩增的抗性。