Herpes Simplex Virus Immediate-early Promoters Research Articles

Zhangfei: a second cellular protein interacts with herpes simplex virus accessory factor HCF in a manner similar to Luman and VP16.

Host cell factor (HCF, C1, VCAF or CFF) is a cellular protein that is required for transcription activation of herpes simplex virus (HSV) immediate-early (IE) genes by the virion protein VP16. The biological function of HCF remains unclear. Recently we identified a cellular transcription activator, Luman. As with VP16, the transactivation function of Luman is also regulated by HCF. Here we report a second human protein, Zhangfei (ZF) that interacts with HCF in a fashion similar to Luman and VP16. Although ZF shares no significant sequence homology with Luman, the two proteins have some structural similarities. These include: a basic domain-leucine zipper (bZIP) region, an acidic activation domain and a consensus HCF-binding motif. Unlike Luman, or most other bZIP proteins, ZF by itself did not appear to bind consensus bZIP-binding sites. It was also unable to activate promoters containing these response elements. Although in transient expression assays ectopically expressed ZF was unable to block transactivation by VP16 of a HSV IE promoter, ZF could prevent the expression of several HSV proteins in cells infected with the virus. The ability of ZF to block the synthesis of the HSV IE protein ICP0 relied on its binding to HCF, since a mutant of ZF that was unable to bind HCF was also unable to prevent viral IE protein expression.

Interdigitated Residues within a Small Region of VP16 Interact with Oct-1, HCF, and DNA

Upon infection, the herpes simplex virus (HSV) activator of immediate-early (IE) gene transcription VP16 forms a multiprotein-DNA complex with two cellular proteins, Oct-1 and HCF. First, VP16 associates with HCF independently of DNA, and this association stimulates subsequent association with Oct-1 on the DNA target of VP16 activation, the TAATGARAT motif found in HSV IE promoters. We have analyzed the involvement of VP16 residues lying near the carboxy-terminal transcriptional activation domain of VP16 in associating with HCF, Oct-1, and DNA. To assay VP16 association with HCF, we developed an electrophoretic mobility retardation assay in which HCF is used to retard the mobility of a hybrid VP16-GAL4 DNA-binding domain fusion protein bound to a GAL4 DNA-binding site. Analysis of an extensive set of individual and combined alanine substitutions over a 61-amino-acid region of VP16 shows that, even within a region as small as 13 amino acids, there are separate residues involved in association with either HCF, DNA, or Oct-1 bound to DNA; indeed, of two immediately adjacent amino acids in VP16, one is important for DNA binding and the other is important for HCF binding. These results suggest that a small region in VP16 is important for linking in close juxtaposition the four components of the VP16-induced complex and support the hypothesis that the structure of the Oct-1-VP16 interaction in this complex is similar to that formed by the yeast transcriptional regulatory proteins MATa1 and MAT alpha2. We propose that HCF stabilizes this Oct-1-VP16 interaction.

Neurons differentially control expression of a herpes simplex virus type 1 immediate-early promoter in transgenic mice

The immediate-early proteins of herpes simplex virus control the cascade of viral gene expression during lytic infection. It is not known which viral or host proteins control the reactivation of the viral genome in latently infected neurons. To determine whether neuronal proteins can regulate a herpes simplex virus immediate-early promoter in vivo, transgenic mice containing the promoter regulatory region of the herpes simplex virus type 1 immediate-early gene (ICP4) fused to the bacterial beta-galactosidase gene were generated. Two lines of mice, in the absence of viral proteins, displayed ICP4 promoter activity in neurons in specific locations in the central nervous system. The anatomic locations of these neurons were the hippocampus, cerebellar cortex, superior colliculus, indusium griseum, mammillary nucleus, cerebral cortex, and the dorsal laminae of the dorsal horns of the spinal cord. Additional subsets of neurons expressed the ICP4 promoter at lower levels; these included trigeminal ganglia and retinas. In a third line of mice, lower levels of expression were present in many of the above-described neurons. Many types of neurons, nearly all nonneuronal cells in the central nervous system, and some non-nervous system tissues were negative. Viral proteins including VP16 are not necessary to induce transcription from the ICP4 promoter in many neurons and some other cell types but may be required in most cells in vivo. An approximately 100-fold-greater number of neurons in the trigeminal ganglia expressed ICP4 promoter activity in newborn mice compared with adults. These data provide direct evidence that host proteins are sufficient to activate a herpes simplex virus immediate-early promoter in neurons in vivo and that a differential expression pattern for this promoter exists within different neuronal phenotypes and between the same neurons in different ages of mice.

Repression of a Herpes Simplex Virus Immediate-Early Promoter by the Oct-2 Transcription Factor Is Dependent on an Inhibitory Region at the N Terminus of the Protein

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein.

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

Functional interaction between different isoforms of the Oct-2 transcription factor expressed in neuronal cells.

The predominant neuronal isoforms of the Oct-2 transcription factor, Oct 2.4 and Oct 2.5, repress the herpes simplex virus immediate-early promoter both in neuronal cells and in fibroblasts normally lacking Oct-2. In contrast, the predominant B lymphocyte form Oct 2.1, which is present at a lower level in neuronal cells, activates the immediate-early promoter in fibroblasts but represses it in neuronal cells. We show here that both Oct 2.4 and Oct 2.5 can functionally interact with Oct 2.1 and convert it from an activator into a repressor. Hence, the cell type-specific activity of Oct 2.1 results from the presence of Oct 2.4 and 2.5 in neuronal cells and their absence in other cell types. The significance of this effect is discussed in terms of the role of Oct-2 in the regulation of viral and cellular gene expression in neuronal cells.

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.

A modular system for the assay of transcription regulatory signals: the sequence TAATGARAT is required for herpes simplex virus immediate early gene activation.

A modular system for assaying the activity of transcriptional regulatory signals based on herpes simplex virus (HSV) promoter and terminator sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene has been used to study activation of HSV immediate early (IE) gene expression. Insertion of the SV40 72 base pair (bp) repeat increased mRNA levels by 15-fold thus demonstrating the ability of the HSV IE promoter to respond to a heterologous enhancer. A fragment containing part of the intergenic region located between HSV-2 immediate early (IE) genes-3 and -4/-5 increased mRNA levels by 5-fold in response to transactivation by an HSV virion structural polypeptide. The HSV activator fragment increased mRNA levels by 2-fold in the absence of transactivation indicating that cellular proteins are involved in IE gene expression. From HSV-1/HSV-2 DNA sequence comparisons we previously proposed that a DNA sequence, consensus TAATGARAT, present upstream of all HSV-1 and HSV-2 IE genes was required for the co-ordinate induction of IE genes. We show here that a synthetic oligonucleotide containing TAATGARAT conferred the ability to stimulate CAT activity only on transactivation: two copies of TAATGARAT stimulated expression by 2-fold while six copies gave an 8-fold increase. This activation, which was not dependent on orientation of the TAATGARAT sequence, directly demonstrates that TAATGARAT is a component of the IE gene activation sequence.