Herpes Simplex Virus Transactivator Research Articles

NMR structure of the human Mediator MED25 ACID domain

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed β-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel, defined by strands B4-B7-B6.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Setting the Stage for S Phase

In a recent issue of Molecular Cell, Tyagi et al. (2007) show that E2F1, a positive regulator of S phase entry, recruits cofactor HCF-1 and associated hSet1/MLL histone H3 lysine 4 methyltransferase complex, facilitating the activation of genes required for proliferation.

Transcriptional Regulation Is Affected by Subnuclear Targeting of Reporter Plasmids to PML Nuclear Bodies

Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

Host Cell Factor-1 and E2F4 Interact Via Multiple Determinants in Each Protein

Host Cell Factor (HCF-1) is a conserved, essential protein initially identified as a co-regulator for the Herpes Simplex Virus transactivator VP16. HCF-1 is variously involved in regulating transcription, splicing, cell proliferation and cytokinesis; however, its mechanisms of action remain unknown. HCF-1 function is manifested through an increasing assortment of cellular factors that target different regions of the protein. Several HCF-1 partners target the amino-terminal kelch domain of HCF-1 (residues 1-380) via a consensus HCF-binding motif (HBM) comprising the tetrapeptide (D/E)HXY. Searches of sequence databases indicated that this motif is present in E2F1 and E2F4, two members of the E2F family of cell cycle regulators. We show here that E2F4 specifically and directly interacts with HCF-1. Mutational analysis showed E2F4 independently targets the kelch domain and the basic domain (residues 450-902) of HCF-1, both of which are required for normal cell-cycle progression via separate determinants. The HBM-containing domain of E2F4 was necessary for interaction with the kelch domain of HCF-1 but not for interaction with the basic domain. Mutations in the HCF-1 kelch domain known to block cell growth abrogated E2F4 binding to the kelch domain in the absence but not in the presence of the juxtaposed basic region. Functionally, HCF-1 co-activated E2F4/DP-1 in transient transfection assays, while E2F4 blocked HCF-1-dependent rescue of a cell line that harbors a temperature sensitive mutant of HCF-1 that causes growth arrest. Our findings show that HCF-1 and E2F4 interact via multiple determinants and suggest a linkage between E2F4 and HCF-1 cell growth pathways.

Host Cell Factor-1 Interacts with and Antagonizes Transactivation by the Cell Cycle Regulatory Factor Miz-1

Human host cell factor-1 (HCF-1) is essential for cell cycle progression and is required, in conjunction with the herpes simplex virus transactivator VP16, for induction of viral immediate-early gene expression. We show here that HCF-1 directly binds to the Myc-interacting protein Miz-1, a transcription factor that induces cell cycle arrest at G(1), in part by directly stimulating expression of the cyclin-dependent kinase inhibitor p15(INK4b). A domain encompassing amino acids 750-836, contained within a subregion of HCF-1 required for cell cycle progression, was sufficient to bind Miz-1. Conversely, HCF-1 interacted with two separate regions in Miz-1: the N-terminal POZ domain and a C-terminal domain (residues 637-803) previously shown to harbor determinants for interaction with c-Myc and the coactivator p300. The latter functioned as a potent transactivation domain when tethered to DNA, indicating that HCF-1 targets a transactivation function in Miz-1. HCF-1 or a Miz-1-binding fragment of HCF-1 repressed transactivation by Gal4-Miz-1 in transfection assays. Moreover, HCF-1 repressed Miz-1-mediated transactivation of a reporter gene linked to the p15(INK4b) promoter. Protein/protein interaction studies and transient transfection assays demonstrated that HCF-1 interferes with recruitment of p300 to Miz-1, similar to what has been reported with c-Myc. Our findings identify Miz-1 as a novel HCF-1-interacting partner and illustrate cross-talk between these two proteins that may be of consequence to their respective functions in gene regulation and their opposing effects on the cell cycle.

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human beta-actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16-responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre-implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2-cell to the 4-cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility.

Herpes simplex virus transactivator VP16 discriminates between HCF-1 and a novel family member, HCF-2.

Herpes simplex virus infection is initiated by VP16, a viral transcription factor that activates the viral immediate-early (IE) genes. VP16 does not recognize the IE gene promoters directly but instead forms a multiprotein complex with Oct-1 and HCF-1, a ubiquitous nuclear protein required for progression through the G1 phase of the cell cycle. The functional significance of recruiting HCF-1 to the VP16-induced complex is not understood. Here we describe the identification of a second HCF-like protein, designated HCF-2. HCF-2 is smaller than HCF-1 but shares three regions of strong amino acid sequence homology, including the beta-propeller domain required for association with VP16. HCF-2 is expressed in many tissues, especially the testis, and shows a more dynamic pattern of subcellular localization than HCF-1. Although HCF-2 associates with VP16 and can support complex assembly with Oct-1 and DNA, it is significantly less efficient than HCF-1. A similar preference is shown by LZIP, a cellular counterpart of VP16. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the beta-propeller domains from HCF-1 and HCF-2 dictate this selectivity. These results reveal an unexpected level of specificity in the recruitment of HCF-1 to the VP16-induced complex, paralleling the preferential selection of Oct-1 rather than the closely related POU domain protein Oct-2. Implications for regulation of the viral life cycle are discussed.

Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16

The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.

Amino Acid Substitutions in the Herpes Simplex Virus Transactivator VP16 Uncouple Direct Protein-Protein Interaction and DNA Binding from Complex Assembly and Transactivation

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional assembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.

Transcriptional Activation by DNA-Binding Derivatives of HSV-1 VP16 That Lack the Carboxyl-Terminal Acidic Activation Domain

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex with cellular components Oct-1 and VCAF-1, contributing a potent carboxyl-terminal acidic activation domain that is essential for activation of gene expression in mammalian cells. We show here that VP16, devoid of this acidic activation domain, functions as a strong transcriptional activator in the yeast Saccharomyces cerevisiae when appended onto a heterologous GAL4 DNA binding domain, as determined by measuring activation of a resident GAL1;lacZ reporter gene. Deletion analysis indicated that sequences contained within the amino-terminal 369 amino acids of VP16 were necessary for transactivation by truncated VP16. Activation by truncated VP16 in yeast was comparable to that observed with a hybrid protein consisting of the GAL4 DNA binding domain linked to the VP16 acidic activation domain. Similar GAL4-VP16 hybrid proteins were only marginally active in mammalian cells. Sequence requirements for transactivation by truncated VP16 can be demarcated from domains of VP16 that are required for interaction with VCAF-1 and for protein-DNA complex formation with Oct-1. Our findings indicate that VP16 contains additional sequences upstream of the acidic activation domain that may play a direct role in transactivation.

An unusual cellular factor potentiates protein-DNA complex assembly between Oct-1 and Vmw65.

The herpes simplex virus trans-activator Vmw65 interacts with the cellular factors Oct-1 and VCAF-1 to generate a multicomponent DNA binding complex that specifically recognizes conserved enhancer elements found upstream of the viral immediate-early genes, resulting in potent stimulation of their transcription. We have identified a HeLa cell factor, distinct from Oct-1 or VCAF-1, which significantly enhances the stability or formation of Vmw65-dependent complexes, as judged by mobility shift analysis using either nuclear extracts or bacterially expressed Oct-1 and Vmw65. This factor, designated SF (stimulatory factor), was partially purified from HeLa cell postnuclear extracts and has an apparent molecular weight of 1500-3000, based on ultrafiltration and size-exclusion chromatography. SF was shown to be resistant to inactivation by heat treatment, protease, nuclease, and phospholipase digestions, and extraction with organic solvents. Pretreatment of SF with beta-glucuronidase did not affect its ability to stimulate Vmw65-dependent complex formation but did reduce the electrophoretic mobility of the resulting complex. These data indicate that SF is probably a component of the Vmw65-induced complex and may be composed, at least partially, of carbohydrate.

Functional Interference between the Ubiquitous and Constitutive Octamer Transcription Factor 1 (OTF-1) and the Glucocorticoid Receptor by Direct Protein-Protein Interaction Involving the Homeo Subdomain of OTF-1

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1.

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

The herpes simplex virus trans-activator VP16 recognizes the Oct-1 homeo domain: evidence for a homeo domain recognition subdomain.

The homeo domain of the Oct-1 transcription factor directs formation of a multiprotein-DNA complex containing Oct-1, the herpes simplex virus (HSV) trans-activator VP16, and a second host cell factor (HCF). This VP16-induced complex alters the regulatory activity of Oct-1, in part, by associating it with the potent VP16 acidic transcriptional activation domain. Here, we show that in the absence of HCF, VP16 can recognize specifically the Oct-1 homeo domain. A region of VP16 near the acidic activation domain appears to be involved exclusively in homeo domain recognition because a 4-amino-acid insertion within this region only affects the ability of VP16 to interact with Oct-1, leaving its DNA- and HCF-binding activities unchanged. A 33-amino-acid peptide containing this region complexes with the Oct-1 POU domain bound to DNA, suggesting that this VP16 region contains an autonomous homeo domain recognition subdomain.

Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.

A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1.

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.

A Cellular Factor Binds to the Herpes Simplex Virus Type 1 Transactivator Vmw65 and Is Required for Vmw65-Dependent Protein-DNA Complex Assembly with Oct-1

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.