title: Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies.
abstract: 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.
authors.affiliation: Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Vadim
authors.initials: V
authors.lastname: Patsalo
authors.forename: Mark A
authors.initials: MA
authors.lastname: Yondola
authors.forename: Bowu
authors.initials: B
authors.lastname: Luan
authors.forename: Ilana
authors.initials: I
authors.lastname: Shoshani
authors.forename: Caroline
authors.initials: C
authors.lastname: Kisker
authors.forename: David F
authors.initials: DF
authors.lastname: Green
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
authors.forename: Patrick
authors.initials: P
authors.lastname: Hearing
mesh_terms: D000256:Adenoviridae / Q000378:metabolism*; D000257:Adenoviridae Infections / Q000378:metabolism*; D017504:Adenovirus E4 Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001703:Biophysics / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology; D019281:Dimerization; D006367:HeLa Cells; D006790:Host-Parasite Interactions / Q000502:physiology; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017504:Adenovirus E4 Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011994:Recombinant Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human