The role of virus structure and host cell membrane trafficking as well as signalling factors in Human Papillomavirus Type 16 endocytosis
Final Report Abstract
In the last few years, our group has made significant progress in the elucidation of cellular processes leading to the entry of Human Papillomaviruses (HPV). First, we found that HPV16 interaction with disaccharide-sequence and sulfation pattern specific domains of glycosaminoglycan chains of heparan sulfate proteoglycans (HSPGs) is required and sufficient to structurally activate the virus for infection independent of the protein moieties of cellular HSPGs. It is thus tempting to speculate that different sulfation patterns of cellular HSPGs could allow for a specific tropism of virus entry. Our work established that after binding HPV16 entry occurs by a novel ligand-induced endocytosis pathway that is independent of clathrin-, caveolin-, lipid-raft-, and dynamin-mediated mechanisms but required regulated actin dynamics. In addition to HPV16, our work suggests that two other high-risk HPVs (18 and 31) use the same pathway. Based on the literature, it is likely that other viruses such as Influenza A viruses and Arenaviruses can make use of similar endocytic mechanisms. To understand this novel mechanism in more detail, a model for the activation of endocytic pit formation was derived from the literature and our experimental data. After internalization, virus particles were delivered to endosomal organelles. Based on electron microscopy data, colocalization analysis, live cell microscopy and cell biological perturbations, our data suggested that HPV16 entered early endosomes or macropinosomelike structures and was delivered to late endosomal/lysosomal compartments as an infectious route. Since sorting was independent of Rab7, a GTPase unaminously required for sorting from early to late endosomes, the viruses are sorted via a different route to late endsosomes. Based on preliminary data, we suspect this side route to involve the GOLGI apparatus. Uncoating occured most likely by a proteolytic L1 cleavage facilitated by an acid-activated but elusive protease. Upon the proposed separation of the minor capsid protein L2 and the viral DNA from the major capsid protein L1, L1 was found to be subjected to cathepsin-mediated proteolysis. L1 degradation, however, was not required for infection. Finally and surprisingly, RNAi screening of 7000 human genes combined with bioinformatic analysis revealed that numerous mitotic factors were involved in the entry of HPV16. Besides providing a long list of potential cellular regulators of HPV16 entry, our follow-up work indicated that nuclear entry of the viral DNA occurred only after nuclear envelope (NE) breakdown during mitosis. This is a unique finding for a DNA virus. Only c-type retroviruses have been proposed to use a similar mechanism for nuclear import of the pre-integration complex. Our findings thus explain, why HPVs exclusively infect stem cells of epidermal tissue as only those are mitotically active.
Publications
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(2010). Come in and take your coat off - how host cells provide endocytosis for virus entry. Cell. Microbiol. 12, 1378-88
Schelhaas, M.
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(2010). Virus entry by endocytosis. Annu. Rev. Biochem. 79, 803-833
Mercer, J., Schelhaas, M., Helenius, A.
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(2012). Analysis of virus entry and cellular membrane dynamics by single particle tracking. Methods Enzymol. 506; 63-80. (Volume 506: P. Michael Conn, ed.: Imaging of Live Cells in Health and Disease)
Ewers, H., Schelhaas, M.
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(2012). Entry of Human Papillomavirus Type 16 by actin-dependent, clathrin-, caveolin-, and lipid raft independent endocytosis. PLoS pathog. 8, e1002657
Schelhaas, M., Shah, B., Holzer, M., Blattmann, P., Kühling, L., Day, P.M., Schiller, J.T., Helenius, A.
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(2012). Principles of Polyoma- and Papillomavirus uncoating. Med. Microbiol. Immun. 201, 427-436
Cerqueira, C., Schelhaas, M.
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(2012). Single-cell analysis of population context advances RNAi screening at multiple levels. Mol. Syst. Biol. 8, 579
Snijder, B., Sacher, R., Rämö, P., Liberali, P., Mench, K., Wolfrum, N., Burleigh, L., Scott, CC., Verheije, MH., Mercer, J., Moese, S., Heger, T., Theusner, C., Jurgeit, A., Lamparter, D., Ballistreri, G., Schelhaas, M., De Haan, CAM, Marjomäki, V., Hyypia, T., Rottier, P., Sodeik, B., Marsh, M.,Gruenberg, J., Amara, A., Greber, U., Helenius, A., Pelkmans, L.
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(2013). Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure. Cell Microbiol. 15, 1818-36
Cerqueira, C., Liu, Y., Kühling, L., Chai, W., Hafezi, W., van Kuppevelt, T., Kühn, J.E., Feizi, T., Schelhaas, M.
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(2013). Human papillomavirus types -16, -18, and -31 share similar requirements for entry. J. Virol. 87, 7765-7773
Spoden, G., Kühling, L., Cordes, N., Frenzel, B. Sapp, M., Boller, K., Florin, L., Schelhaas, M.
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(2014) Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses. PLoS pathog. 10, e1004162
Aydin, I., Weber, S., Snijder, B., Samperio Ventayol, P., Kühbacher, A., Becker, M., Day, P.M., Schiller, J.T., Kann, M., Pelkmans, L., Helenius, A., Schelhaas, M.
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(2014). Concepts of papillomavirus entry. Curr. Opin. Virol. 4, 24- 31
Day, P.M. Schelhaas, M.
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(2015). Fluorescently labeled human papillomaviruses in virus entry experiments. Curr Protocol Microbiol, 14B, 4.1-14B, 4.22
Samperio Ventayol, P, Schelhaas, M.
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(2015). Kallikrein-8 proteolytically processes human papillomaviruses in the extracellular space to facilitate virus entry. J. Virol. 89, 7038-7052
Cerqueira, C., Samperio Ventayol, P., Vogeley, C., Schelhaas, M.
