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Journal of Virology, June 2005, p. 6732-6740, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6732-6740.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of the E1E4 Protein in the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 31

Regina Wilson, Frauke Fehrmann, and Laimonis A. Laimins^*

Department of Microbiology-Immunology, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received 4 November 2004/ Accepted 1 February 2005

The most highly expressed protein in the productive life cycle of human papillomaviruses (HPVs) is E1E4, but its function is not well understood. To investigate the role of E1E4, we undertook a genetic analysis in the context of the complete HPV type 31 (HPV31) genome. A mutant HPV31 genome (E4M9) was constructed that contained a stop codon in the E4 open reading frame at amino acid 9 and was silent in the overlapping E2 coding sequence. Wild-type and mutant genomes were transfected into normal human foreskin keratinocytes (HFKs) and selected for drug resistance, and pooled cultures were examined for effects of E1E4 on viral functions. Southern blot analyses of transfected HFKs demonstrated that cells carrying the E4M9 mutant genomes were maintained as episomes at copy numbers similar to those in keratinocytes transfected with wild-type HPV31. Both sets of cells grew at similar rates, exhibited comparable extensions of life spans, and had equivalent levels of early transcripts. Following suspension of the cells in a semisolid medium, differentiation-dependent genome amplification and late gene expression were significantly decreased in cells maintaining the E4M9 mutant genome compared to those with wild-type HPV31. One explanation for these effects could be a reduction in the number of cells harboring mutant genomes that enter S phase upon differentiation. An analysis of cells containing E4M9 mutant genomes in organotypic raft cultures indicated a reduction in bromodeoxyuridine incorporation in differentiated suprabasal cells compared to that seen in wild-type rafts. Our results indicate that the HPV31 E1E4 protein plays a significant role in promoting HPV genome amplification and S phase maintenance during differentiation.

These authors contributed equally to this work.

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