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。高风险的人乳头瘤病毒导致全球超过50万例宫颈癌，这是女性第四大常见癌症（2012年估计）。人乳头瘤病毒是一种小的DNA病毒，必须将其基因组传递到宿主细胞核才能成功地引发感染。像所有其他非包膜病毒一样，hpv必须将其遗传物质（vDNA）运输过细胞内限制膜，这是一个由次要衣壳蛋白L2介导的关键事件。我们之前的工作表明L2的n端结构域是这种膜渗透活性的关键区域。宿主细胞蛋白酶furin对L2的N端切割和保守的跨膜结构域（TMD）是vDNA易位的必要条件。在病毒粒子进入过程中，细胞表面furin对L2的切割似乎调节了一种构象变化，使L2能够相互作用并跨越局部膜，我们称之为“突出”。在这种膜突出的构象中，L2能够进入细胞质吸收细胞分选因子，同时保持与管腔vDNA的络合。细胞质分选连接蛋白和逆转录酶的参与使vDNA高效的l2依赖性运输到高尔基体。我们最近的研究表明，这种突出L2/vDNA复合体的膜在有丝分裂开始之前一直存在于高尔基体中，当高尔基体随着染色体凝聚、中心粒迁移、纺锤体组装和核膜破裂而自然分散时。在这个短暂的动态变化期间，囊泡结合膜突出的L2/管腔vDNA复合体从囊泡高尔基体中出来，在中期定位于中心体附近，并沿着纺锤体穿越，在中期到达凝聚的染色体。此时，我们认为L2使用染色质结合结构域来物理地连接宿主染色体，这是vDNA从后高尔基囊泡中完全易位所必需的功能。然后，这种染色体系住的L2/vDNA分裂成子细胞，建立基底角化细胞的持续感染。在此，我们提出了旨在了解L2亚细胞运输和有丝分裂依赖易位的迷人生物学机制和含义的研究。具体来说，我们的目标是了解膜突出的L2/vDNA复合体的性质和furin在实现这种构象中的作用，以及确定有丝分裂依赖的L2/vDNA从后高尔基囊泡到中期染色体易位的时间和位置。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先天免疫反应，并可能影响持续感染的建立-致瘤性hpv的标志，无疑有助于这些病毒的致瘤性。

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.