Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1

Wang Jing Liu, Yun Shiang Chang, Hao Ching Wang, Jiann Horng Leu, Guang Hsiung Kou, Chu Fang Lo

Research output: Contribution to journalArticle

29 Citations (Scopus)

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 32P-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.

Original languageEnglish
Pages (from-to)11362-11373
Number of pages12
JournalJournal of Virology
Volume82
Issue number22
DOIs
Publication statusPublished - Nov 2008
Externally publishedYes

Fingerprint

White spot syndrome virus 1
Immediate-Early Proteins
White spot syndrome virus
dimerization
Dimerization
transcriptional activation
Transcriptional Activation
DNA
proteins
Nucleopolyhedrovirus
Amino Acids
TATA Box
assays
Trans-Activators
Zinc Fingers
DNA-Binding Proteins
Glutaral
Electrophoretic Mobility Shift Assay
Autographa californica multiple nucleopolyhedrovirus
Glutathione Transferase

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1. / Liu, Wang Jing; Chang, Yun Shiang; Wang, Hao Ching; Leu, Jiann Horng; Kou, Guang Hsiung; Lo, Chu Fang.

In: Journal of Virology, Vol. 82, No. 22, 11.2008, p. 11362-11373.

Research output: Contribution to journalArticle

Liu, Wang Jing ; Chang, Yun Shiang ; Wang, Hao Ching ; Leu, Jiann Horng ; Kou, Guang Hsiung ; Lo, Chu Fang. / Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1. In: Journal of Virology. 2008 ; Vol. 82, No. 22. pp. 11362-11373.
@article{858116a2a3cc47b4b1e7b58667b64744,
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 32P-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.",
author = "Liu, {Wang Jing} and Chang, {Yun Shiang} and Wang, {Hao Ching} and Leu, {Jiann Horng} and Kou, {Guang Hsiung} and Lo, {Chu Fang}",
year = "2008",
month = "11",
doi = "10.1128/JVI.01244-08",
language = "English",
volume = "82",
pages = "11362--11373",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "22",

}

TY - JOUR

T1 - Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1

AU - Liu, Wang Jing

AU - Chang, Yun Shiang

AU - Wang, Hao Ching

AU - Leu, Jiann Horng

AU - Kou, Guang Hsiung

AU - Lo, Chu Fang

PY - 2008/11

Y1 - 2008/11

N2 - 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 32P-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.

AB - 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 32P-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.

UR - http://www.scopus.com/inward/record.url?scp=55549111874&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=55549111874&partnerID=8YFLogxK

U2 - 10.1128/JVI.01244-08

DO - 10.1128/JVI.01244-08

M3 - Article

VL - 82

SP - 11362

EP - 11373

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 22

ER -