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

• 批准号: 9249481
• 负责人: Daniel C. Dimaio
• 金额: $ 41.63万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9249481
• 关键词: Anogenital cancer; Antiviral Agents; Biochemical; Biological; Capsid; Capsid Proteins; Cell physiology; Cells; Cellular biology; Cervical; Chemicals; Complex; DNA Viruses; Disease; Endosomes; Etiology; Event; Functional disorder; Future; Genes; Genetic; Golgi Apparatus; Hand; Human; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 16; Image; Income; Infection; L2 viral capsid protein; Life Cycle Stages; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mediating; Medical; Microscopy; Minor; Minority; Molecular; Molecular Machines; Mutation; Mutation Analysis; Oncogenic Viruses; Papillomaviridae; Papillomavirus; Pathway interactions; Population; Preventive vaccine; Process; Proteins; Proteomics; RNA Interference; RNA interference screen; Recruitment Activity; Research Design; Resolution; Role; System; Techniques; Testing; Vaccines; Viral; Virus; Virus Diseases; burden of illness; cancer cell; cost; experimental study; functional genomics; genome-wide; high risk; insight; keratinocyte; knock-down; malignant oropharynx neoplasm; novel; novel strategies; pathogen; public health relevance; retrograde transport; small molecular inhibitor; trafficking; trans-Golgi Network; vaccine efficacy; virology

DESCRIPTION (provided by applicant): High-risk types of human papillomaviruses (HPV) are small, non-enveloped DNA viruses etiologically associated with 5% of human cancers, including essentially all cervical cancer, most other anogenital cancer, and an increasing fraction of oropharyngeal cancer. Vaccines have been developed that target some types of HPV, but the great majority of people will remain unvaccinated for the foreseeable future. Little is known about the intracellular events occurring during HPV entry into cells. To understand this important process in molecular detail, we performed a genome-wide RNA interference screen for genes required for infection of cervical cancer cells by HPV16 pseudovirus. The screen was technically robust and successfully identified several factors known to be required for HPV infection. In addition, the screen identified numerous novel essential factors, including several factors involved in retrograde transport to the Golgi apparatus. Prominent validated hits encode components of the retromer, a molecular machine involved in transport of cargo from endosomes to Golgi. In addition, incoming, partially disassembled HPV16 capsids localize to the trans-Golgi network (TGN) in a retromer-dependent fashion, and components of the retromer are in a physical complex with HPV capsid proteins in infected cells. Finally, a small molecular inhibitor of retrograde transport also specifically inhibits infection. Notably, the retromer has nt been previously implicated in entry of any virus. On the basis of these exciting discoveries, we hypothesize that retromer and associated pathways directly transport HPV into the TGN during virus entry. We propose experiments to test this hypothesis and identify the intracellular trafficking pathway(s) utilized by HPV16. In aim 1, we will conduct genetic and cell localization experiments to explore the role of the endosome-to-Golgi retromer transport system during HPV infection and identify additional essential factors. In aim 2, we will conduct biochemical and imaging experiments to identify and characterize proteins that associate with incoming HPV16 capsids, with a focus on retrograde pathway components. In aim 3, we will conduct mutational and other analyses to test our hypothesis that retromer supports HPV infection by participating in the transfer of HPV constituents to the TGN. Overall, these experiments will elucidate the mechanistic details of HPV entry, provide new insights into fundamental cell biology, and suggest novel anti-viral strategies.

描述（由申请人提供）：高危类型的人乳头瘤病毒 (HPV) 是小型无包膜 DNA 病毒，在病因学上与 5% 的人类癌症相关，包括基本上所有宫颈癌、大多数其他肛门生殖器癌症以及越来越多的口咽癌。针对某些类型 HPV 的疫苗已经开发出来，但在可预见的未来，绝大多数人仍不会接种疫苗。对于 HPV 进入细胞期间发生的细胞内事件知之甚少。为了从分子细节上了解这一重要过程，我们对 HPV16 假病毒感染宫颈癌细胞所需的基因进行了全基因组 RNA 干扰筛选。该筛查在技术上非常可靠，并成功识别出 HPV 感染所需的几个已知因素。此外，筛选还发现了许多新的重要因素，包括与逆行运输至高尔基体有关的几个因素。重要的经过验证的命中编码了逆转录酶的成分，逆转录酶是一种参与将货物从内体运输到高尔基体的分子机器。此外，传入的部分分解的 HPV16 衣壳以逆转录酶依赖性方式定位于跨高尔基体网络 (TGN)，并且逆转录酶的成分与受感染细胞中的 HPV 衣壳蛋白形成物理复合物。最后，逆行转运的小分子抑制剂也能特异性抑制感染。值得注意的是，此前并未发现逆转录酶与任何病毒的进入有关。基于这些令人兴奋的发现，我们假设逆转录酶和相关途径在病毒进入过程中直接将 HPV 转运到 TGN 中。我们提出实验来检验这一假设并确定 HPV16 利用的细胞内运输途径。在目标 1 中，我们将进行遗传和细胞定位实验，以探索内体到高尔基体逆转录体转运系统在 HPV 感染过程中的作用，并确定其他重要因素。在目标 2 中，我们将进行生化和成像实验，以识别和表征与传入 HPV16 衣壳相关的蛋白质，重点关注逆行途径成分。在目标 3 中，我们将进行突变和其他分析来检验我们的假设，即逆转录酶通过参与 HPV 成分向 TGN 的转移来支持 HPV 感染。总体而言，这些实验将阐明 HPV 进入的机制细节，提供对基础细胞生物学的新见解，并提出新的抗病毒策略。