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）是一种小的、非包膜的DNA病毒，与5%的人类癌症（包括基本上所有的宫颈癌、大多数其他肛门生殖器癌和越来越多的口咽癌）的病因学相关。针对某些类型的人乳头瘤病毒的疫苗已经开发出来，但在可预见的未来，绝大多数人仍未接种疫苗。人们对HPV进入细胞时发生的细胞内事件知之甚少。为了从分子细节上理解这一重要过程，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。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，筛选还发现了许多新的基本因素，包括涉及逆行运输到高尔基体的几个因素。突出的验证点编码逆转录体的成分，逆转录体是一种参与货物从核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衣壳以反转录依赖的方式定位于反式高尔基网络（TGN），并且在感染细胞中，反转录物的组分与HPV衣壳蛋白处于物理复合物中。最后，一种逆行运输的小分子抑制剂也能特异性地抑制感染。值得注意的是，该逆转录酶以前没有涉及任何病毒的进入。基于这些令人兴奋的发现，我们假设逆转录物和相关途径在病毒进入时直接将HPV转运到TGN。我们提出实验来验证这一假设，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HPV感染过程中内核体到高尔基逆转录体运输系统的作用，并确定其他必要因素。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，重点是逆行途径成分。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.