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J. Virol. doi:10.1128/JVI.01481-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The HPV-16 E8^E2 Gene Product Represses Early Transcription and Replication but is Dispensable for Viral Plasmid Persistence in Keratinocytes

Department of Pathology, Veterans Affairs Medical Center, and The University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA

^* To whom correspondence should be addressed. Email: thomas-haugen{at}uiowa.edu.

	Abstract

A conserved E8^E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus (HPV)–type 16 plasmid DNA. Expression of HPV-16 E8^E2 is independent of the major early promoter, P97, and is modulated both by specific splicing events and conserved cis elements in the upstream regulatory region (URR) in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8^E2 message also increase initial HPV-16 plasmid amplification 8-15 fold and major early gene (P97) transcription 4-5 fold compared to wt. Expressing the HPV-16 E8^E2 gene product from the CMV promoter represses HPV-16 early gene transcription from the major early gene promoter (P97, E6/E7) in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8^E2 also inhibits amplification of an HPV-16 plasmid and a heterologous SV40 ori plasmid that contains E2 binding sites in cis. In contrast, co-transfections with HPV-16 wt genomes that express physiologic levels of 16-E8^E2 are sufficient to repress HPV-16 plasmid amplification, but are limiting and insufficient for repression of SV40 amplification. 16-E8^E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. In contrast to other PVs, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain >8 fold elevated levels of unintegrated plasmid compared to wt, demonstrating that 16-E8^E2 limits viral copy number but is not required for plasmid persistence and maintenance.