TAATGARAT Elements Research Articles

The Neuronal Host Cell Factor-Binding Protein Zhangfei Inhibits Herpes Simplex Virus Replication

During lytic infection in epithelial cells the expression of herpes simplex virus type 1 (HSV-1) immediate-early (IE) genes is initiated by a multiprotein complex comprising the virion-associated protein VP16 and two cellular proteins, host cellular factor (HCF) and Oct-1. Oct-1 directly recognizes TAATGARAT elements in promoters of IE genes. The role of HCF is not clear. HSV-1 also infects sensory neurons innervating the site of productive infection and establishes a latent infection in these cells. It is likely that some VP16 is retained by the HSV-1 nucleocapsid as it reaches the neuronal nucleus. Its activity must therefore be suppressed for successful establishment of viral latency. Recently, we discovered an HCF-binding cellular protein called Zhangfei. Zhangfei, in an HCF-dependent manner, inhibits Luman/LZIP/CREB3, another cellular HCF-binding transcription factor. Here we show that Zhangfei is selectively expressed in human neurons. When delivered to cultured cells that do not normally express the protein, Zhangfei inhibited the ability of VP16 to activate HSV-1 IE expression. The inhibition was specific for HCF-dependent transcriptional activation by VP16, since a Gal4-VP16 chimeric protein was inhibited only on a TAATGARAT-containing promoter and not a on a Gal4-containing promoter. Zhangfei associated with VP16 and inhibited formation of the VP16-HCF-Oct-1 complex on TAATGARAT motifs. Zhangfei also suppressed HSV-1-induced expression of several cellular genes including topoisomerase IIalpha, suggesting that in addition to suppressing IE expression Zhangfei may have an inhibitory effect on HSV-1 DNA replication and late gene expression.

Distinct responses of the herpes simplex virus and varicella zoster virus immediate early promoters to the cellular transcription factors Brn-3a and Brn-3b

The related viruses herpes simplex virus (HSV) and varicella zoster virus (VZV) show distinct but related patterns of latent infection and reactivation in human sensory ganglia. The cellular POU family transcription factors Brn-3a and Brn-3b are expressed in sensory ganglia and bind to the TAATGARAT (R stands for purine) regulatory motifs in the immediate–early gene promoters of these viruses. We show that Brn-3a activates the full length HSV IE1 promoter whereas Brn-3b represses its activity. In contrast both Brn-3a and Brn-3b activate the full length VZV IE promoter. The response of the full length VZV promoter to Brn-3b is not observed with a minimal VZV immediate–early promoter lacking any TAATGARAT elements and cannot be restored by addition of either the upstream TAATGARAT-containing region of the HSV IE promoter or a VZV TAATGARAT-like element to this minimal promoter. The unique effect of Brn-3b on the full length VZV immediate early gene promoter may play a key role in the distinct pattern of latent infection and reactivation observed with this virus in vivo.

Cellular transcription factors enhance herpes simplex virus type 1 oriS-dependent DNA replication.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains three binding sites for the viral origin binding protein (OBP) flanked by transcriptional regulatory elements of the immediate-early genes encoding ICP4 and ICP22/47. To assess the role of flanking sequences in oriS function, plasmids containing oriS and either wild-type or mutant flanking sequences were tested in transient DNA replication assays. Although the ICP4 and ICP22/47 regulatory regions were shown to enhance oriS function, most individual elements in these regions, including the VP16-responsive TAATGARAT elements, were found to be dispensable for oriS function. In contrast, two oriS core-adjacent regulatory (Oscar) elements, OscarL and OscarR, at the base of the oriS palindrome were shown to enhance oriS function significantly and additively. Specifically, mutational disruption of either element reduced oriS-dependent DNA replication by 60 to 70%, and disruption of both elements reduced replication by 90%. The properties of protein-DNA complexes formed in gel mobility shift assays using uninfected and HSV-1-infected Vero cell nuclear extracts demonstrated that both OscarL and OscarR are binding sites for cellular proteins. Whereas OscarR does not correspond to the consensus binding site of any known transcription factor, OscarL contains a consensus binding site for the transcription factor Sp1. Gel mobility shift and supershift experiments using antibodies directed against members of the Sp1 family of transcription factors demonstrated the presence of Sp1 and Sp3, but not Sp2 or Sp4, in the protein-DNA complexes formed at OscarL. The abilities of OscarL and OscarR to bind their respective cellular proteins correlated directly with the efficiency of oriS-dependent DNA replication. Cooperative interactions between the Oscar-binding factors and proteins binding to adjacent OBP binding sites were not observed. Notably, Oscar element mutations that impaired oriS-dependent DNA replication had no detectable effect on either basal or induced levels of transcription from the ICP4 and ICP22/47 promoters, as determined by RNase protection assays. The Oscar elements thus appear to provide binding sites for cellular proteins that facilitate oriS-dependent DNA replication but have no effect on transcription of oriS-flanking genes.

