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

Pathakamuri, Joseph; Theilmann, David A.

doi:10.1128/jvi.76.11.5598-5604.2002

Cited by 25 publications

(21 citation statements)

References 54 publications

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“…Our on May 10, 2018 by guest http://jvi.asm.org/ data support a model in which basic domain I functions to concentrate IE1 at the hr enhancers to stimulate transcription by a DNA-binding-dependent mechanism. By virtue of its hrbinding function, we predict that basic domain I is also required for IE1's role in DNA replication (17,35,40). Participation of basic domain I in hr enhancer binding.…”

Section: Discussionmentioning

confidence: 99%

“…Wild-type and mutated IE1s were synthesized for 2 h at 30°C by using 30-l coupled in vitro transcriptiontranslation reaction mixtures (TNT system; Promega) programmed with plasmid DNA and SP6 RNA polymerase. 35 S-labeled methionine-cysteine (NEN) was included only when levels of in vitro-synthesized protein were quantified by denaturing SDS-10% polyacrylamide gel electrophoresis. A 61-bp DNA probe containing the leftmost hr5 28-mer was generated by FokI and HinfI digestion of a PCR-derived fragment from plasmid phr5.1 (43).…”

Section: Plasmids (I) Ie1 Substitutions and Deletions Pie1mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Olson

Wetter

Friesen

2003

J Virol

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Plasmids (I) Ie1 Substitutions and Deletions Pie1mentioning

confidence: 99%

mentioning

confidence: 99%

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

Olson

Wetter

Friesen

2003

J Virol

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“…Forsythe et al (1998) performed a mutational analysis of OpMNPV IE1 AAD and showed that it comprised two activation subdomains. More recently, Pathakamuri & Theilmann (2002) showed that the AAD of OpMNPV IE1 also contains an essential DNA replication subdomain that is separate from the transcriptional activation domain.…”

Section: Introductionmentioning

confidence: 99%

The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells

Dai

Willis

Huijskens

et al. 2004

Journal of General Virology

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The acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0 are essential for transcriptional transactivation. To compare the relative transcriptional activation potentials of IE1 and IE0 AADs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV), we constructed two ecdysone receptor (EcR)-based inducible expression systems to analyse six baculovirus AADs in two insect cell lines (Ld652Y and Sf9) and two mammalian cell lines (NIH-3T3 and CHO). For insect cell expression, the AADs were fused to the C, D, E and F domains of the spruce budworm Choristoneura fumiferana EcR. For mammalian cell expression the AADs were fused to the E and F domains of mammalian Mus musculus retinoid X receptor. In Ld652Y and Sf9 cells, chimeric proteins containing the AcMNPV AADs activated gene expression to higher levels than those containing the OpMNPV AADs. In NIH-3T3 cells, chimeras containing AcMNPV IE1 and IE0 AADs consistently activated gene expression to higher levels than the archetypal mammalian herpesvirus VP16 AAD. In contrast, OpMNPV AADs only activated expression by 5-15 % relative to the VP16 AAD. In CHO cells, both AcMNPV and OpMNPV AADs exhibited intermediate transactivation levels relative to VP16 AAD. These results show that the baculovirus AADs are functional for transcriptional activation in mammalian cells and that AcMNPV AADs generally appear to be more potent than OpMNPV AADs in both insect and mammalian cells.

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“…Previous studies of the alphabaculovirus Orgyia pseudotsugata MNPV (OpMNPV) revealed that the N-terminal 65 residues of OpMNPV IE1 are required for plasmid DNA replication assays in transfected cells (32). However, this N-terminal region and the complete transactivation domain of OpMNPV IE1 exhibit little if any sequence identity with AcMNPV IE1.…”

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confidence: 92%

A Conserved N-Terminal Domain Mediates Required DNA Replication Activities and Phosphorylation of the Transcriptional Activator IE1 of Autographa californica Multicapsid Nucleopolyhedrovirus

Taggart

Mitchell

Friesen

2012

J Virol

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IE1 is the principal transcriptional regulator of the baculoviruses. Like multifunctional transcription factors of other large DNA viruses, IE1 is an essential, site-specific DNA-binding phosphoprotein that activates virus gene expression and promotes genome replication. To define the poorly understood mechanisms by which IE1 achieves its diverse functions, we identified IE1 domains that contribute to productive infection of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the baculovirus prototype. Site-directed mutagenesis revealed that the N-terminal 23 residues of IE1 are required for origin-specific DNA replication and AcMNPV propagation, but not for DNA-binding-dependent transcriptional activation. Within this defined replication domain, we identified an invariant TPXR/H motif that resembles a consensus cyclin-dependent kinase phosphorylation site. Amino acid substitutions of potential phosphorylation sites within or near this motif caused loss of IE1-mediated DNA replication activity. Remarkably, substitution of the single threonine (residue 15) within the TPXR/H motif caused complete loss of AcMNPV multiplication. The replication domain was required for IE1 phosphorylation. It was also sufficient for conferring phosphorylation of a heterologous protein. Importantly, IE1 hyperphosphorylation coincided exclusively with AcMNPV DNA replication. The temporal regulation of IE1 phosphorylation and the essential nature of the TPXR/H motif suggest that phosphorylation critically alters and possibly activates DNA replication activity of IE1 during infection. The striking conservation of the TPXR/H motif among IE1 proteins further suggests that this molecular switch may be a common mechanism by which the alphabaculoviruses coordinate DNA replication and gene expression by using a single regulator.

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

Cited by 25 publications

References 54 publications

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

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

The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells

A Conserved N-Terminal Domain Mediates Required DNA Replication Activities and Phosphorylation of the Transcriptional Activator IE1 of Autographa californica Multicapsid Nucleopolyhedrovirus

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