Human papillomavirus entry: late trafficking and establishment of infection

人乳头瘤病毒进入：晚期贩运和感染建立

基本信息

批准号：
10316816
负责人：
Martin Sapp
金额：
$ 44.5万
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：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10316816
关键词：
Anaphase Basal Ganglia Body Composition Cell Culture Techniques Cell Nucleus Cells Chromosomes Cytoplasmic Organelle Data Deposition Dynein ATPase Electron Microscopy Endosomes Enzymes Epithelial Event Genetic Transcription Genome Homeostasis Human Papillomavirus Human papillomavirus 16 Human papillomavirus 18 Image Immune Impairment Infection JAK1 gene Kinesin Kinetics Knock-out Knowledge L2 viral capsid protein Lead Life Cycle Stages Link Lipase Maintenance Mediating Membrane Metaphase Microscopy Microtubule Proteins Microtubule-Organizing Center Microtubules Minor Mitosis Mitotic Mitotic Chromosome Mitotic spindle Modeling Motor Mucous Membrane Natural Immunity Nuclear Nuclear Envelope Pathway interactions Pharmacology Phase Phospholipase Phospholipase C Play Prevention Primary Infection Prophase Protein Isoforms Proteins Quality Control Reporting Research Role Signal Transduction Skin Small Interfering RNA Stains Stratified Epithelium Techniques Testing Time Transcriptional Regulation Vesicle Viral Viral Genome Virion Virus Diseases base follow-up inhibitor/antagonist keratinocyte knock-down live cell imaging mutant novel nucleocytoplasmic transport prevent recruit sensor small molecule inhibitor trafficking trans-Golgi Network tumor vesicle transport

项目摘要

The trans-golgi network (TGN) serves as intermediate target organelle of the cytoplasmic transport during HPV infectious entry. It has previously been established that nuclear envelope breakdown is required for viral genome to enter the nucleus. We recently found that viral genome dissociates from TGN in prophase and associates with microtubules to accumulate at the microtubule-organizing center. At later stages of mitosis (metaphase), viral genome moves away from the mitotic spindle poles towards chromosomes. Our data point to a switch in the directionality of microtubule mediated transport from minus end- to plus end-directed during mitosis. Preliminary inhibitor studies point to dynein and Kif11 as motor proteins involved in the transport. Shift in directionality and the role of motor proteins for microtubule mediated transport will be further studied in Aim 1. Rather than egressing from the endocytic compartment in prophase, viral genome is still found in a membrane-bound vesicular compartment throughout mitotic transport. Viral genome egresses from membrane vesicles after nuclear envelope reformation. These are paradigm shifting findings, which have recently been confirmed by Day et al using electron microscopy. It is unclear, how HPV achieves egress from the nuclear transport vesicles. We hypothesized that HPV utilizes a cellular pathway to dissolve vesicles ending up in the nucleus, whether on purpose (e.g. viral infection) or accidentally. Indeed, preliminary data suggest that specific isoforms of phospholipase C, whose knockdown severely impairs HPV16 infection at a stage after nuclear delivery, may be involved in dissolving the vesicular membrane. The hypothesis will be tested in Aim 2 using knockdown and knockout approaches combined with differential staining techniques and EM. Release from transport vesicles is preceded by PML protein recruitment. Recruitment of another component of PML nuclear bodies (NB), Sp100, is specifically delayed in PML NB assembling around incoming genome. In cells deficient for PML protein, viral genome is lost and therefore transcriptionally inactive. In Aim 2, we will test our hypothesis that the recruitment of specific PML isoforms is the underlying reason for delayed recruitment of Sp100 and that HPV-induced PML NB reorganization is important for transcriptional regulation of the incoming genome. We have recently described a novel cell culture model that allows efficient infection of primary keratinocytes for investigating the role of PML NB components in a relevant cell culture model. Overall, our proposed studies will fill a huge gap in the understanding of microtubule mediated transport during late HPV intracellular trafficking and will help us unravel the role of PML NB during the immediate early events of the HPV life cycle essential for establishing infection. Furthermore, the proposal has the potential to uncover a hitherto unrecognized cellular pathway to dissolve vesicles ending up in the nucleus. Even though speculative at this time, the observations may trigger a rethinking of possible roles of vesicular cargo delivery to the nucleus during mitosis and its role in nucleus homeostasis.

跨高尔基体网络 (TGN) 作为 HPV 期间细胞质运输的中间靶细胞器传染性进入。此前已确定病毒基因组需要核膜破裂进入细胞核。我们最近发现病毒基因组在前期与TGN分离并与微管在微管组织中心聚集。在有丝分裂的后期（中期），病毒基因组从有丝分裂纺锤体极移向染色体。我们的数据指向一个开关有丝分裂期间微管介导的从负端到正端的运输的方向性。初步的抑制剂研究表明动力蛋白和 Kif11 是参与运输的运动蛋白。方向性的转变和运动蛋白在微管介导的运输中的作用将在目标 1 中进一步研究。在前期从内吞区室中出来后，病毒基因组仍然存在于膜结合的结构中。整个有丝分裂运输过程中的囊泡区室。病毒基因组从膜囊泡中逸出核膜改造。这些是范式转变的发现，最近已被 Day 证实等人使用电子显微镜。目前尚不清楚 HPV 如何从核转运囊泡中排出。我们假设 HPV 利用细胞途径溶解最终进入细胞核的囊泡，无论是在出于目的（例如病毒感染）或意外。事实上，初步数据表明，特定亚型磷脂酶 C 的敲低会严重损害核交付后阶段的 HPV16 感染，可能是参与溶解囊泡膜。该假设将在目标 2 中使用击倒和敲除方法结合差异染色技术和 EM。从运输囊泡的释放是先于 PML 蛋白募集。招募 PML 核体 (NB) 的另一个组成部分，Sp100， PML NB 在传入基因组周围的组装被特别延迟。在缺乏 PML 蛋白的细胞中，病毒基因组丢失，因此转录失活。在目标 2 中，我们将检验我们的假设：招募特定 PML 亚型的缺失是 Sp100 募集延迟的根本原因，并且 HPV 诱导的 PML NB 重组对于传入基因组的转录调控非常重要。我们最近描述了一种新型细胞培养模型，可有效感染原代角质形成细胞，以研究 PML 的作用相关细胞培养模型中的 NB 组件。总的来说，我们提出的研究将填补对微管介导的运输的理解的巨大空白晚期 HPV 细胞内运输，将帮助我们揭示 PML NB 在早期事件中的作用 HPV 生命周期对于建立感染至关重要。此外，该提案还有可能揭示迄今为止尚未认识到的溶解囊泡并最终进入细胞核的细胞途径。尽管是投机性的此时，这些观察结果可能会引发人们重新思考囊泡货物向细胞核输送的可能作用有丝分裂期间及其在细胞核稳态中的作用。