Structural Organization and Chromosomal Assignment of the Mouse Embryonic TEA Domain-Containing Factor (ETF) Gene

Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed thatEtdfspans approximately 17.9 kb and consists of 12 exons. The exon–intron structure ofEtdfclosely resembles that of theDrosophila scallopedgene, indicating that these genes may have evolved from a common ancestor. The multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in the 5′-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. TheEtdflocus was assigned to the proximal region of mouse chromosome 7 using fluorescencein situhybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation,in vivofunction, and evolution ofEtdf.

Proteins and cis-acting elements associated with transactivation of the varicella-zoster virus (VZV) immediate-early gene 62 promoter by VZV open reading frame 10 protein.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein, the homolog of herpes simplex virus type 1 (HSV-1) VP16, is a virion-associated transactivator of the VZV immediate-early (IE) gene 62 (IE62) promoter. VP16 forms a complex with cellular factors (Oct1 and host cell factor [HCF]) and TAATGARAT elements (found in all HSV-1 IE promoter/enhancer sequences) to mediate stimulation of IE transcription. The VZV IE62 promoter also contains three TAATGARAT-like elements. Mutagenesis studies of the VZV IE62 promoter indicated that TAATGARAT-like elements contribute to transactivation of the VZV IE62 promoter by ORF10 protein. Other cis-acting elements such as GA-rich and cyclic AMP-responsive elements were also needed for full transactivation by ORF10 protein. In mobility shift assays, ORF10 protein formed a complex with either of two TAATGARAT-like elements that lack an overlapping octamer-binding motif (octa-/TAATGARAT) but not with a TAATGARAT element with an overlapping octamer-binding motif (octa+/TAATGARAT). In contrast, VP16 formed a high-affinity ternary complex with an octa+/TAATGARAT element and a low-affinity complex with octa-/TAATGARAT elements. Addition of antibodies to ORF10 protein, Oct1, or HCF disrupted the complexes, demonstrating that ORF10 protein interacts with Oct1 and HCF. These results suggest that transactivation of the VZV IE62 gene by ORF10 protein and HSV IE genes by VP16 require similar cellular proteins but distinct cis-acting elements.

Mechanisms for Flexibility in DNA Sequence Recognition and VP16-Induced Complex Formation by the Oct-1 POU Domain

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

A cellular factor binding to the TAATGARAT DNA sequence prevents the expression of the HSV immediate-early genes following infection of nonpermissive cell lines derived from dorsal root ganglion neurons

Cell lines derived from dorsal root ganglion neurons are nonpermissive for HSV infection and do not transcribe the viral immediate-early genes following infection. The lack of immediate-early gene transcription in these cells is caused by the presence of a neuronal cell specific inhibitory factor which binds to the TAATGARAT elements in the promoters of these genes and prevents their transcription. The significance of these results for an understanding of the processes regulating the interaction of HSV with neuronal cell types and the establishment of latent infections in vivo is discussed.

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.

Control of expression of the varicella-zoster virus major immediate early gene

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.

Overlapping octamer and TAATGARAT motifs in the VF65-response elements in herpes simplex virus immediate-early promoters represent independent binding sites for cellular nuclear factor III

