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

• 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
• 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

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。