Mechanisms and Consequences of L2-Dependent Subcellular Trafficking of the HPV Genome.

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。高风险致癌HPV在全球范围内导致超过50万例宫颈癌，这是女性第四大常见癌症（2012年估计）。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必须将其遗传物质（vDNA）运输穿过细胞内限制膜，这是由次要衣壳蛋白L2介导的关键事件。我们先前的工作已经暗示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和保守的跨膜结构域（TMD）对L2的N-末端切割对于vDNA易位是必需的。在病毒体进入期间，细胞表面弗林蛋白酶对L2的裂解似乎调节构象变化，使L2能够相互作用并跨越局部膜，我们称之为“突出”的活动。在这种膜突出的构象中，L2能够进入胞质溶胶以募集细胞分选因子，同时保持与内腔vDNA复合。胞质分选连接蛋白和逆转录聚合物的参与使得vDNA能够有效地L2依赖性转运到高尔基体。我们最近的工作表明，这种膜突出的L2/vDNA复合物仍然在高尔基体，直到有丝分裂的开始，当高尔基体自然分散与染色体凝聚，中心粒迁移，纺锤体组装，和核膜破裂。在这个短暂的动态变化期间，囊泡结合的膜突出的L2/内腔vDNA复合物从泡状高尔基体流出，通过前中期定位在中心体附近，并且似乎沿沿着纺锤体穿过，通过中期到达浓缩的染色体。此时，我们相信L2使用染色质结合结构域物理地拴系到宿主染色体，这是vDNA完全易位出高尔基体后囊泡所必需的功能。然后，这种染色体束缚的L2/vDNA分配到子细胞中，以建立基底角质形成细胞的持续感染。在此，我们提出的研究，旨在了解迷人的生物学机制和L2亚细胞运输和有丝分裂依赖易位的影响。具体而言，我们的目标是了解膜突出L2/vDNA复合物的性质和弗林蛋白酶在实现这种构象中的作用，以及确定L2/vDNA从高尔基体后囊泡到中期染色体的有丝分裂依赖性易位的时间和地点。最后，我们将探讨这种独特的贩运和易位与先天免疫监视方面的生物学后果。这些基本过程可能有助于病毒逃避早期先天免疫应答，并可能影响持续感染的建立-致癌HPV的标志，这无疑有助于这些病毒的致癌性质。

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. High risk oncogenic HPVs caused over 500,000 cases of cervical cancer worldwide, the fourth most common cancer in women (2012 estimates). 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 must transport their genetic material (vDNA) across an intracellular limiting membrane, a critical event mediated by the minor capsid protein L2. Our prior work has 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 transmembrane domain (TMD) are essential for vDNA translocation. During virion entry, cleavage of L2 by cell surface furin appears to modulate a conformational change enabling L2 to interact and span across local membranes, an activity we call “protrusion”. In this membrane-protruding conformation, L2 is able to access the cytosol to recruit cellular sorting factors while remaining complexed to the lumenal vDNA. Engagement of cytosolic sorting nexins and retromer enable efficient L2-dependent transport of vDNA to the Golgi. Our recent work reveals that this membrane protruding L2/vDNA complex remains at the Golgi until the onset of mitosis, when the Golgi naturally disperses coincident with chromosome condensation, centriole migration, spindle assembly, and nuclear envelope breakdown. During this brief period of dynamic changes, the vesicle-bound membrane-protruding L2/lumenal vDNA complex egresses from the vesiculating Golgi, localizes near centrosomes by prometaphase, and appears to traverse along the spindle, reaching the condensed chromosomes by metaphase. At this time, we believe L2 uses a chromatin- binding domain to physically tether to host chromosomes, a function that is essential for full translocation of the vDNA out of the post-Golgi vesicle. This chromosome-tethered L2/vDNA then partitions into daughter cells to establish a persistent infection of basal keratinocytes. Herein, we propose studies aimed at understanding the fascinating biological mechanisms and implications of L2 subcellular trafficking and mitosis-dependent translocation. Specifically, we aim to understand the nature of the membrane-protruding L2/vDNA complex and the role of furin in achieving this conformation, as well as determine the timing and locale of mitosis- dependent translocation of L2/vDNA from post-Golgi vesicle to metaphase chromosome. Finally, we will explore the biological consequences of this unique trafficking and translocation with regards to innate immune surveillance. These fundamental processes likely contribute towards viral evasion of early innate immune responses and may influence the establishment of persistent infections- hallmarks of oncogenic HPVs that undoubtedly contributes to the oncogenic nature of these viruses.