Expression of the immediate-early (IE) genes of herpes simplex virus (HSV) is specifically stimulated by a 65-kilodalton virion transcription factor (VF65 or VP16) that is introduced as a component of infecting virions. In both the IE175(ICP4) and IE110(ICP0) promoters, this activation requires an upstream cis-acting target response element that contains a single TAATGARAT consensus element. Furthermore, many HSV IE TAATGARAT elements overlap with ATGCTAAT octamer motifs that are similar to the OTF-1-binding sites found in both immunoglobulin and histone H2b genes and to the nuclear factor III (NFIII)-binding site within the adenovirus type 2 origin of DNA replication. Purified HeLa cell NFIII protein proved to form specific DNA-protein complexes with several upstream regions from both the IE110 and IE175 promoters, and this interaction was subject to efficient competition with an adenovirus type 2 DNA fragment containing an intact NFIII-binding site. Surprisingly, the NFIII protein bound to synthetic oligonucleotides containing only the TAATGARAT consensus elements as well as to those containing the ATGCTAAT octamer sequence, although the former exhibited lower affinity and gave complexes with slightly different electrophoretic mobility. The ATGCTAAT oligonucleotide also competed more efficiently than the TAATGARAT sequence itself for binding to a TAATGARAT probe, indicating that the same protein species binds to both sites. The oligonucleotides also formed novel supershifted complexes with lysed virion proteins, but only in the presence of a crude nuclear extract and not with affinity-purified NFIII alone. We conclude that the cellular NFIII protein can recognize both the ATGCTAAT and TAATGARAT elements independently but that only the interaction with TAATGARAT represents an intermediate step in the transcriptional stimulation of IE genes by the HSV virion factor.

The different competitive abilities of viral TAATGARAT elements and cellular octamer motifs, mediate the induction of viral immediate-early genes and the repression of the histone H2B gene in herpes simplex virus infected cells

An HSV virion component, Vmw65, interacts with cellular transcription factors to transactivate TAATGARAT-containing viral genes and some cellular genes containing the related octamer element. We show that the octamer-containing histone H2B promoter can be trans-activated by transfection of Vmw65 but not by viral infection. The induction of H2B transcription by Vmw65 can be abolished by co-transfection of excess amounts of either a TAATGARAT element or a Vmw65 responsive octamer element. This effect cannot be overcome by addition of increasing amounts of Vmw65. The H2B promoter and TAATGARAT-containing viral promoters therefore compete for limiting cellular factors required for induction by Vmw65 resulting in repression of the H2B gene during lytic infection. The competitive effect of TAATGARAT elements on the H2B gene is not observed in the absence of Vmw65, but can be produced in the presence of a truncated form of Vmw65 lacking the acidic tail required for transcriptional activation. Hence a domain of Vmw65 distinct from that involved in transcriptional induction interacts with cellular octamer binding proteins favouring binding to the TAATGARAT motif.

A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins.

Immediate early genes of herpes simplex viruses contain one or more copies of the conserved TAAT-GARAT (where R is purine) DNA motif. A virus-encoded regulatory protein (Vmw65) is believed to stimulate transcription via this element, although the protein does not bind directly to DNA. Overlapping the TAATGARAT element in many cases is an octamer sequence (ATGCAAAT) that is involved both in transcription by RNA polymerases II and III and in adenovirus DNA replication. So far at least two proteins (OTF-1 and OTF-2) have been identified that bind to the octamer. We show that both affinity-purified OTF-1 and OTF-2 bind to the TAATGARAT sequence and that Vmw65 induces the formation of an additional complex that involves OTF-1 and that is further retarded in a band-shift gel assay. Complementation experiments involving addition of purified OTF-1 to nuclear extracts that have been depleted of endogenous OTF-1 show that at least one other cellular factor(s) is required for complex formation. This cellular factor may be involved in recognition of the GARAT sequence.

Sp1 binds to promoter sequences and activates herpes simplex virus 'immediate-early' gene transcription in vitro.

During a herpes simplex virus (HSV-1) lytic infection, three classes of viral genes are transcribed in a temporally regulated manner. The HSV-1 'immediate-early' (IE) promoter sequences contain multiple copies of a hexanucleotide sequence, GGGCGG, known as a GC box, and one or more copies of an 11-base pair (bp) conserved A + T-rich element, designated TAATGARAT. The TAATGARAT elements are thought to mediate the trans-activation of IE RNA synthesis by a virion-associated protein(s), and the flanking G + C-rich sequences appear both to potentiate this induction and to direct IE promoter activity in vivo. The similarity of the herpesvirus GC box repeats to those of the simian virus 40 (SV40) early promoter prompted the in vitro analysis of HSV IE transcription reported here. We show that the mammalian gene-specific transcription factor Sp1 binds to eight distinct regions of the HSV short terminal repeat and stimulates transcription 25-fold from the divergent IE-3 (ICP-4) and IE-4/5 promoters.