Abstract
Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches.
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Acknowledgements
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). ML was supported by a studentship from the Fonds de recherche du Québec—Santé (FRQS) and a CIHR Frederick Banting and Charles Best doctoral scholarship award. AFT was supported by a studentship from the FRQS.
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Lehoux, M., Fradet-Turcotte, A., Archambault, J. (2015). Methods to Assess the Nucleocytoplasmic Shuttling of the HPV E1 Helicase and Its Effects on Cellular Proliferation and Induction of a DNA Damage Response. In: Keppler, D., Lin, A. (eds) Cervical Cancer. Methods in Molecular Biology, vol 1249. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2013-6_5
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DOI: https://doi.org/10.1007/978-1-4939-2013-6_5
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