ICP4 Binding Research Articles

Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells

G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

Binding of ICP4, TATA-binding protein, and RNA polymerase II to herpes simplex virus type 1 immediate-early, early, and late promoters in virus-infected cells.

The binding of herpes simplex virus type 1 ICP4, TATA-binding protein (TBP), and RNA polymerase II (polII) to the promoter regions of representative immediate-early (IE) (ICP0), early (E) (thymidine kinase [tk]), and late (L) (glycoprotein C [gC]) genes on the viral genome was examined as a function of time postinfection, viral DNA replication, cis-acting sites for TFIID in the tk and gC promoters, and genetic background of ICP4. The binding of TBP and polII to the IE ICP0 promoter was independent of the presence of ICP4, whereas the binding of TBP and polII to the tk and gC promoters occurred only when ICP4 also bound to the promoters, suggesting that the presence of ICP4 at the promoters of E and L genes in virus-infected cells is crucial for the formation of transcription complexes on these promoters. When the TATA box of the tk promoter or the initiator element (INR) of the gC promoter was mutated, a reduction in the amount of TBP and polII binding was observed. However, a reduction in the amount of ICP4 binding to the promoters was also observed, suggesting that the binding of TBP-containing complexes and ICP4 is cooperative. The binding of ICP4, TBP, and polII was also observed on the gC promoter at early times postinfection or when DNA synthesis was inhibited, suggesting that transcription complexes may be formed early on L promoters and that additional events or proteins are required for expression. The ability to form these early complexes on the gC promoter required the DNA-binding domain but in addition required the carboxyl-terminal 524 amino acids of ICP4, which is missing the virus n208. This region was not required to form TBP- and polII-containing complexes on the tk promoter. n208 activates E but not L genes during viral infection. These data suggest that a region of ICP4 may differentiate between forming TBP- and polII-containing complexes on E and L promoters.

DNA-dependent oligomerization of herpes simplex virus type 1 regulatory protein ICP4.

The human herpes simplex virus type 1 regulatory protein ICP4 binds DNA as a dimer and forms a single protein-DNA complex (A complex) with short DNA probes. ICP4 oligomerized in a DNA-dependent manner, forming two or more protein-DNA complexes with longer DNA fragments containing a single DNA binding site. When resolved electrophoretically, one or more low-mobility DNA-protein complexes follow the fast-moving A complex. The major protein-DNA complex (B complex) formed by ICP4 with long DNA probes migrates just behind the A complex in the electric field, implying the oligomerization of ICP4 on the DNA. Binding experiments with circularly permutated DNA probes containing one ICP4 binding site revealed that about 70 bp of nonspecific DNA downstream of the cognate ICP4 binding site was required for efficient B complex formation. In addition, the C-terminal domain of ICP4 was found to be required for DNA-dependent oligomerization and B complex formation. Gel mobility shift analysis of protein-DNA complexes, combined with supershift analysis using different monoclonal antibodies, indicated that the B complex contained two ICP4 dimers. DNase I footprinting of ICP4-DNA complexes showed that one ICP4 dimer contacts the specific binding site and another ICP4 dimer contacts nonspecific DNA in the B complex. DNA-dependent oligomerization increased the affinity of ICP4 for relatively weak binding sites on large DNA molecules. The results of this study suggest how ICP4 may use multiple weak binding sites to aid in transcription activation.

Temperature-dependent conformational changes in herpes simplex virus ICP4 that affect transcription activation.

