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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.

这是对“逆转录酶介导的逆行转运在HPV进入中的作用”5 R 01-AI 102876的第二个五年资助的更新申请的重新提交。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，占所有癌症的5%，数百万例生殖器疣，以及无数其他类型的皮肤和粘膜疣（大多数由非疫苗HPV类型引起）。目前还没有针对HPV的具体治疗方法。在这笔赠款的支持下，我们为了解HPV在入境期间的细胞内运输做出了重要贡献。我们发现了逆行转运在HPV感染中的核心作用，表明逆转录酶是将进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，并且在L2衣壳蛋白上发现了一种新的细胞穿透肽（CPP），其将其驱动到细胞质中以接合逆转录酶。这是第一个逆转录酶或CPP在病毒进入中发挥作用的例子。这些结果已发表在顶级期刊上，并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分裂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，即现存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，映射暴露在细胞质中的L2片段，并测试它是否依次突出穿过膜。我们将继续分离和表征抑制HPV进入的人工小TM蛋白，并使用它们来鉴定新的HPV进入因子，并剖析它们对逆行运输的贡献，我们将利用我们对HPV进入的新发现的机制理解来设计抑制肽，该抑制肽利用CPP的膜穿透活性将逆转录酶结合位点递送到细胞质中，在细胞质中它取代来自传入HPV的逆转录酶。这些实验将阐明HPV进入的重要机制方面，导致预防和治疗HPV感染的新方法的开发，并为基础细胞生物学提供重要的新见解。