Mechanisms of human papillomavirus entry

人乳头瘤病毒的侵入机制

• 批准号: 10240651
• 负责人: Daniel C. Dimaio
• 金额: $ 100.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-17 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10240651
• 关键词: Adopted; Amaze; Anogenital venereal warts; Binding; Binding Sites; Biology; Cancer Etiology; Cell Nucleus; Cell physiology; Cells; Cellular biology; Cytoplasm; Funding; Future; Genetic; Grant; HIV; Human; Human Papilloma Virus-Related Malignant Neoplasm; Human Papillomavirus; Human papilloma virus infection; Infection; Integral Membrane Protein; Journals; L2 viral capsid protein; Malignant Neoplasms; Maps; Membrane; Minority Groups; National Cancer Institute; Oncogenic Viruses; Papilloma; Papillomavirus; Pathway interactions; Peptides; Phenotype; Play; Proteins; Public Health; Publishing; Role; Seminal; Sorting - Cell Movement; System; Testing; Time; Vaccination; Vaccines; Virion; Virus; Virus Diseases; anticancer research; design; experimental study; genetic analysis; genetic approach; innovation; insight; mutant; novel; novel strategies; pathogen; prevent; protein aminoacid sequence; receptor; retrograde transport; trafficking; tumor; vaccine acceptance; virology; virome

This is a revised application for an R35 grant from the National Cancer Institute entitled, “Mechanisms of human papillomavirus entry.” HPV is responsible for 5% of human cancer, millions of cases of genital warts, and countless cases of other types of papillomas (most caused by non-vaccine HPV types). Despite the existence of effective vaccines, HPV infection and the cancers it causes will remain a major public health problem for decades because vaccine uptake is poor and vaccination does not clear established infections. There are no specific treatments for HPV or HPV-associated cancers. The DiMaio lab has received more than 35 years of continuous funding from the NCI, during which time it has made seminal contributions to tumor virology. We recently discovered that HPV traffics via the retrograde transport pathway during infection, showed that retromer is required for sorting of the incoming virus particle into this pathway and that HPV is a novel type of retromer cargo, and discovered a cell-penetrating peptide (CPP) that drives the HPV L2 capsid protein into the cytoplasm to engage retromer. This is the first example where either retromer or a CPP has been shown to play a role in virus entry. These results have been paradigm-shifting in the field and have been published since 2013 in Cell, PLoS Pathogens, mBio, PNAS, and Journal of Cell Biology, and they have fundamentally changed our understanding of HPV entry and the role of retromer and CPPs in biology. Here, we will discover how HPV accomplishes these amazing feats. We will determine the requirements for L2 membrane protrusion and establish how sequences flanking the core CPP modulate its activity. We will test whether the abundance of CPP sequences in the extant papillomavirus virome reflects their membrane- penetrating activity, determine whether L2 truly adopts a transmembrane existence, and map L2 segments exposed in the cytoplasm. We have designed an innovative new functional genetics approach and used it to isolate artificial small transmembrane proteins that inhibit HPV entry, and we will use these artificial proteins to identify new HPV entry factors and dissect their role in HPV entry. We will exploit our 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 to compete for binding with incoming HPV, validating an entirely new approach to prevent virus infections. We will develop the first genetic system to select HPV mutants with informative phenotypes, attempt to identify the HPV entry receptor, and extend these studies to additional tumor viruses and HIV. These experiments will elucidate important aspects of the mechanisms of HPV entry, validate new approaches to prevent and treat HPV infection, and revolutionize genetic analysis of HPV. Critically, our studies will provide new insights into fundamental cell biology. If this proposal is funded, we will continue to make novel and important contributions to virology and cancer research for many years.

这是一份来自国家癌症研究所的R35资助的修订申请，题为“癌症的机制”。 人乳头瘤病毒进入。”HPV是导致5%人类癌症的原因，数百万例生殖器疣， 以及无数其他类型的乳头状瘤病例（大多数由非疫苗HPV类型引起）。尽管 有效疫苗的存在、HPV感染及其引起的癌症仍将是一个主要的公共卫生问题 这是一个几十年来一直存在的问题，因为疫苗接种率很低，而且疫苗接种不能清除已建立的感染。 没有针对HPV或HPV相关癌症的特定治疗方法。DiMaio实验室已经收到了超过 35年来NCI的持续资助，在此期间，它为肿瘤治疗做出了开创性的贡献。 病毒学我们最近发现HPV在感染期间通过逆行运输途径运输， 表明逆转录酶是进入该途径的病毒颗粒分选所必需的，并且HPV是一种 一种新型的逆转录聚合物货物，并发现了一种驱动HPV L2衣壳的细胞穿透肽（CPP） 蛋白质进入细胞质以接合retromer。这是第一个例子，其中无论是retromer或CPP具有 在病毒入侵中发挥作用。这些结果已经在该领域的范式转变，并已被 自2013年以来发表在Cell，PLoS Pathogens，mBio，PNAS和Journal of Cell Biology上， 从根本上改变了我们对HPV进入以及逆转录酶和CPP在生物学中的作用的理解。在这里， 我们将发现HPV是如何完成这些惊人的壮举的。我们将确定L2的要求 膜突起，并建立侧接核心CPP的序列如何调节其活性。我们将测试 在现存的乳头瘤病毒病毒组中CPP序列的丰度是否反映了它们的膜- 穿透活性，确定L2是否真正采取跨膜存在，并映射L2片段 暴露在细胞质中。我们设计了一种创新的新功能遗传学方法，并将其用于 分离抑制HPV进入的人工小跨膜蛋白，我们将使用这些人工蛋白， 识别新的HPV进入因子并剖析它们在HPV进入中的作用。我们将利用我们对HPV的理解 进入设计抑制肽，利用CPP的膜穿透活性，以提供 逆转录酶结合位点进入细胞质，与进入的HPV竞争结合，验证了一种全新的 防止病毒感染的方法。我们将开发第一个选择HPV突变体的遗传系统， 信息表型，试图确定HPV进入受体，并将这些研究扩展到其他 肿瘤病毒和HIV。这些实验将阐明HPV进入机制的重要方面， 验证预防和治疗HPV感染的新方法，并彻底改变HPV的遗传分析。 重要的是，我们的研究将为基础细胞生物学提供新的见解。如果这项建议获得资助，我们将 多年来继续为病毒学和癌症研究做出新的重要贡献。