The C-terminal 500 amino acids of herpes simplex virus type 1 ICP4 are required for full activator function and viral growth and are known to participate in interactions consistent with the role of ICP4 as an activator of transcription. Oligonucleotide mutagenesis was used to target stretches of amino acids that are conserved with the ICP4 analogs of other alphaherpesviruses and were also predicted to be exposed on the surface of the molecule. Seven mutants were isolated that possessed one to three amino acid changes to the residue alanine in four regions between residues 1000 and 1200. The mutants generated were analyzed first in transfection assays and subsequently after introduction into the viral genome. A number of phenotypes representing different degrees of functional impairment were observed. In transient assays conducted at 37 degrees C, mutant M2 was indistinguishable from wild-type ICP4. Mutants M6 and M7 were marginally impaired. M3, M4, and M5 were more significantly impaired but still able to activate transcription, and M1 was completely impaired. In the context of the viral genome, M1, M3, and M7 were found to be temperature sensitive for growth. All three overproduced immediate-early (IE) proteins at the nonpermissive temperature (NPT). M3 and M7 produced early but not late proteins, and M1 produced neither early nor late proteins, at the NPT. The ICP4 proteins synthesized by all of the mutants tested were able to bind to specific ICP4 binding sites in electrophoretic mobility shift experiments. However, the DNA-protein complexes formed with the ICP4 from M1, M3, or M7 produced at the NPT possessed altered mobility. These complexes were not supershifted by a monoclonal antibody that recognizes an epitope in the C terminus; however, they were supershifted by a monoclonal antibody that recognizes the N terminus. The results suggest that the mutant forms of ICP4, while able to bind to DNA, are conformationally altered at the NPT, thus impairing the ability of the protein to activate transcription to different extents. The complete lack of ICP4 function characteristic of the M1 protein, and the inability of all the mutants to attenuate IE gene expression, suggest that the mutations additionally affect functions of the N terminus to different extents.

The Equine Herpesvirus 1 Immediate-Early Protein Interacts with EAP, a Nucleolar-Ribosomal Protein

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein–Barr virus-encoded small nuclear RNA-associated protein), a 15-kDa nucleolar-ribosomal protein (R. Leopardi, and B. Roizman, Proc. Natl. Acad. Sci. USA 93, 4572–4576, 1996). DNA binding assays revealed that (i) glutathione S-transferase (GST)-EAP disrupted the binding of HSV-1 ICP4 to its cognate DNA in a dose-dependent manner, (ii) GST-EAP interacted with the EHV-1 IE protein, but did not disrupt its binding to its cognate site in viral DNA. GST-pulldown assays indicated that the SRT of the IE protein is required for physical interaction with EAP. The IE protein and EAP colocalized in the cytoplasm of the infected equine ETCC cells at late times of the infection cycle. This latter finding may be important in EHV-1 gene regulation since late viral gene expression is greatly influenced by the EICP0 trans-activator protein whose function is antagonized by the IE protein.

Herpes simplex virus 1 open reading frames O and P are not necessary for establishment of latent infection in mice.

Open reading frame (ORF) O and ORF P partially overlap and are located antisense to the gamma(1)34.5 gene within the domain transcribed during latency. In wild-type virus-infected cells, ORF O and ORF P are completely repressed during productive infection by ICP4, the major viral transcriptional activator/repressor. In cells infected with a mutant in which ORF P was derepressed there was a significant delay in the appearance of the viral alpha-regulatory proteins ICP0 and ICP22. The ORF O protein binds to and inhibits ICP4 binding to its cognate DNA site in vitro. These characteristics suggested a role for ORF O and ORF P in the establishment of latency. To test this hypothesis, two recombinant viruses were constructed. In the first, R7538(P-/O-), the ORF P initiator methionine codon, which also serves as the initiator methionine codon for ORF O, was replaced and a diagnostic restriction endonuclease was introduced upstream. In the second, R7543(P-/O-)R, the mutations were repaired to restore the wild-type virus sequences. We report the following. (i) The R7538(P-/O-) mutant failed to express ORF O and ORF P proteins but expressed a wild-type gamma(1)34.5 protein. (ii) R7538(P-/O-) yields were similar to that of the wild type following infection of cell culture or following infection of mice by intracerebral or ocular routes. (iii) R7538(P-/O-) virus reactivated from latency following explanation and cocultivation of murine trigeminal ganglia with Vero cells at a frequency similar to that of the wild type, herpes simplex virus 1(F). (iv) The amount of latent R7538(P-/O-) virus as assayed by quantitative PCR is eightfold less than that of the repair virus. The repaired virus could not be differentiated from the wild-type parent in any of the assays done in this study. We conclude that ORF O and ORF P are not essential for the establishment of latency in mice but may play a role in determining the quantity of latent virus maintained in sensory neurons.

