Human papillomavirus entry: late trafficking and establishment of infection

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The trans-golgi network (TGN) serves as intermediate target organelle of the cytoplasmic transport during HPV infectious entry. It has previously been established that nuclear envelope breakdown is required for viral genome to enter the nucleus. We recently found that viral genome dissociates from TGN in prophase and associates with microtubules to accumulate at the microtubule-organizing center. At later stages of mitosis (metaphase), viral genome moves away from the mitotic spindle poles towards chromosomes. Our data point to a switch in the directionality of microtubule mediated transport from minus end- to plus end-directed during mitosis. Preliminary inhibitor studies point to dynein and Kif11 as motor proteins involved in the transport. Shift in directionality and the role of motor proteins for microtubule mediated transport will be further studied in Aim 1. Rather than egressing from the endocytic compartment in prophase, viral genome is still found in a membrane-bound vesicular compartment throughout mitotic transport. Viral genome egresses from membrane vesicles after nuclear envelope reformation. These are paradigm shifting findings, which have recently been confirmed by Day et al using electron microscopy. It is unclear, how HPV achieves egress from the nuclear transport vesicles. We hypothesized that HPV utilizes a cellular pathway to dissolve vesicles ending up in the nucleus, whether on purpose (e.g. viral infection) or accidentally. Indeed, preliminary data suggest that specific isoforms of phospholipase C, whose knockdown severely impairs HPV16 infection at a stage after nuclear delivery, may be involved in dissolving the vesicular membrane. The hypothesis will be tested in Aim 2 using knockdown and knockout approaches combined with differential staining techniques and EM. Release from transport vesicles is preceded by PML protein recruitment. Recruitment of another component of PML nuclear bodies (NB), Sp100, is specifically delayed in PML NB assembling around incoming genome. In cells deficient for PML protein, viral genome is lost and therefore transcriptionally inactive. In Aim 2, we will test our hypothesis that the recruitment of specific PML isoforms is the underlying reason for delayed recruitment of Sp100 and that HPV-induced PML NB reorganization is important for transcriptional regulation of the incoming genome. We have recently described a novel cell culture model that allows efficient infection of primary keratinocytes for investigating the role of PML NB components in a relevant cell culture model. Overall, our proposed studies will fill a huge gap in the understanding of microtubule mediated transport during late HPV intracellular trafficking and will help us unravel the role of PML NB during the immediate early events of the HPV life cycle essential for establishing infection. Furthermore, the proposal has the potential to uncover a hitherto unrecognized cellular pathway to dissolve vesicles ending up in the nucleus. Even though speculative at this time, the observations may trigger a rethinking of possible roles of vesicular cargo delivery to the nucleus during mitosis and its role in nucleus homeostasis.