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Role of Retromer-mediated Retrograde Transport in HPV Entry

逆转录酶介导的逆行转运在 HPV 进入中的作用

基本信息

批准号：
10020312
负责人：
Daniel C. Dimaio
金额：
$ 50.25万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-18 至 2020-09-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10020312
关键词：
Adopted Amaze Amino Acid Sequence Anogenital venereal warts Antiviral Agents Binding Binding Proteins Binding Sites Biological Biological Assay Biological Models Biology Carrier Proteins Cations Cell physiology Cells Cellular biology Cessation of life Chimeric Proteins Cutaneous Cytoplasm DNA Viruses Dependence Development Disease Endosomes Etiology Funding Future Genetic Screening Grant Human Human Papillomavirus Human papilloma virus infection Infection Infection prevention Integral Membrane Protein Journals Knowledge L2 viral capsid protein Lead Malignant Neoplasms Maps Mediating Membrane Mucous Membrane Mutation Nature Oncogenic Viruses Papillomavirus Pathway interactions Peptides Physiological Play Process Protein Engineering Proteins Public Health Publishing Role Sorting - Cell Movement Testing Underserved Population Vaccinated Vaccination Vaccines Virion Virus design experimental study human pathogen inhibitor/antagonist insight knock-down novel novel strategies prevent protein aminoacid sequence protein complex protein transport retrograde transport sexually transmitted virus trafficking trans-Golgi Network virome

项目摘要

This is a resubmission of a renewal application for a second five years of funding for “Role of Retromer-mediated Retrograde Transport in HPV Entry,” 5R01-AI102876. Despite effective vaccines, HPV infection and HPV-induced diseases remain a major public health problem worldwide. HPV is the most common sexually transmitted virus and is responsible for 5% of all cancer, millions of cases of genital warts, and countless cases of other types of cutaneous and mucosal warts (most caused by non- vaccine HPV types). There are no specific treatments for HPV. With the support of this grant, we have made important contributions to understanding intracellular trafficking of HPV during entry. We discovered the central role of retrograde transport in HPV infection, showed that retromer is required for sorting of the incoming virus particle into the retrograde pathway and that HPV is a new class of retromer cargo, and discovered a novel cell-penetrating peptide (CPP) on the L2 capsid protein that drives it into the cytoplasm to engage the retromer. This is the first example where retromer or a CPP has been shown to play a role in virus entry. These results have been published in top journals and have fundamentally changed our understanding of HPV entry and challenged dogma concerning the biological roles of retromer and CPPs and the definition of transmembrane proteins. Here, we will study how the L2 protein accomplishes these amazing feats. We will determine the sequence and physiological requirements for L2 protrusion using a novel split-GFP assay we developed. We will test our hypothesis that the abundance of CPP sequences in the extant papillomavirus virome reflects their membrane- penetrating activity and we will establish how sequences flanking the core CPP modulate its activity. We will establish the nature and extent of L2 membrane protrusion and determine if L2 truly adopts a TM orientation, map L2 segments exposed in the cytoplasm, and test whether it protrudes through the membrane sequentially. We will continue to isolate and characterize artificial small TM proteins that inhibit HPV entry and use them to identify novel HPV entry factors and dissect their contribution to retrograde trafficking, and we will exploit our new-found mechanistic understanding of HPV entry to design inhibitory peptides that harness the membrane-penetrating activity of CPPs to deliver the retromer binding site into the cytoplasm where it displaces retromer from incoming HPV. These experiments will elucidate important mechanistic aspects of HPV entry, lead to the development of new approaches to prevent and treat HPV infection, and provide important new insights into fundamental cell biology.

这是重新提交第二个五年资助“逆转录酶介导的逆行转运在 HPV 进入中的作用”5R01-AI102876 的续展申请。尽管有有效的疫苗，HPV 感染和 HPV 引起的疾病仍然是全球主要的公共卫生问题。 HPV 是最常见的性传播病毒，导致 5% 的癌症、数百万生殖器疣病例以及无数其他类型的皮肤和粘膜疣病例（大多数由非疫苗 HPV 类型引起）。没有针对 HPV 的具体治疗方法。在这笔赠款的支持下，我们为了解 HPV 进入细胞内的情况做出了重要贡献。我们发现了逆行转运在 HPV 感染中的核心作用，表明逆转录酶是将传入的病毒颗粒分选到逆行途径中所必需的，并且 HPV 是一类新的逆转录酶货物，并在 L2 衣壳蛋白上发现了一种新型细胞穿透肽 (CPP)，可驱动其进入细胞质以与逆转录酶结合。这是逆转录酶或 CPP 被证明在病毒进入中发挥作用的第一个例子。这些结果已发表在顶级期刊上，从根本上改变了我们对 HPV 进入的理解，并挑战了有关逆转录酶和 CPP 的生物学作用以及跨膜蛋白定义的教条。在这里，我们将研究 L2 蛋白如何完成这些惊人的壮举。我们将使用我们开发的新型 split-GFP 测定法确定 L2 突出的序列和生理要求。我们将检验我们的假设，即现有乳头瘤病毒病毒组中 CPP 序列的丰度反映了它们的膜穿透活性，并且我们将确定核心 CPP 侧翼的序列如何调节其活性。我们将确定 L2 膜突出的性质和程度，并确定 L2 是否真正采用 TM 方向，绘制暴露在细胞质中的 L2 片段图，并依次测试其是否突出穿过膜。我们将继续分离和表征抑制 HPV 进入的人工小 TM 蛋白，并使用它们来识别新的 HPV 进入因子并剖析它们对逆行贩运的贡献，我们将利用我们新发现的对 HPV 进入机制的理解来设计抑制肽，利用 CPP 的膜穿透活性将逆转录酶结合位点传递到细胞质中，并在细胞质中取代逆转录酶。即将到来的 HPV。这些实验将阐明 HPV 进入的重要机制，导致开发预防和治疗 HPV 感染的新方法，并为基础细胞生物学提供重要的新见解。