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Journal of Virology, September 2008, p. 9056-9064, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.01080-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells {triangledown}

Sally Roberts,1,{dagger}* Sarah R. Kingsbury,2,{dagger} Kai Stoeber,2 Gillian L. Knight,1 Phillip H. Gallimore,1 and Gareth H. Williams2*

Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TT, United Kingdom,1 Wolfson Institute for Biomedical Research and Research Department of Pathology, University College London, Gower Street, London, WC1E 6BT, United Kingdom2

Received 22 May 2008/ Accepted 3 July 2008

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

* Corresponding author. Mailing address for Sally Roberts: CR-UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Birmingham B15 2TT, United Kingdom. Phone: 44(0)121-4147459. Fax: 44(0)121-4144486. E-mail: s.roberts{at}bham.ac.uk. Mailing address for Gareth Williams: Wolfson Institute for Biomedical Research and Department of Pathology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Phone: 44(0)207-6796304. Fax: 44(0)207-3384408. E-mail: gareth.williams{at}ucl.ac.uk

{triangledown} Published ahead of print on 16 July 2008.

{dagger} These authors contributed equally to this work.

Journal of Virology, September 2008, p. 9056-9064, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.01080-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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