Mechanisms of L2-Mediated Membrane Translocation of the Papillomaviral Genome

描述（由申请人提供）：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中感染和复制，诱导细胞增殖作为其复制生命周期的一部分。高 高危致癌HPV在全球范围内导致25万例宫颈癌，这是女性第二大常见癌症。HPV是小DNA病毒，必须将其基因组递送到宿主细胞核以启动成功感染。与所有其他无包膜病毒一样，HPV面临着将其遗传物质（vDNA）转移穿过限制膜的任务，这是由次要衣壳蛋白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渗透活性的关键区域。宿主细胞蛋白酶弗林蛋白酶对L2的N-末端切割以及Cys 22和Cys 28之间的保守二硫键对于vDNA易位是必不可少的，并且我们最近鉴定并暗示N-末端跨膜结构域（TMD）也参与了该过程。在此，我们提出了旨在了解L2依赖的vDNA易位的研究，特别是旨在开发一个结构和机制的理解弗林蛋白酶裂解的作用，二硫键，和TMD的L2，除了相互作用的细胞因子的参与。我们将使用各种结构，生物化学和基于细胞的实验方法来获得有关这个知之甚少的过程的知识。这些拟议的研究将进一步推进我们对致癌HPV感染生命周期关键过程的理解，因此代表了开发低成本抗HPV药物的潜在药物靶点，这些药物可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性表明内源性细胞蛋白可能利用类似的途径。因此，这些研究有可能揭示细胞生物学中的新途径和转运机制。最后，vDNA易位的具体机制的知识也将是重要的，以了解宿主细胞是否和如何通过激活先天免疫应答途径在病毒入侵和膜渗透的HPV感染“的感觉”。因此，这些基本过程可能有助于病毒逃避早期先天免疫反应，并可能影响持续感染的建立-致癌HPV的标志和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.