Mechanisms of L2-Mediated Membrane Translocation of the Papillomaviral Genome

描述(申请人提供)：人乳头瘤病毒(HPV)是最常见的性传播感染。这些病毒在粘膜和皮肤上皮细胞中感染和复制，作为其复制生命周期的一部分，诱导细胞增殖。高潮 风险致癌的HPV导致全球25万例宫颈癌，这是女性第二常见的癌症。HPV是一种小的DNA病毒，必须将其基因组运送到宿主细胞核才能启动成功的感染。像所有其他无包膜病毒一样，HPV面临着通过限制膜转移其遗传物质(VDNA)的任务，这是一个由次要衣壳蛋白L2介导的关键事件。最近的研究表明，L2的N-末端结构域是这种膜穿透活性的关键区域。宿主细胞蛋白酶Furin对L2的N端切割和Cys22和Cys28之间保守的二硫键是VDNA易位所必需的，我们最近发现并推测N端跨膜区(TMD)也参与了这一过程。在此，我们提出了旨在了解L2依赖的VDNA易位的研究，特别是旨在发展对L2的Furin裂解、二硫键和TMD的作用的结构和机制的理解，以及相互作用的细胞因素的参与。我们将使用各种结构、生化和基于细胞的实验方法来获得关于这个鲜为人知的过程的知识。这些拟议的研究将进一步促进我们对致癌HPV感染生命周期关键过程的理解，从而为开发低成本的抗HPV预防性药物提供潜在的药物靶点，这些药物可以应用于避孕套或润滑剂中，以进一步减少这些潜在致命病毒的传播。已知某些病毒和细菌毒素具有这些膜穿透能力，但它们对特定宿主细胞蛋白的依赖表明，内源性细胞蛋白可能也可以利用类似的途径。因此，这些研究有可能揭示细胞生物学中的新途径和运输机制。最后，了解VDNA易位的具体机制对于理解宿主细胞是否以及如何通过激活病毒入侵和穿膜过程中的先天免疫反应通路来“感知”HPV感染也是重要的。因此，这些基本过程可能有助于病毒逃避早期的先天免疫反应，并可能影响持续感染的建立--致癌HPV的特征，以及无疑有助于这些病毒的致癌性质的特征。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are the most common sexually transmitted infection. These viruses infect and replicate in mucosal and cutaneous epithelium, inducing cell proliferation as part of their replicative life cycle. The high risk oncogenic HPVs cause 250,000 cases of cervical cancer worldwide, the second most common cancer in women. HPVs are small DNA viruses and must deliver their genomes to the host cell nucleus to initiate a successful infection. Like all other non-enveloped viruses, HPVs are faced with the task of transferring their genetic material (vDNA) across a limiting membrane, a critical event mediated by the minor capsid protein L2. Recent studies have implicated the N-terminal domain of L2 as a crucial region for this membrane penetration activity. N-terminal cleavage of L2 by the host cell protease furin and a conserved disulfide bond between Cys22 and Cys28 are essential for vDNA translocation and we recently identified and implicated an N-terminal transmembrane domain (TMD) in this process as well. Herein, we propose studies aimed at understanding L2-dependent vDNA translocation, specifically aimed at developing a structural and mechanistic comprehension of the roles of furin cleavage, the disulfide bond, and the TMD of L2, in addition to the involvement of interacting cellular factors. We will use a variet of structural, biochemical, and cell-based experimental approaches to gain knowledge about this poorly understood process. These proposed studies will further advance our understanding of processes critical to the infectious life cycle of the oncogenic HPVs and thus represent potential drug targets for the development of low cost anti-HPV prophylactics that could be applied on condoms or within lubricants to further decrease transmission of these potentially deadly viruses. Certain viruses and bacterial toxins are known to possess these membrane penetration capabilities but their reliance on specific host cellular proteins suggest it is possibe that endogenous cellular proteins could utilize similar pathways. Thus, these studies have potential to unveil novel pathways and transport mechanisms in cell biology. Lastly, knowledge of the specific mechanisms of vDNA translocation will also be important towards understanding if and how host cells "sense" infection by HPVs through activation of innate immune response pathways during viral invasion and membrane penetration. These fundamental processes may therefore contribute towards viral evasion of early innate immune responses and could influence the establishment of persistent infection--hallmarks of oncogenic HPVs and a feature that undoubtedly contributes to the oncogenic nature of these viruses.