title: Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1.
abstract: Immediate-early proteins from many viruses function as transcriptional regulators and exhibit transactivation activity, DNA binding activity, and dimerization. In this study, we investigated these characteristics in white spot syndrome virus (WSSV) immediate-early protein 1 (IE1) and attempted to map the corresponding functional domains. Transactivation was investigated by transiently expressing a protein consisting of the DNA binding domain of the yeast transactivator GAL4 fused to full-length IE1. This GAL4-IE1 fusion protein successfully activated the Autographa californica multicapsid nucleopolyhedrovirus p35 basal promoter when five copies of the GAL4 DNA binding site were inserted upstream of the TATA box. A deletion series of GAL4-IE1 fusion proteins suggested that the transactivation domain of WSSV IE1 was carried within its first 80 amino acids. A point mutation assay further showed that all 12 of the acidic residues in this highly acidic domain were important for IE1's transactivation activity. DNA binding activity was confirmed by an electrophoresis mobility shift assay using a probe with (32)P-labeled random oligonucleotides. The DNA binding region of WSSV IE1 was located in its C-terminal end (amino acids 81 to 224), but mutation of a putative zinc finger motif in this C-terminal region suggested that this motif was not directly involved in the DNA binding activity. A homotypic interaction between IE1 molecules was demonstrated by glutathione S-transferase pull-down assay and a coimmunoprecipitation analysis. A glutaraldehyde cross-linking experiment and gel filtration analysis showed that this self-interaction led to the formation of stable IE1 dimers.
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei 106, Taiwan. gracelow@ntu.edu.tw
authors.forename: Wang-Jing
authors.initials: WJ
authors.lastname: Liu
authors.forename: Yun-Shiang
authors.initials: YS
authors.lastname: Chang
authors.forename: Hao-Ching
authors.initials: HC
authors.lastname: Wang
authors.forename: Jiann-Horng
authors.initials: JH
authors.lastname: Leu
authors.forename: Guang-Hsiung
authors.initials: GH
authors.lastname: Kou
authors.forename: Chu-Fang
authors.initials: CF
authors.lastname: Lo