Role of Retromer-mediated Retrograde Transport in HPV Entry

描述（由申请人提供）：高危型人乳头瘤病毒（HPV）是一种小型无包膜DNA病毒，与5%的人类癌症病因相关，包括基本上所有宫颈癌、大多数其他肛门生殖器癌和越来越多的口咽癌。已经开发出针对某些类型HPV的疫苗，但在可预见的未来，绝大多数人仍将未接种疫苗。关于HPV进入细胞期间发生的细胞内事件知之甚少。为了从分子上详细了解这一重要过程，我们对HPV 16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。该筛查在技术上是可靠的，并成功地确定了已知HPV感染所需的几个因素。此外，筛选确定了许多新的必需因子，包括参与逆行转运到高尔基体的几个因子。突出的经验证的命中编码逆转录聚合物的组分，逆转录聚合物是参与将货物从内体运输到高尔基体的分子机器。此外，进入的部分分解的HPV 16衣壳以逆转录酶依赖的方式定位于高尔基体网络（TGN），并且逆转录酶的组分与感染细胞中的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特异性抑制感染。值得注意的是，逆转录酶体以前没有涉及任何病毒的进入。在这些令人兴奋的发现的基础上，我们假设逆转录酶和相关途径在病毒进入期间直接将HPV转运到TGN中。我们提出实验来验证这一假设，并确定细胞内运输途径所利用的HPV 16。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转运系统在HPV感染过程中的作用，并确定其他必要因素。在目标2中，我们将进行生物化学和成像实验，以识别和表征与传入的HPV 16衣壳相关的蛋白质，重点是逆行途径组分。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。总的来说，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。

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.