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

人类乳头瘤病毒(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。高危致癌HPV导致全球超过50万例宫颈癌，是女性第四大常见癌症(2012年估计)。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。像所有其他非包膜病毒一样，HPV必须通过细胞内的限制膜运输它们的遗传物质(VDNA)，这是一个由次要衣壳蛋白L2介导的关键事件。我们先前的工作表明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端切割和保守的跨膜区(TMD)是VDNA易位所必需的。在病毒粒子进入期间，L2被细胞表面的Furin裂解似乎调制了一种构象变化，使L2能够相互作用并跨越局部膜，我们称这种活动为“突起”。在这种膜突起的构象中，L2能够进入胞浆以招募细胞分选因子，同时保持与腔内VDNA的络合。胞质分选连接蛋白和逆转聚体的结合使VDNA能够有效地依赖于L2转运到高尔基体。我们最近的工作表明，这种突起L2/VDNA复合体的膜一直保持在高尔基体中，直到有丝分裂开始，高尔基体自然分散，同时伴随着染色体凝聚、中心粒迁移、纺锤体组装和核膜破裂。在这一短暂的动态变化期间，囊泡结合的L2/腔内VDNA复合体从囊泡形成的高尔基体出来，通过前中期定位在中心体附近，并似乎沿着纺锤体横穿，通过中期到达浓缩的染色体。此时，我们认为L2使用染色质结合域来物理地拴住宿主染色体，这一功能对于VDNA完全移位出高尔基体后的小泡是必不可少的。这种染色体拴系的L2/VDNA然后分裂成子细胞，以建立对基底角质形成细胞的持续感染。在此，我们建议进行一些研究，旨在了解L2亚细胞运输和依赖有丝分裂的易位的有趣的生物学机制和意义。具体地说，我们的目标是了解膜突出的L2/VDNA复合体的性质和Furin在实现这一构象中的作用，以及确定依赖有丝分裂的L2/VDNA从高尔基体后小泡到中期染色体的转位的时间和地点。最后，我们将探讨这种独特的贩运和易位在先天免疫监测方面的生物学后果。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.