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: 8577201
• 负责人: Daniel C. Dimaio
• 金额: $ 39.11万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8577201
• 关键词: Anogenital cancer; Antiviral Agents; Biochemical; Biological; Capsid; Capsid Proteins; Cell physiology; Cells; Cellular biology; Cervical; Chemicals; Complex; DNA Viruses; Disease; Endosomes; Event; Functional disorder; Future; Genes; Genetic; Golgi Apparatus; HPV-High Risk; Hand; Human; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 16; Image; Infection; L2 viral capsid protein; Life Cycle Stages; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mediating; Medical; Microscopy; Minor; Minority; Molecular; Molecular Machines; Oncogenic Viruses; Papillomavirus; Pathway interactions; Population; Process; Proteins; Proteomics; RNA Interference; Recruitment Activity; Research Design; Resolution; Role; System; Techniques; Testing; Vaccines; Viral; Virus; Virus Diseases; base; burden of illness; cancer cell; cost; functional genomics; genome-wide; high risk; inhibitor/antagonist; insight; keratinocyte; knock-down; malignant oropharynx neoplasm; novel; novel strategies; pathogen; prophylactic; public health relevance; research study; retrograde transport; 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(申请人提供)：高危类型的人乳头瘤病毒(HPV)是一种小的、无包膜的DNA病毒，在病因学上与5%的人类癌症有关，包括基本上所有的宫颈癌、大多数其他肛门生殖器癌，以及越来越多的口咽癌。针对某些类型的HPV的疫苗已经开发出来，但在可预见的未来，绝大多数人仍将没有接种疫苗。人们对HPV进入细胞过程中发生的细胞内事件知之甚少。为了在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干扰筛查。该筛查在技术上是可靠的，并成功地确定了HPV感染所需的几个已知因素。此外，屏幕确定了许多新的基本因素，包括涉及到逆行运输到高尔基体的几个因素。突出的验证命中编码了逆转聚体的组件，逆转聚体是一种分子机器，参与将货物从内体运输到高尔基体。此外，进入的、部分分解的HPV16衣壳以逆转录依赖的方式定位于反式高尔基体网络(TGN)，并且逆转聚体的组成部分与感染细胞中的HPV衣壳蛋白形成物理复合体。最后，一种逆行转运的小分子抑制剂也能特异性地抑制感染。值得注意的是，该反转录聚体以前并未与任何病毒的进入有关。在这些令人兴奋的发现的基础上，我们假设逆转录和相关的途径在病毒进入TGN期间直接将HPV运送到TGN。我们建议用实验来验证这一假说，并确定HPV16利用的细胞内转运途径(S)。在目标1中，我们将进行遗传和细胞定位实验，以探索内小体到高尔基体逆转录转运系统在HPV感染过程中的作用，并确定其他基本因素。在目标2中，我们将进行生化和成像实验，以鉴定和表征与进入HPV16衣壳相关的蛋白质，重点是逆行途径的组成部分。在目标3中，我们将进行突变和其他分析，以验证我们的假设，即逆转录病毒通过参与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，为基础细胞生物学提供新的见解，并提出新的抗病毒策略。