Herpes Simplex Virus Immediate-early Genes Research Articles

The octamer-binding protein Oct-2 represses HSV immediate-early genes in cell lines derived from latently infectable sensory neurons

Transcription of herpes simplex virus (HSV) immediate-early (IE) genes does not occur in sensory neurons latently infected with the virus or following infection of neuronal cell lines. In neuronal cell lines this inability results from the weak activity of the viral IE promoters, which is caused by a neuron-specific repressor factor that binds specifically to the TAATGARAT motif in these promoters and to related octamer elements. Cells expressing this repressor contain an additional octamer-binding protein that is absent from permissive cells. We identify this factor as the lymphocyte- and neuron-specific octamer-binding protein Oct-2 and show that Oct-2 mRNA is also present in dorsal root ganglion neurons, the natural site of HSV latency in vivo. Moreover, artificially elevated expression of Oct-2 can repress the IE promoter. The potential role of Oct-2 in the initiation and maintenance of in vivo latent infection with HSV is discussed.

Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis

Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis

Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells

The non-permissivity of C1300 mouse neuroblastoma cells for herpes simplex virus (HSV) infection is due to a failure of such cells to transcribe the immediate-early (IE) genes following viral infection. We have transfected both C1300 cells and permissive cells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.

The herpes simplex virus protein Vmw65 can trans-activate both viral and cellular promoters in neuronal cells

Transcription of the herpes simplex virus (HSV) immediate-early (IE) genes in lytic infection is dependent upon the formation of a complex between the cellular transcription factor Oct-1 and the HSV virion protein Vmw65. This complex then binds to the TAATGARAT sequence in the IE promoters and trans-activates the IE genes. Following infection of neuronal cells such as the C1300 neuroblastoma cell line, however, the viral (IE) genes are not transcribed and the lytic cycle is aborted at an early stage. We show here that the cellular factors necessary to form a trans-activating complex with Vmw65 are present in C1300 cells and that trans-activation of both viral and cellular promoters by Vmw65 can be observed in these cells. In contrast with permissive cells, however, trans-activation is only observed in C1300 cells at a high concentration of the target viral promoter and not at a low concentration of the target promoter, regardless of the amount of Vmw65 transfected. The significance of these effects for the regulation of latent infection and cellular gene expression in neuronal cells is discussed.

Regulated transcription of herpes simplex virus immediate-early genes in neuroblastoma cells

C1300 neuroblastoma cells are nonpermissive for infection with herpes simplex virus but can be rendered permissive by pretreatment with sodium butyrate. This increased permissivity which is specific for HSV is caused by increased transcription of the viral immediate-early genes following infection of butyrate-treated cells and can be observed for at least 24 hr following withdrawal of butyrate. The use of C1300 cells as a model system for studying the regulation of immediate-early gene expression in neuronal cells in vitro and its possible relevance to the study of the processes regulating latent infection in vivo is discussed.

ICP4-binding sites in the promoter and coding regions of the herpes simplex virus gD gene contribute to activation of in vitro transcription by ICP4

The herpes simplex virus immediate-early gene product ICP4 activates the transcription of viral early and late genes. We characterized the DNA sequence elements of the early glycoprotein D (gD) gene that play a role in the response to ICP4 in vitro. Using gel mobility shift assays and DNase I footprinting, we identified three ICP4-binding sites, two 5' to the mRNA start site and a third within the coding region. Site II, which gave a footprint between nucleotides -75 and -111 relative to the RNA start site, was previously identified by Faber and Wilcox and contained the reported consensus ICP4-binding site. Site III, which was located between nucleotides +122 and +163, was very similar to the site II sequence, including a core consensus binding sequence, TCGTC. The site I sequence (nucleotides -308 to -282), however, did not share significant homology with either site II or site III. In vitro transcription experiments from mutant constructs of the gD promoter indicated that all three ICP4-binding sites contribute to the stimulation of transcription by ICP4. DNase I footprinting of the gD promoter with uninfected nuclear extracts of HeLa cells showed protection of two very G-rich sequences between nucleotides -33 and -75. We propose that optimal transcription of the gD gene depends on the interaction of ICP4 with multiple binding sites across the gene and cellular factors that recognize specific sequence elements in the promoter.

Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene.

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately -100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.

Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation

Functional upstream activator sequences (TAATGARAT motifs) of herpes simplex virus immediate-early genes were identified and shown both to bind a factor (TRF) present in uninfected HeLa cells and to confer inducibility by the virus regulatory protein, Vmw65, on a normally nonresponsive promoter. Point-mutation analyses demonstrated binding specificity and correlated binding with Vmw65 induction. Furthermore, the octamer domains of the adenovirus DNA replication origin, the histone H2B, and the immunoglobulin light chain genes bound and competed for TRF. The immunoglobulin octamer also conferred Vmw65 inducibility on the TK promoter. In addition, a modified form of TRF was specifically detected in infected cells. We conclude that TRF is similar or identical to the previously described octamer binding protein and is likely to be the target for coordinate induction of immediate-early gene expression by Vmw65.

