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.
This proposed work has relevance to host-pathogen interactions of a cancer-causing virus and to protein- membrane interactions and translocation mechanisms- fundamental processes to cell biology. These general membrane translocation processes are common to other viruses and bacterial toxins and the knowledge gained from these studies will provide insight into these systems as well. Lastly, the findings from this project will contribute to our understanding about how a persistent virus like HPV may avert host innate immune sensory systems by virtue of its unique trafficking and translocation mechanisms during initial infection.
