Herpes Simplex Virus Immediate-early Research Articles

Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency

The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) gene enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the super elongation complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation, and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and invivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes.

HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus.

ABSTRACTEpstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation.IMPORTANCE EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACTCellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

The RING Finger Domain of Varicella-Zoster Virus ORF61p Has E3 Ubiquitin Ligase Activity That Is Essential for Efficient Autoubiquitination and Dispersion of Sp100-Containing Nuclear Bodies

Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli β-galactosidase induced durable β-gal-specific IgG and CD8 + T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.

Combinatorial Transcription of Herpes Simplex Virus and Varicella Zoster Virus Immediate Early Genes Is Strictly Determined by the Cellular Coactivator HCF-1

The mammalian transcriptional coactivator host cell factor-1 (HCF-1) functions in concert with Oct-1 and VP16 to assemble the herpes simplex virus (HSV) immediate early (IE) transcription enhancer core complexes that mediate the high level transcription of these genes upon infection. Although this transcriptional model has been well characterized in vitro, the requirements and significance of the components have not been addressed. Oct-1 was previously determined to be critical but not essential for HSV IE gene expression. In contrast, RNA interference-mediated depletion of HCF-1 resulted in abrogation of HSV IE gene expression. The HSV IE gene enhancer domain is a model of combinatorial transcription and consists of the core enhancer and multiple binding sites for factors such as Sp1 and GA-binding protein. It was striking that HCF-1 was strictly required for VP16-mediated transcriptional induction via the core enhancer as well as for basal level transcription mediated by GA-binding protein and Sp1. HCF-1 was also found to be essential for the induction of varicella zoster virus IE gene expression by ORF10, the VZV ortholog of the HSV IE transactivator VP16, and the autostimulatory IE62 protein. The critical dependence upon HCF-1 demonstrates that this cellular component is a key factor for control of HSV and VZV IE gene expression by functioning as the common element for distinct factors cooperating at the IE gene enhancers. The requirements for this protein supports the model whereby the regulated transport of HCF-1 from the cytoplasm to the nucleus in sensory neurons may control IE gene expression and reactivation of these viruses from the latent state.

Spontaneous Reversion of tsBN67 Cell Proliferation and Cytokinesis Defects in the Absence of HCF-1 Function

Mammalian HCF-1 is a highly conserved and abundant chromatin-bound protein that plays a role in both herpes simplex virus (HSV) immediate-early (IE) gene transcription and cell proliferation. Its role in cell proliferation has been evidenced through the analysis of a temperature-sensitive hamster cell line called tsBN67. When placed at nonpermissive temperature, tsBN67 cells undergo a stable and reversible proliferation arrest after a lag of 36–48 h. This phenotype results from a single point mutation in HCF-1, which disrupts HCF-1 association with both chromatin and the HSV IE transactivator VP16 at nonpermissive temperature. Here, we report the isolation and characterization of spontaneous tsBN67 growth-revertant cells that are able to proliferate at nonpermissive temperatures. These cells retain the tsBN67 HCF-1 point mutation and grow in the absence of HCF-1 chromatin association, demonstrating that complete restoration of tsBN67 HCF-1 functions is not essential for cell proliferation. Phenotypic analysis of both mutant and revertant tsBN67 cells shows that, in addition to a cell proliferation defect, these cells display a conspicuous multinucleated phenotype in a significant population of arrested cells. This defect in cytokinesis is also a result of loss of HCF-1 function, suggesting that HCF-1 plays a role in cell exit from mitosis. The revertant tsBN67 cells display a coincident restoration of cell proliferation and suppression of the cytokinetic defect, suggesting that HCF-1 plays a shared role in cell proliferation and cytokinesis.

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.

HCF-dependent nuclear import of VP16.

Transactivation by VP16 requires the formation of a multicomponent complex, the TAATGAAAT recognition factor complex (TRF.C), that contains in addition to VP16, two cellular proteins, Oct-1 and HCF. HCF binds directly to VP16 and this promotes subsequent interaction of the VP16-HCF complex with the POU DNA-binding domain of Oct-1 and selective assembly onto target sites. Here we demonstrate a novel role of HCF in the intracellular compartmentalization of VP16. We show that while VP16 does not contain a consensus nuclear localization signal (NLS) and is largely cytoplasmic, co-expression with HCF resulted in VP16 nuclear accumulation. A candidate NLS within the C-terminus of HCF was identified and insertion of this motif into green fluorescent protein (GFP) promoted nuclear accumulation. Conversely, removal of this signal from HCF (HCFDeltaNLS) resulted in its cytoplasmic accumulation. Co-expression of HCFDeltaNLS with wild-type (wt) VP16, or of wt HCF with VP16 mutants lacking HCF-binding activity failed to promote the nuclear enrichment of VP16. These results indicate that in addition to its role in stabilizing TRF.C, HCF acts as a nuclear import factor for VP16.

