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J Biol Chem, Vol. 275, Issue 20, 14969-14978, May 19, 2000
From the Department of Molecular Microbiology and Immunology, Johns
Hopkins University School of Public Health,
Baltimore, Maryland 21205
E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral
DNA replication, apoptosis, double strand break repair, and
transformation through multiple interactions with components in
infected and transformed cells. Conservation of several cysteine and
histidine residues among E4 34k sequences from a variety of adenovirus
serotypes suggests the presence of a zinc binding domain important for
function. Consistent with the hypothesis that E4 34k is a zinc
metalloprotein, zinc binding by baculovirus-expressed E4 34k protein
was demonstrated in a zinc blotting assay. To investigate the
relationship between the potential zinc-binding region and E4 34k
function, a series of mutant genes containing single amino acid
substitutions at each of the conserved cysteine and histidine residues
in E4 34k were constructed. The mutant proteins were examined for the
ability to complement the late protein synthetic defect of an E4
deletion mutant, to physically interact with the viral E1b 55-kDa
protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of
cysteine and histidine residues required for stimulation of late gene
expression, physical interaction with E1b 55k, and p53 destabilization.
These data suggest that a zinc-binding domain participates in the
formation of the E4 34k-E1b 55k physical complex and
that the complex is required in late gene expression and for p53 destabilization.
Genetic Analysis of a Potential Zinc-binding Domain of the
Adenovirus E4 34k Protein*
and
*
This work was supported by U. S. Public Health Service
Grants AI26239 and T32AI07417.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Microbiology, Columbia University
College of Physicians and Surgeons, 701 W. 168th St., New York, NY
10032
§
To whom correspondence should be addressed: Dept. of Molecular
Microbiology and Immunology, Johns Hopkins University School of Public
Health, 615 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3776; Fax:
410-955-0105; E-mail: gketner@jhsph.edu.
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