Characterization of the Acidic Domain of the IE1 Regulatory Protein fromOrgyia pseudotsugataMulticapsid Nucleopolyhedrovirus

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.White spot syndrome virus (WSSV) is the causative agent of a disease that has led to severe mortalities of cultured shrimps all over the world (10,14,23,53). WSSV is a large doublestranded DNA virus which is extremely virulent (23,38,39), has a wide host range (14, 33), and targets various tissues (32, 59). It was recently erected as the type species of genus Whispovirus in the family Nimaviridae (56). Although the complete sequence of the WSSV genome has been known for several years (7,55,60), knowledge of the biological functions of the viral proteins is still quite poor. The WSSV immediate-early gene ie1 (31) was recently shown to use a shrimp signal transducer and activator of transcription (STAT) as a transcription factor to enhance its expression and contribute to its high promoter activity in host cells (30). In the present study, we further investigate the characteristics of WSSV IE1. This is made more difficult by the fact that no continuous shrimp cell line is currently available, and while bearing in mind that a heterologous system might introduce experimental artifacts, here we follow previous studies (22,30,34) and use the Sf9 insect cell system. Many viral immediate-early genes encode multifunctional transcriptional regulators that both positively and negatively modulate gene expression (26,52,57). These transcriptional regulators must po...

Section: Wssv Ie1 Contains Tadsmentioning

confidence: 95%

Transactivation, Dimerization, and DNA-Binding Activity of White Spot Syndrome Virus Immediate-Early Protein IE1

Liu

Chang

Wang

et al. 2008

“…1a), IE1 N-terminal and C-terminal acidic domain deletion constructs (see Fig. 3), and the p39CAT-E3 reporter have been described previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, it was shown that the AAD could be replaced by the archetype VP16 AAD from herpes simplex virus type 1 and the AcMNPV IE1 AAD. Chimeric proteins containing the VP16 and AcMNPV AADs appeared to be more potent activators of gene expression than the wildtype (WT) IE1 (17).…”

mentioning

confidence: 97%

See 1 more Smart Citation

The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication

Pathakamuri

Theilmann

2002

Self Cite

IE1 is a potent transcriptional transactivator of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and has been shown to be essential for viral DNA replication. IE1 contains an acidic activation domain (AAD) at the N terminus that is essential for transcriptional transactivation, but its role in viral DNA replication is unknown. In this study the role of the IE1 AAD in DNA replication is investigated. We have determined that deletion of the AAD eliminates the ability of IE1 to support DNA replication, showing that the AAD is essential for DNA replication as well as transcriptional transactivation. Replacement of the AAD with the archetype domain from herpesvirus VP16 and the evolutionarily related domain from Autographa californica MNPV (AcMNPV) IE1 produces chimeric proteins that are potent transactivators. Surprisingly, however, these chimeric proteins were unable to support DNA replication, indicating that there is a host-or virus-specific replication subdomain in the AAD that was not functionally replaced by the VP16 or AcMNPV AAD. Using N-and C-terminal deletion mutants, the region of the AAD that was essential for DNA replication was mapped to amino acids 1 to 65. AAD deletion mutants also showed that an IE1 that is functional for transcriptional transactivation is not required for viral DNA replication. The IE1 AAD therefore contains an essential replication domain that is separable from the transcriptional activation domains. Our results suggest that IE1 specifically interacts with a component of the viral replication complex, supporting the view that it acts as a nucleating factor by binding to the viral replication origins.The baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) has a genome of 131,990 bp and encodes approximately 150 genes of 50 amino acids (aa) or larger (5). Using transient-replication assays, it has been shown that six genes, lef-1, lef-2, lef-3, dnapol, p143, and ie1, are essential for replication and that three genes, ie2, p34, and iap-1, are stimulatory (4). Based on studies with OpMNPV and the related viruses Autographa californica MNPV (AcMNPV) and Bombyx mori NPV, data suggest that lef-1 encodes a possible primase and interacts directly with lef-2, which has an unknown function (14). lef-3 encodes a single-stranded DNA binding (SSB) protein, forms homotrimers, and is essential for transport of P143 into the nucleus (1, 15, 56). P143 is a possible helicase, and dnapol is a DNA polymerase (2). IE1 is a primary transcriptional transactivator known to activate early-and lategene expression during baculovirus infections (19, 54). The role of IE1 in baculovirus replication has been hypothesized to be transactivation of other replication genes, or it may play a role as an origin binding protein (OBP). However, Rapp et al.(45) expressed all late expression factors under control of the heat shock promoter and showed that IE1 transcriptional transactivation may not be required for replication. The stimulatory genes IE2 and P34 (PE38 in AcMN...

