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Journal of Virology, June 1999, p. 4600-4610, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

Joseph S. Orlando and David A. Ornelles*

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064

Received 9 November 1998/Accepted 23 February 1999

A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic alpha  helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this alpha  helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic alpha  helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Bowman Gray Campus, Winston-Salem, NC 27157-1064. Phone: (336) 716-9332. Fax: (336) 716-9928. E-mail: ornelles{at}wfubmc.edu.

Journal of Virology, June 1999, p. 4600-4610, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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