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Journal of Virology, October 2002, p. 9806-9818, Vol. 76, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.19.9806-9818.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of a G₂ Arrest Domain in the E1E4 Protein of Human Papillomavirus Type 16

Divisions of Virology,¹ Yeast Genetics, National Institute for Medical Research, London NW7 1AA,⁴ OSI Pharmaceuticals, Oxford OX4 6LY, United Kingdom,³ Department of Virology, Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland²

Received 5 April 2002/ Accepted 19 June 2002

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1E4 arrests HeLa cervical epithelial cells in G₂. 16E1E4 also caused a G₂ arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1E4-mediated G₂ arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G₂ arrest was also mediated by the E1E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G₂ arrest. We conclude that, like other papillomavirus proteins, 16E1E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.

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