Immediate early events of the HPV life cycle

人乳头瘤病毒（HPV）在皮肤和粘膜的分层上皮中复制，并需要终末细胞的免疫应答。 差异化计划，以完成其生命周期。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。基底细胞 在生产性感染的隔室中，基因组水平得以维持，并且早期病毒转录物水平较低。 当受感染的细胞进入宿主细胞时，早期、中期和晚期转录物以及基因组水平增加。 终端差异化程序E6和E7癌蛋白阻止分化的细胞离开细胞 周期，导致通常与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。但也有 包括HPV 16在内的HPV类型与恶性肿瘤如宫颈癌有关。hpv诱导 转化是随着长寿命的、有复制能力的细胞中癌基因表达的失调而开始的。 基底层的细胞。虽然我们对E6和E7的功能有很好的了解， 由于缺乏合适的细胞培养模型，我们无法立即早期研究 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来源的 从低度病变或在将病毒基因组转染到原代角质形成细胞后建立的细胞系 和含有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，这是永生化细胞的必需条件。 永生对这些细胞系的研究使我们能够理解分化诱导的变化， 病毒基因表达、基因组扩增和早期病毒蛋白在此过程中的作用。然而，在这方面， 关于HPV感染建立期间的早期事件以及 病毒式转变仍然没有答案。 基于我们对HPV 16病毒粒子通过角质形成细胞的附着、结合和内化的深入研究， 我们的能力，产生病毒粒子使用异源表达系统，我们现在已经建立了一个 允许用HPV 16有效感染原代人包皮角质形成细胞（HFK）的感染模型 准病毒体该模型模拟自然感染，因为（i）它利用病毒体与细胞外基质的预结合， 基底膜等同物;（ii）允许病毒基因组有效递送至PML核体，（iii） 在未分化的HFK中，只有早期启动子而不是晚期启动子是有活性的;（iv）早期和晚期启动子是 响应于由甲基纤维素或器官型筏培养物中的生长引发的分化，导致高的 晚期转录物水平;（V）病毒基因组保持游离型并且在分化时扩增;（vi）以及 衣壳蛋白在来自HPV 16感染的细胞的器官型筏培养物的上层中表达， 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。以来 早期启动子在分化时上调，我们假设感染模型也模拟早期启动子， 在自然感染的病变中观察到启动子抑制。我们已经收集了初步证据， 第二代RNA测序表明，只有非常有限的宿主细胞基因子集在细胞中差异表达。 HPV 16感染的HFK，而不是HPV 16永生化的HFK中的数千个基因。因为感染 模型使用在293 TT生产细胞系中产生的准病毒体，其不依赖于任何非 用于病毒生产的结构性HPV因子，可以进行广泛的基因操作。反过来，这使得 参与HPV 16感染的立即早期事件的病毒因素的表征。我们有证据- 原则上，突变病毒可以产生，并发现证据表明，E7敲除影响早期和晚期 分别在单层和分化的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控中的作用， 描述E6和E7对HPV 16生命周期的贡献（Aim 2）;以及 比较E6和E7对低风险和高风险HPV类型生命周期的贡献（目标3）。的 拟议的研究将有助于填补理解HPV生命周期的即时早期事件的巨大空白。 它们还将有助于更好地理解经常假设但从未经过实验验证的 自然感染时基底细胞层癌基因表达的抑制和病毒蛋白的作用 在这个过程中玩。在未来，该模型将允许研究病毒癌基因的失调 在转化的初始事件中表达。感染模型有可能与 研究HPV 16生命周期的立即早期事件和HPV建立时的转化- 窝藏角质形成细胞是为了了解晚期分化诱导阶段。

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.