Immediate early events of the HPV life cycle

HPV 生命周期的早期事件

• 批准号: 9358047
• 负责人: Martin Sapp
• 金额: $ 33.17万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9358047
• 关键词: Address; Affect; Alpha Cell; Appearance; Basal Cell; Basement membrane; Benign; Binding; Cancerous; Capsid Proteins; Cell Compartmentation; Cell Culture Techniques; Cell Cycle; Cell Differentiation process; Cell Line; Cell Nucleus; Cells; Cervix carcinoma; Clone Cells; DNA; Deposition; Dermal; Development; Early Promoters; Epithelial; Epithelial Cells; Epithelium; Event; Extracellular Matrix; Future; Gene Expression; Generations; Genes; Genetic Screening; Genome; Growth; Human; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 16; Income; Infection; Investigation; Knock-out; Late Promoters; Lesion; Life Cycle Stages; Maintenance; Malignant Neoplasms; Measures; Methylcellulose; Modeling; Mucous Membrane; Nuclear; Oncogene Deregulation; Oncogenes; Oncoproteins; Papilloma; Play; Premalignant; Primary Infection; Process; Production; Regulation; Repression; Role; Skin; Stratified Epithelium; Stratum Basale; System; Testing; Tonsil; Transcript; Transcriptional Regulation; Transfection; Undifferentiated; Viral; Viral Genes; Viral Genome; Viral Oncogene; Viral Physiology; Viral Proteins; Virion; Virus; base; differential expression; established cell line; genetic analysis; genetic manipulation; high risk; immortalized cell; improved; innovation; keratinocyte; monolayer; mutant; next generation; penis foreskin; prevent; programs; promoter; therapy development; tool; transcriptome sequencing; tumor

Human papillomaviruses (HPV) replicate in stratified epithelia of the skin and mucosa and require the terminal differentiation program to complete their lifecycle. HPV access the basal cells of these epithelia through lesions and establish infection after genome delivery to the nucleus and initial genome amplification. In the basal cell compartment of productive infections, genome levels are maintained and early viral transcript levels are low. Early, intermediate and late transcripts as well as genome levels increase when infected cells enter the terminal differentiation program. The E6 and E7 oncoproteins prevent differentiated cells from exiting the cell cycle, resulting in the appearance of the benign lesions typically associated with HPV infection (warts, papillomas) and allowing genome amplification, late gene expression and virus production. However, some HPV types including HPV16 are associated with malignancies such as cervical carcinoma. HPV-induced transformation is initiated with the deregulation of oncogene expression in the long-lived, replication-competent cells of the basal layer. While we do have a very good understanding of E6 and E7 function during transformation, the lack of appropriate cell culture models have prevented us from studying immediate early events following infection of basal cells. Most studies of the HPV life cycle depend on keratinocytes-derived cell lines established from low-grade lesions or after transfection of viral genome into primary keratinocytes and outgrowth of HPV DNA-containing cell clones. In contrast to basal cells in productive lesions, HPV- immortalized cell lines express high levels of the E6 and E7 oncoproteins, which is a requirement for immortalization. The study of such cell lines allowed an understanding of differentiation-induced changes to viral gene expression, genome amplification and the role early viral proteins play in this process. However, many questions regarding early events during the establishment of HPV infection as well as early events of viral transformation remain unanswered. Based on our intimate studies of attachment, binding and internalization of HPV16 virions by keratinocytes and our capability to generate virions using heterologous expression systems, we have now established an infection model that allows efficient infection of primary human foreskin keratinocytes (HFK) with HPV16 quasivirions. The model mimics natural infection in that (i) it utilizes prebinding of virions to extracellular matrix, the basement membrane-equivalent; (ii) allows efficient delivery of viral genome to PML nuclear bodies, (iii) only the early but not the late promoter is active in undifferentiated HFK; (iv) early and late promoter are responsive to differentiation triggered by growth in methylcellulose or organotypic raft cultures resulting in high levels of late transcripts; (v) viral genome remains episomal and is amplified upon differentiation; (vi) and capsid proteins are expressed in the upper layers of organotypic raft cultures derived from HPV16-infected HFK. Thus, the infection model is the first cell culture model to recapitulate the complete viral lifecycle. Since the early promoter is upregulated upon differentiation, we assume that the infection model also mimics early promoter repression observed in naturally infected lesions. We have gathered preliminary evidence using next generation RNA sequencing that only a very limited subset of host cell genes is differentially expressed in HPV16-infected HFK as opposed to thousands of genes in HPV16-immortalized HFK. Because the infection model uses quasivirions generated in the 293TT production cell line, which does not depend on any non- structural HPV factor for virus production, it is amenable to extensive genetic manipulations. In turn, this allows the characterization of viral factors involved in the immediate early events of HPV16 infection. We have prove- of-principal that mutant viruses can be generated and found evidence that E7 knockout affects early and late viral promoter activity in monolayer and differentiated HFK cells, respectively. We propose to utilize the infection model to determine the role of viral and host cell factors in genome amplification and regulation of early promoter activity (Aim1); delineate the contributions of E6 and E7 to the HPV16 lifecycle (Aim 2); and compare the contributions of E6 and E7 to the lifecycles of low- and high-risk HPV types (Aim 3). The proposed studies will help fill huge gaps in the understanding of immediate early events of the HPV lifecycle. They will also help to gain a better understanding of the often hypothesized but never experimentally tested repression of oncogene expression in the basal cell layer during natural infection and the role viral proteins play during this process. In future, this model will allow investigating the deregulation of viral oncogene expression during initial events of transformation. The infection model has the potential to be as important for the study of immediate early events of the HPV16 lifecycle and transformation as the establishment of HPV- harboring keratinocytes was for understanding the late differentiation-induced stages.