“…9B). Indeed, the acidic nature of the transactivation domains is conserved among baculovirus IE1s and is required for transcriptional stimulation (8). However, upon direct interaction with negatively charged 28-mer DNA, basic domain I may pull away from the transactivation domains (Fig.…”

Section: Vol 77 2003mentioning

confidence: 99%

The Highly Conserved Basic Domain I of Baculovirus IE1 Is Required for hr Enhancer DNA Binding and hr -Dependent Transactivation

Olson

Wetter

Friesen

2003

The immediate-early protein IE1 is the principal transcriptional regulator of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is dramatically stimulated by cis linkage of the affected promoter to AcMNPV homologous region (hr) elements that contain palindromic 28-bp repeats (28-mers) with enhancer activity. This hr-dependent transcriptional enhancement requires binding of the 28-mer by dimeric IE1. Here, we have defined IE1 domains required for this DNA binding in order to investigate the mechanism of IE1 function. Analysis of a panel of IE1 insertion mutations indicated that disruption of a highly conserved domain (residues 152 to 161) consisting of mostly positive-charged residues (basic domain I) abolished hr-dependent transactivation. Targeted mutagenesis of basic residues within basic domain I caused loss of hr-dependent transactivation but had no effect on IE1 oligomerization, nuclear localization, or hr-independent transactivation of viral promoters. Alanine substitutions of K 152 and K 154 or K 160 and K 161 impaired IE1 binding to 28-mer DNA as a homodimer, indicating that these basic residues are required for enhancer binding. Consistent with a DNA-binding defect, 28-mer interaction was improved by heterodimerization with wild-type IE1 or by increasing mutated IE1 concentrations. DNA binding mediated by basic domain I was also required for IE1 transactivation that occurred through physically separated, unlinked hr elements. We concluded that basic domain I is the enhancer-binding domain for IE1. Our data also suggest that DNA binding activates IE1 for transcriptional enhancement, possibly through a conformational change involving basic domain I.The major transcriptional regulator of Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) is the immediate-early protein IE1. Highly conserved among members of Baculoviridae, this 67-kDa DNA-binding protein is postulated to participate in critical processes during baculovirus infection, including the initiation of DNA replication and transregulation of transcription from early and late viral promoters (1, 13, 15, 17-20, 22, 23, 26, 28, 30, 34, 35, 39-41). Current evidence suggests that IE1's regulatory functions are mediated through its interaction with homologous region (hr) sequences that are repeated throughout the circular DNA genome of AcMNPV (reviewed in references 9 and 27). The baculovirus hrs are transcription enhancers and possible origins of DNA replication (11,12,14,20,22,30,37,41,42). In transfection assays, cis linkage of the hr element to baculovirus promoters can boost IE1-mediated transactivation as much as 200-fold (30, 39, 41). During infection, IE1 also colocalizes with viral DNA replication factories in the nucleus, where it may function as a DNA origin (hr)-binding protein (31,35,40). Despite these roles in transcription and DNA replication, the molecular mechanisms by which IE1 functions through DNA binding are unknown.AcMNPV IE1 is a 582-residue nuclear phosphoprotein wit...