Immediate early events of the HPV life cycle

HPV 生命周期的早期事件

基本信息

批准号：
10163808
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
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163808
关键词：
Address Affect 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 Event Extracellular Matrix Future Gene Expression Generations Genes Genetic Screening Genome Growth Human Human Papillomavirus Human papilloma virus infection Human papillomavirus 16 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 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 cell immortalization differential expression established cell line genetic analysis genetic manipulation high risk improved innovation keratinocyte monolayer mutant next generation penis foreskin premalignant prevent programs promoter therapy development tool transcriptome sequencing tumor wound

项目摘要

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) 在皮肤和粘膜的复层上皮细胞中复制，需要末端差异化计划来完成其生命周期。 HPV 通过病变进入这些上皮的基底细胞并在基因组递送至细胞核和初始基因组扩增后建立感染。在基底细胞中在生产性感染区室中，基因组水平得以维持，早期病毒转录水平较低。当受感染的细胞进入细胞时，早期、中期和晚期转录物以及基因组水平都会增加终端分化计划。 E6 和 E7 癌蛋白阻止分化细胞离开细胞周期，导致出现通常与 HPV 感染相关的良性病变（疣、乳头状瘤）并允许基因组扩增、晚期基因表达和病毒产生。然而，一些包括 HPV16 在内的 HPV 类型与宫颈癌等恶性肿瘤有关。 HPV诱导的转化是随着长寿、具有复制能力的细胞中癌基因表达的失调而开始的。基底层细胞。虽然我们在使用过程中确实对E6和E7的功能有了很好的了解由于缺乏合适的细胞培养模型，我们无法立即进行转化研究基底细胞感染后的事件。大多数 HPV 生命周期的研究依赖于角质形成细胞衍生的从低度病变或将病毒基因组转染至原代角质形成细胞后建立的细胞系以及含有 HPV DNA 的细胞克隆的生长。与生产性病变中的基底细胞相比，HPV- 永生化细胞系表达高水平的 E6 和 E7 癌蛋白，这是永生化。对此类细胞系的研究使我们能够了解分化诱导的变化病毒基因表达、基因组扩增以及早期病毒蛋白在此过程中发挥的作用。然而，关于 HPV 感染期间的早期事件以及 HPV 感染的早期事件的许多问题病毒转化仍然没有答案。基于我们对 HPV16 病毒颗粒通过角质形成细胞的附着、结合和内化的深入研究，由于我们有能力使用异源表达系统产生病毒粒子，我们现在已经建立了一个允许用 HPV16 有效感染原代人包皮角质形成细胞 (HFK) 的感染模型准病毒粒子。该模型模拟自然感染，因为（i）它利用病毒粒子与细胞外基质的预结合，基底膜等效物； (ii) 允许将病毒基因组有效递送至 PML 核体，(iii) 在未分化的 HFK 中，只有早期启动子有活性，而晚期启动子则没有活性； (iv) 早期和晚期启动子对甲基纤维素或器官筏培养物中的生长引发的分化作出反应，从而导致高晚期成绩单的水平； (v) 病毒基因组保持游离状态并在分化时扩增； (六) 和衣壳蛋白在 HPV16 感染的器官型筏培养物的上层表达 HFK。因此，感染模型是第一个概括完整病毒生命周期的细胞培养模型。自从早期启动子在分化时上调，我们假设感染模型也模仿早期在自然感染的病变中观察到启动子抑制。我们已经使用下一步收集了初步证据一代 RNA 测序表明，只有非常有限的宿主细胞基因子集在 HPV16 感染的 HFK 与 HPV16 永生化 HFK 中的数千个基因不同。因为感染模型使用 293TT 生产细胞系中生成的准病毒粒子，该细胞系不依赖于任何非作为病毒产生的 HPV 结构因子，它适合广泛的基因操作。反过来，这允许 HPV16 感染早期事件中涉及的病毒因素的特征。我们已经证明—— 原则上可以产生突变病毒，并发现 E7 敲除影响早期和晚期的证据分别在单层和分化的 HFK 细胞中病毒启动子活性。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性（目标1）；描述 E6 和 E7 对 HPV16 生命周期的贡献（目标 2）；和比较 E6 和 E7 对低危和高危 HPV 类型生命周期的贡献（目标 3）。这拟议的研究将有助于填补对 HPV 生命周期早期事件理解的巨大空白。它们还将有助于更好地理解经常假设但从未经过实验测试的问题自然感染过程中基底细胞层癌基因表达的抑制以及病毒蛋白的作用在此过程中进行游戏。将来，该模型将允许研究病毒癌基因的失调转化初始事件期间的表达。感染模型有可能同样重要随着HPV-16的建立，研究HPV16生命周期和转化的早期事件含有角质形成细胞是为了了解晚期分化诱导阶段。