DNA sequences which regulate the expression of the pseudorabies virus major immediate early gene

It has been shown previously that the transcription of herpes simplex virus (HSV) immediate early (IE) genes is trans-activated by a component of the virus particle. The trans-inducing factor (TIF) is known to be polypeptide Vmw65. Infection with pseudorabies virus (PRV), a related herpesvirus, does not increase expression from HSV IE regulatory sequences (W. Batterson and B. Roizman, 1983, J. Virol. 46, 371–377). To examine the control of the PRV IE gene and possible sequence specificity of a TIF, the 5′ terminus of the PRV major IE transcript was mapped and hybrid plasmids containing PRV upstream sequences linked to the HSV-1 TK gene were constructed. Gene expression under the control of PRV IE or HSV-1 IE gene 3 upstream regions were compared using transient expression assays. It was found that infection with uv-irradiated PRV did not stimulate expression from PRV IE or from HSV-1 IE gene 3 upstream regions, indicating that PRV did not possess an effective TIF. Infection with uv-treated HSV-1, or cotransfection with a plasmid which encodes Vmw65, stimulated expression from both PRV and HSV IE gene upstream regions. The nucleotide sequence of the 5′ end of the PRV transcript and its upstream region was determined. This region was, in overall structure, unlike the upstream regions of HSV IE genes but showed a strong similarity to the enhancers of human and murine cytomegaloviruses (HCMV and MCMV). In particular, a reiterated 15-bp element of the PRV upstream region was homologous to a conserved, repeated sequence element found in both HCMV and MCMV enhancer regions and was also related to the “TAATGARATTC” motif found upstream of all HSV IE genes. Thus a conserved sequence element occurs upstream of IE genes in four herpesviruses with different genome structures and diverse biological properties.

Binding of the herpes simplex virus immediate-early gene product ICP4 to its own transcription start site.

A gel electrophoresis DNA-binding assay was used to detect proteins from herpes simplex virus type 1-infected and uninfected cells that specifically bind the upstream region of immediate-early (IE) gene 3. The assay is based on the altered electrophoretic mobility of DNA-protein complexes relative to that of free DNA in native gels. A series of end-labeled overlapping DNA fragments spanning a region from -272 to +27 (relative to the 5' terminus of the IE gene 3 mRNA) were used as probes. Two complexes were identified (referred to as A and B) which were driven by different protein factors. Formation of the A complex required infected-cell proteins extracted at any time from 2 to 16 h postinfection; a 0.5 to 1 M NaCl extract of infected cells, and a DNA probe that contained the sequences from -4 to +27 (relative to the 5' terminus of IE gene 3 mRNA). The protein that drove the formation of the A complex is not related to transcription factors TFIIIA or Sp1 or their cognate binding domains since neither the 5S RNA gene nor the GC box of simian virus 40 could compete for proteins that induced formation of the A complex. Through the use of monoclonal antibodies, the complex was shown to contain the IE gene 3 product, ICP4. A more detailed localization of the DNA-binding site in vitro by using chemical footprinting revealed that binding occurs over the sequence from -10 to +3 relative to the mRNA terminus. The binding of ICP4 to its own transcription start site may explain the repression of IE gene transcription which attends the onset of early (beta) gene expression and suggests an autoregulatory mechanism for gene control in herpes simplex virus type 1. The B complex was readily detected in uninfected cells (of a number of different cell lines), as well as in infected cells, with a probe containing the IE consensus sequence TAATGARATTC (where R is a purine) and two nested copies of the Sp1 binding motif GGGCGG; however, complexes were also detected with probes that lack the IE consensus sequence but contain Sp1 sites. These data suggest that the B complex contains the promoter-specific factor Sp1, and competition experiments with the clustered Sp1 binding domains from simian virus 40 confirmed this idea.

Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded trans-acting factors

Using a short-term cotransfection system with recombinant chloramphenicol acetyltransferase (CAT) target genes and intact genes for regulatory proteins, we previously demonstrated that expression from the promoter-regulatory region of the gene for the immediate-early 175,000-molecular-weight (IE175K) protein of herpes simplex virus type 1 was subject to trans-acting effects by three different virus-encoded components. In the present work we have attempted to delineate the upstream cis-acting requirements within the IE175K promoter-regulatory region for stimulation by the late structural protein Vmw65, stimulation by the IE110K protein, and repression by its own gene product, the IE175K protein. Our results augment previous reports of others by demonstrating that a construct containing only the single TAATGARAT consensus sequence, TAATGGAAT, between -115 and -106 was efficiently induced by Vmw65. Deletion to -108 effectively abolished the response to Vmw65. However, this latter construct remained responsive to IE110K stimulation and was induced as efficiently as the parental construct which contained sequences to -1900. Furthermore, not only basal levels of expression, but also Vmw65 activation of the parental construct and deletion mutants delta 380, delta 330, delta 300, and delta 160 and IE110K-activated expression of the delta 108 construct were all subject to dominant repression by the IE175K protein. Finally, we show that expression from each of the deletions was open to stimulation by linkage to the simian virus 40 enhancer region. Enhancer-stimulated expression from each construct, including the -108 deletion, was efficiently repressed by the IE175K protein. In contrast, expression from the simian virus 40 enhancer when linked to its own promoter was unaffected by IE175K. These results place sequence requirements for both IE110K stimulation and IE175K autoregulation within the minimal promoter region -108 to +30, separate from the major requirements for Vmw65 activation located further upstream.