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Journal of Virology, February 2009, p. 1689-1699, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01703-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Localization and Importance of the Adenovirus E4orf4 Protein during Lytic Infection ^,

Marie-Joëlle Miron,^1, Paola Blanchette,^1, Peter Groitl,² Frederic Dallaire,¹ Jose G. Teodoro,^1,3 Suiyang Li,¹ Thomas Dobner,² and Philip E. Branton^1,3*

Department of Biochemistry,¹ Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Canada,³ Heinrich Pette Institute for Experimental Virology and Immunology, Martinistrasse 52, 20251 Hamburg, Germany²

Received 11 August 2008/ Accepted 26 November 2008

The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells (C. O'Shea, et al., EMBO J. 24:1211-1221, 2005). Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.

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* Corresponding author. Mailing address: Department of Biochemistry, McGill University, McIntyre Medical Bldg., Rm. 823, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada. Phone: (514) 398-8350. Fax: (514) 398-7384. E-mail: Philip.branton{at}mcgill.ca

Published ahead of print on 10 December 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

These authors contributed equally to this work.

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Journal of Virology, February 2009, p. 1689-1699, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01703-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.