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.

Enhanced infectivity of herpes simplex virus type 1 viral DNA in a cell line expressing the trans-inducing factor Vmw65

Vmw65 is a structural component of herpes simplex virus (HSV) which is involved in transactivating the expression of the viral immediate-early (IE) genes. To gain further insight into the function of this protein, a cell line, BSV65, was established which expresses biologically active Vmw65 under control of the Moloney leukemia virus long terminal repeat. This cell line was shown to specifically activate IE genes as demonstrated by transient transfection assays with reporter genes linked to HSV IE or delayed-early promoter-regulatory regions. Furthermore, by using mobility shift assays, cell extracts were shown to be capable of forming a Vmw65-containing complex with oligonucleotides that contained a TAATGARAT motif, a conserved cis-acting IE regulatory element which is required for Vmw65-mediated trans induction. BSV65 cells were able to complement HSV type 1 in 1814, a mutant which is unable to trans-induce IE gene expression and whose growth is impaired at low multiplicities of infection. Transfection of purified HSV type 1 viral DNA into BSV65 cells resulted in an approximately 200-fold increase in virus production compared with the parental cell line. In addition, in comparison to wild-type cells, infectious virus production occurred sooner and efficiency of plaque formation was higher in BSV65 cells following transfection of viral DNA but not following infection with virus. Northern (RNA) dot blot analysis of cells transfected with viral DNA showed that transcription of the IE gene Vmw175 was approximately 10-fold greater in BSV65 cells compared with wild-type cells. These results indicate that, in the presence of functional Vmw65, there is a greater probability that transfected viral DNA will lead to a productive infection.

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.

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.

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.

Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription

Herpes simplex virus (HSV) immediate early (IE) transcription is known to be stimulated by a structural component of the virion which interacts, either directly or indirectly, with specific regulatory sequences located far upstream from IE messenger RNA 5′-termini. The aim of the work described in this paper is the mapping and identification of the virion component. Cloned HSV DNA fragments derived from various parts of the genome were cotransfected into BHK cells together with chimaeric plasmids which contained the thymidine kinase gene under IE control. Stimulation of thymidine kinase synthesis was elicited by cloned EcoRIi (0.63 to 0.72 map units), BamHIf (0.64 to 0.69) or EcoRIb (0.72 to 0.87). Cloned BamHIf had the same specificity as the virion component, since it stimulated thymidine kinase expression only from chimaeric plasmids which contained functional IE-specific regulatory sequences. The effect of EcoRIb was not confined to plasmids with IE-specific regulatory regions, suggesting a more general stimulatory role for one or more of the polypeptides encoded by this fragment. A subclone containing a 2.7 × 10 3 base-pair fragment of BamHIf (pMCl) was active in the cotransfection assay, and the effect was abolished by an eight base-pair insertion into the middle of this fragment. The only polypeptide known to map entirely within the HSV genome region defined by pMCl was identified as the major tegument species Vmw65. The results therefore suggest that Vmw65 is the virion component which trans-activates HSV IE transcription.

Analysis of DNA sequences which regulate the transcription of a herpes simplex virus immediate early gene.

The locations and functions of DNA sequences involved in transcription of the gene encoding herpes simplex virus type 1 immediate early (IE) mRNAs 4 and 5 were analyzed by use of a transient-expression assay. The region upstream of the genes encoding IE mRNAs 4 and 5 was fused to the thymidine kinase gene coding sequences, and production of enzyme or RNA was measured after transfection of plasmids into BHK cells. The effect of deletions in the upstream region was determined in the absence or presence of a virus structural component which stimulates herpes simplex virus IE transcription. Two distinct units were identified. One of these was a promoter which required not more than 69 base pairs of DNA specific for the genes encoding IE mRNAs 4 and 5 upstream from the mRNA 5' terminus. The other was a far-upstream region which mediated the response to the virion component and had an upstream boundary between nucleotides -347 and -335. An origin of DNA replication was interposed between these two units. The element TAATGAGATAC , which represents a consensus sequence present in the upstream regions of all herpes simplex type 1 IE genes, appeared to be essential for stimulation by the virion component. The activity of this element was modulated by the sequences which flank it, especially by regions having extremely high contents of guanine plus cytosine and which contain a conserved unit CCCGCCC or its complement GGGCGGG .