人乳头瘤病毒（HPV）在皮肤和粘膜的分层上皮中复制，需要末端 分化计划以完成其生命周期。 HPV通过病变进入这些上皮的基础细胞 并在基因组传递到细胞核和初始基因组扩增后建立感染。在基础细胞中 生产性感染，基因组水平的腔室保持不变，早期病毒转录水平较低。 早期，中间和晚成绩单以及受感染细胞进入的基因组水平增加 终端分化计划。 E6和E7癌蛋白可以防止分化细胞退出细胞 循环，导致通常与HPV感染相关的良性病变的出现（疣， 乳头状瘤）并允许基因组扩增，晚期基因表达和病毒产生。但是，有些 包括HPV16在内的HPV类型与宫颈癌等恶性肿瘤有关。 HPV引起的 转化是通过在长寿，能力的癌症表达的失调中引发的 基础层的细胞。虽然我们对E6和E7功能的理解确实很好 转型，缺乏适当的细胞培养模型使我们无法立即研究 基础细胞感染后的事件。 HPV生命周期的大多数研究取决于角质形成细胞的衍生 从低度病变或将病毒基因组转染后建立的细胞系 和含HPV DNA的细胞克隆的产物。与生产性病变中的基底细胞相反，HPV- 永生的细胞系表达高水平的E6和E7癌蛋白，这是对 永生化。对此类细胞系的研究允许了解分化引起的变化 病毒基因表达，基因组扩增和早期病毒蛋白在此过程中起着作用。然而， 有关HPV感染期间早期事件以及早期事件的许多问题 病毒转化仍未得到解答。 基于我们对角质形成细胞HPV16病毒体的依恋，结合和内在化的深入研究 我们使用异源表达系统生成病毒体的能力，我们现在已经建立了一个 感染模型，可有效感染HPV16的原代人皮角质形成细胞（HFK） 排序。该模型模仿自然感染，因为（i）它利用了病毒体对细胞外基质的预订， 地下膜等效； （ii）允许将病毒基因组有效地传递到PML核体，（iii） 只有早期但不是晚期启动子在未分化的HFK中活跃。 （iv）早期和晚期发起人是 对甲基纤维素或器官筏培养物的生长触发的分化有效的反应，导致高 晚成绩单的水平； （v）病毒基因组仍然是偶发性的，并在分化后被扩增； （vi）和 衣壳蛋白在源自HPV16感染的器官型筏培养物的上层表达 HFK。因此，感染模型是概括完整病毒生命周期的第一个细胞培养模型。自从 早期启动子在分化后被上调，我们假设感染模型也早期模仿 在自然感染的病变中观察到启动子抑制。我们已经收集了使用下一步的初步证据 在宿主细胞基因的子集中只有非常有限的子集的生成RNA测序在 HPV16感染的HFK与HPV16降低的HFK中的数千个基因相反。因为感染 模型使用293TT生产细胞系中产生的准段，这不取决于任何非非 - 病毒生产的结构HPV因子，它可以接受广泛的遗传操作。反过来，这允许 涉及HPV16感染的早期事件的病毒因素的表征。我们已经证明了 可以产生突变病毒的主要概念，并发现证据表明E7敲除会影响早期和晚期 单层和分化的HFK细胞中的病毒启动子活性。我们建议利用 感染模型，以确定病毒和宿主细胞因子在基因组扩增和调节中的作用 早期启动子活动（AIM1）；描述E6和E7对HPV16生命周期的贡献（AIM 2）；和 比较E6和E7对低风险HPV类型的生命周期的贡献（AIM 3）。这 拟议的研究将有助于填补对HPV生命周期早期事件的理解的巨大空白。 他们还将有助于更好地了解经常假设的，但从未经过实验测试 自然感染期间基底细胞层中癌基的表达和作用病毒蛋白的抑制 在此过程中播放。将来，该模型将允许调查病毒果仁的放松管制 在转换的初始事件中的表达。感染模型有可能对 将HPV16生命周期的早期事件和转化作为HPV-的建立研究 携带角质形成细胞是为了理解晚期分化引起的阶段。