Immediate early events of the HPV life cycle

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 差异化计划以完成其生命周期。HPV通过病变进入这些上皮细胞的基底细胞 并在基因组送入细胞核和初始基因组扩增后建立感染。在基底细胞中 在生产性感染中，基因组水平保持不变，早期病毒转录物水平较低。 早期、中期和晚期转录本以及基因组水平在感染细胞进入 终端分化程序。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。然而，一些人 包括HPV16在内的HPV类型与宫颈癌等恶性肿瘤有关。人乳头瘤病毒感染 在长寿的、有复制能力的人中，癌基因表达的解除调控启动了转化 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，我们无法立即进行早期研究。 基底细胞感染后发生的事件。大多数对HPV生命周期的研究依赖于角质形成细胞来源的 从低级别皮损或将病毒基因组导入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。与生殖性病变中的基底细胞相比，HPV- 永生化细胞株表达高水平的E6和E7癌蛋白，这是 永垂不朽。对这些细胞系的研究有助于理解分化诱导的变化。 病毒基因表达、基因组扩增以及早期病毒蛋白在这一过程中所起的作用。然而， 关于HPV感染早期事件以及HPV感染早期事件的许多问题 病毒式转变仍然没有答案。 基于我们对角质形成细胞对HPV16病毒粒子的附着、结合和内化的深入研究 我们使用异源表达系统产生病毒粒子的能力，我们现在已经建立了一种 HPV16感染原代人包皮角质形成细胞模型的建立 准病毒子。该模型模拟了自然感染，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地输送到PML核体，(Iii) 在未分化的HfK中，只有早期启动子活性，而不是晚期启动子活性；(4)早期启动子和晚期启动子 对由甲基纤维素或器官型树枝培养物中的生长引发的分化作出反应，从而导致高 晚期转录本的水平；(V)病毒基因组保持异构体，并在分化后扩增；(Vi)和 衣壳蛋白在感染HPV16的器官型RAFT培养物的上层表达 赫芬顿。因此，感染模型是第一个概括整个病毒生命周期的细胞培养模型。自.以来 早期启动子在分化后上调，我们假设感染模型也模仿早期 在自然感染的皮损中观察到启动子抑制。我们已经用NEXT收集了初步证据 一代RNA测序，只有非常有限的宿主细胞基因子集在 HPV16感染的HfK与HPV16永生化的HfK中的数千个基因相反。因为感染 模型使用了293TT生产细胞系中产生的准病毒粒子，它不依赖于任何非 结构HPV因子用于病毒生产，它易于进行广泛的遗传操作。反过来，这又允许 与HPV16感染早期事件有关的病毒因素的特征。我们已经证明了- 主要是可以产生突变病毒，并发现E7基因敲除对早期和晚期影响的证据 病毒启动子在单层和分化的HfK细胞中的活性。我们建议利用 确定病毒和宿主细胞因子在基因组扩增和调节中的作用的感染模型 早期启动子活性(Aim1)；描述E6和E7对HPV16生命周期的贡献(Aim 2)；以及 比较E6和E7对低危型和高危型HPV生命周期的贡献(目标3)。这个 拟议的研究将有助于填补对HPV生命周期中直接早期事件的理解方面的巨大空白。 它们还将有助于更好地理解通常是假设的但从未经过实验检验的 自然感染过程中基底层癌基因表达的抑制及其在病毒蛋白中的作用 在这一过程中发挥作用。在未来，这个模型将允许研究病毒致癌基因的放松管制。 在转化的初始事件中的表达。感染模型有可能对 随着HPV-16的建立，HPV16生命周期和转化的早期事件的研究 角质形成细胞的存在是为了了解分化后期的诱导阶段。

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.