Mechanisms of human papillomavirus entry

人乳头瘤病毒的侵入机制

• 批准号: 10042803
• 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/10042803
• 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; National Cancer Institute; Oncogenic Viruses; Papilloma; Papillomavirus; Pathway interactions; Peptides; Phenotype; Play; Population; 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; uptake; 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.

这是美国国家癌症研究所 R35 拨款的修订申请，题为“Mechanisms of 人乳头瘤病毒进入。” HPV 导致 5% 的人类癌症、数百万生殖器疣病例、 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 几十年来这个问题一直存在，因为疫苗接种率很低，而且疫苗接种不能清除既定的感染。 对于 HPV 或 HPV 相关癌症没有具体的治疗方法。 DiMaio 实验室已收到超过 NCI 持续资助 35 年，期间为肿瘤领域做出了开创性贡献 病毒学。我们最近发现 HPV 在感染过程中通过逆行运输途径进行运输， 研究表明，需要逆转录酶将传入的病毒颗粒分类到该途径中，并且 HPV 是一种 新型逆转录酶货物，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逆转录酶。这是retromer 或 CPP 的第一个例子 已被证明在病毒进入中发挥作用。这些结果已经改变了该领域的范式，并已被 自 2013 年起在 Cell、PLoS Pathogens、mBio、PNAS 和 Journal of Cell Biology 上发表，并且 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。这里， 我们将了解 HPV 如何完成这些惊人的壮举。我们将确定 L2 的要求 膜突出并确定核心 CPP 侧翼的序列如何调节其活性。我们将测试 现存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是否真正采用跨膜存在，并绘制L2片段 暴露在细胞质中。我们设计了一种创新的功能遗传学方法，并将其用于 分离出抑制 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 进入因素并剖析它们在 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 的膜穿透活性来传递 逆转录酶结合位点进入细胞质，与传入的 HPV 竞争结合，验证了一种全新的 预防病毒感染的方法。我们将开发第一个基因系统来选择 HPV 突变体 信息表型，尝试识别 HPV 进入受体，并将这些研究扩展到其他 肿瘤病毒和艾滋病毒。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 的基因分析。 至关重要的是，我们的研究将为基础细胞生物学提供新的见解。如果该提案获得资助，我们将 多年来继续为病毒学和癌症研究做出新颖且重要的贡献。