The E1and E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus

doi:10.1128/JVI.74.21.10081-10095.2000

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Journal of Virology, November 2000, p. 10081-10095, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The E1E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus

John Doorbar,¹^,^* Robert C. Elston,¹ Sawsan Napthine,² Kenneth Raj,³ Elizabeth Medcalf,² Deborah Jackson,¹ Nick Coleman,² Heather M. Griffin,⁴ Philip Masterson,¹ Simon Stacey,⁵ Yohannes Mengistu,⁶ and Julia Dunlop⁷

Division of Virology, National Institute for Medical Research, Mill Hill, London,¹ Department of Pathology, University of Cambridge,² and MRC Centre for Protein Engineering,⁴ Cambridge, Paterson Institute for Cancer Research, Manchester,⁵ and Institute of Virology, University of Glasgow, Glasgow,⁷ United Kingdom; Department of Virology, Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland³; and Department of Microbiology, University of Addis Ababa, Addis Ababa, Ethiopia⁶

Received 11 April 2000/Accepted 20 July 2000

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers.The E1 $and$ E4 protein of HPV16 but not those of HPV1 or HPV6 was foundto associate with a novel member of the DEAD box protein familyof RNA helicases through sequences in its C terminus. This protein,termed E4-DBP (E4-DEAD box protein), has a molecular weight of66,000 (66K) and can shuttle between the nucleus and the cytoplasm.It binds to RNA in vitro, including the major HPV16 late transcript(E1 $and$ E4.L1), and has an RNA-independent ATPase activity which canbe partially inhibited by E1 $and$ E4. E4-DBP was detectable in thecytoplasm of cells expressing HPV16 E1 $and$ E4 (in vivo and in vitro)and could be immunoprecipitated as an E1 $and$ E4 complex from cervicalepithelial cell lines. In cell lines lacking cytoplasmic intermediatefilaments, loss of the leucine cluster-cytoplasmic anchor regionof HPV16 E1 $and$ E4 resulted in both proteins colocalizing exclusivelyto the nucleoli. Two additional HPV16 E1 $and$ E4-binding proteins,of 80K and 50K, were identified in pull-down experiments but werenot recognized by antibodies to E4-DBP or the conserved DEAD boxmotif. Sequence analysis of E4-DBP revealed homology in its E4-bindingregion with three Escherichia coli DEAD box proteins involvedin the regulation of mRNA stability and degradation (RhlB, SrmB,and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae,which is involved in ribosome biogenesis. The synthesis of HPV16coat proteins occurs after E1 $and$ E4 expression and genome amplificationand is regulated at the level of mRNA stability and translation.Identification of E4-DBP as an HPV16 E1 $and$ E4-associated proteinindicates a possible role for E1 $and$ E4 in virussynthesis.

^* Corresponding author. Mailing address: Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill,London NW7 1AA, United Kingdom. Phone: 44-208-913-8677. Fax: 44-208-906-4477.E-mail: jdoorba{at}nimr.mrc.ac.uk.

Journal of Virology, November 2000, p. 10081-10095, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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