Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies.

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

Manipulation of early region 4.

Adenovirus early region 4 (E4) regulates processes in infected cells that include viral late gene expression, nonhomologous end joining, responses to DNA damage, and apoptosis. E4 is essential for viral growth in most cell lines. In this chapter, the current knowledge of the functions of six E4 products is summarized briefly. Protocols are presented for manipulation of E4, incorporation of E4 mutations into the viral genome, and growth of E4 mutants on complementing cell lines. A compilation of the described E4-complementing cell lines is included.

p53 is involved in the p120E4F-mediated growth arrest.

Control of cell growth and division by the p53 tumor suppressor protein requires its abilities to transactivate and repress specific target genes and to associate in complex with other proteins. Here we demonstrate that p53 binds to the E1A-regulated transcription factor p120E4F, a transcriptional repressor of the adenovirus E4 promoter. The interaction involves carboxy-terminal half of p120E4F and sequences located at the end of the sequence-specific DNA-binding domain of p53. Ectopic expression of p120E4F leads to a block of cell proliferation in several human and murine cell lines and this effect requires the association with wild-type (wt) p53. Although p120E4F can also bind to mutant p53, the growth suppression induced by overexpression of the protein is severely reduced in a cell line that contains mutant p53. These data suggest that p120E4F may represent an important element within the complex network of p53 checkpoint functions.

Large, persistent epidemic of adenovirus type 4-associated acute respiratory disease in U.S. army trainees.

In May 1997, a large, persistent epidemic of adenovirus type 4-associated acute respiratory disease began at Fort Jackson, South Carolina, the largest army basic training center. The epidemic lasted until December and declined when vaccine administration resumed. More than 1,000 male and female trainees were hospitalized; 66.1% of those hospitalized had an adenovirus type 4 isolate.