The Host-Cell Architectural Protein HMG I(Y) Modulates Binding of Herpes Simplex Virus Type 1 ICP4 to Its Cognate Promoter

The productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site inin vitroassays and to enhance the activity of this protein in short-term transient expression assays.

A virus with a mutation in the ICP4-binding site in the L/ST promoter of herpes simplex virus type 1, but not a virus with a mutation in open reading frame P, exhibits cell-type-specific expression of gamma(1)34.5 transcripts and latency-associated transcripts.

The herpes simplex virus type 1 L/S junction-spanning transcripts (L/STs) are a family of multisized transcripts expressed at high levels in cells infected with mutant viruses that (i) do not express ICP4, (ii) specify forms of ICP4 unable to bind to the consensus ICP4 binding site, or (iii) contain mutations in the ICP4 binding site located at the transcriptional start site of the L/STs. By extension, the failure to detect the L/STs in wild-type virus-infected cells is due to the repressive effect of ICP4 bound to its cognate binding site upstream of the L/ST transcription initiation site. ORF-P, the first and largest open reading frame (ORF) encoded by the L/STs, overlaps >90% of the ORF encoding ORF-34.5, a putative neurovirulence factor, which is transcribed from the opposite DNA strand. Viruses with mutations in the overlapping region of ORF-P and ICP34.5 exhibit premature shutoff of infected-cell protein synthesis and are highly attenuated following intracranial inoculation of juvenile mice. To determine whether the premature protein shutoff and neuroattenuated phenotypes of ORF-P ORF-34.5 double mutants are a consequence of alterations in ORF-P, ORF-34.5, or both, viruses containing mutations only in ORF-P or only in the ICP4 binding site in the L/ST promoter were isolated and characterized. Mutant virus L/ST-n38 contains a single-base-pair transition mutation in ORF-P codon 38, resulting in translational termination of the ORF-P protein (OPP). This mutation does not alter the amino acid sequence of ICP34.5. Expression of a truncated form of OPP by mutant virus L/ST-n38 did not result in premature shutoff of infected-cell protein synthesis and produced no other observable phenotype relative to wild-type virus in in vitro tests. Moreover, the 50% lethal dose (LD50) of L/ST-n38 was comparable to that of wild-type virus following intracranial inoculation of 3-week-old mice, as were the latency and reactivation phenotypes of the virus. These properties of L/ST-n38 indicate that the attenuated phenotype of ORF-P ORF-34.5 double mutants is a consequence of mutations that affect the function of ICP34.5 and not the function of OPP. Mutant virus LST-4BS contains four single-base-pair substitutions in the ICP4 binding site in the L/ST promoter that abrogate the binding of ICP4 to this site, leading to high-level expression of the L/STs and OPP. LST-4BS induced premature shutoff of viral and cellular protein synthesis and was slightly growth restricted in cells of neural lineage (SK-N-SH human neuroblastoma cells) but was wild type for these two parameters in cells of nonneural lineage (immortalized primate Vero cells). Of particular interest was the observation that L/ST-4BS exhibited cell-type-specific expression of both the gamma(1)34.5 transcripts and the latency-associated transcripts (LATs). Thus, expression of these transcripts was barely detectable in cells of neural lineage (NB41A3 mouse neuroblastoma cells) but was wild type in Vero cells. In vivo, L/ST-4BS was reactivated from mouse trigeminal ganglia with reduced efficiency and delayed kinetics relative to wild-type virus. L/ST-4BS was completely attenuated for neurovirulence (LD50 > 10(6) PFU) relative to wild-type virus (LD50 < 900 PFU), although the four single-base-pair substitutions lie outside the coding region for the neurovirulence factor, ICP34.5. Collectively, the complex in vitro and in vivo phenotypes of L/ST-4BS can be attributed to (i) disruptions of the ICP4 binding site in the L/ST promoter and subsequent overexpression of the L/STs and OPP; (ii) alterations in ORF-O, which is also mutated in L/ST-4BS; or (iii) alterations in other cryptic genes or cis-acting elements.

Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.

Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection.

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the alpha4 gene, binds to its transcription initiation site and represses the accumulation of alpha4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3' from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5' of TATA boxes, as in the case of the alpha0 gene, has not been adequately addressed. To this end, recombinant alpha0 promoters with various arrays of ICP4 binding sites flanking the alpha0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous alphaO ICP4 binding site, located 5' of the TATA box, results in derepression of alpha0 protein and RNA accumulation in infected Vero cells. (ii) The degree of alpha0 derepression is equivalent to that reported for the alpha4 gene following destruction of the ICP4 binding site at the alpha4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the alpha0 mRNA cap site represses the accumulation of alpha0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of alpha0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that alpha0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5' of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5', as well as 3', of virus gene TATA boxes.

Characterization of the feline herpesvirus type 1 immediate early gene expression.

We analyzed the mechanism for feline herpesvirus type 1 (FHV-1) immediate early (IE) gene expression. We demonstrated that (i) the transcription initiation site of the FHV-1 IE transcript lies in the putative FHV-1 ICP4 binding site, (ii) the FHV-1 IE transcript is spliced to remove 906 bp intron from the leader region, (iii) cis-acting elements of the FHV-1 IE promoter map both down- and upstream of the transcription initiation site, and (iv) a deletion of a 58 bp sequence which includes the putative FHV-1 ICP4 binding site in the IE promoter resulted in significant induction of promoter activity by a FHV-1 IE gene product although the FHV-1 IE gene product slightly stimulates the FHV-1 IE promoter, indicating that the FHV-1 IE gene product down-regulates its own promoter via the region.

The Role of ICP4 Repressor Activity in Temporal Expression of the IE-3 and Latency-Associated Transcript Promoters during HSV-1 Infection

The herpes simplex virus (HSV) type 1 immediate-early protein ICP4 represses transcription of its own gene and possibly that of other immediate-early genes. In the present study, we analyzed the role that an ICP4 binding site present in two HSV-1 promoters plays in the level and kinetics of expression during HSV-1 infection. Wild-type and mutant forms of the IE-3 (ICP4) promoter and the latency associated promoter (LAP) were fused to the thymidine kinase (tk) coding sequences and transferred to the genome of an ICP4-deficient virus Promoter mutants were constructed to assess the effect of the ICP4 binding site in the presence and absence of defined binding sites for cellular and other viral factors in the promoters. The activities of the promoter constructs were inferred from the level of tk mRNA seen following viral infection in the absence of ICP4, in Vero cells and in the presence of ICP4 in ICP4 expressing E5 cells. Kinetics of expression and the dependence on DNA synthesis for expression were examined following infection of E5 cells. In the presence of the ICP4 binding site in LAP and in IE3 promoters lacking TAATGARAT motifs, expression was maximal late after infection and was greatly reduced when DNA synthesis was inhibited. When the ICP4 binding site was removed, both LAP and the IE3 construct retaining an Sp1 site were more abundantly expressed and exhibited kinetics of expression indistinguishable from that of the tk promoter. In vitro, ICP4 repressed LAP transcriptor mediated by the general transcription factors and upstream activating proteins. Deletion of the ICP4 binding site not only relieved repression, but in the presence of USF activity, ICP4 further induced LAP transcription. The results of these experiments suggest a role for the repressor activity of ICP4 in the temporal regulation of HSV-1 transcription.

A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome

A novel family of transcripts that span the junction between the long and short segments of the herpes simplex virus type 1 genome has been identified. These transcripts, designated L/S junction-spanning transcripts (L/STs), are synthesized in abundance in a variety of cells infected with mutant viruses defective in the gene for ICP4, the major transcriptional regulatory protein of the virus. Transcription of abundant 2.3- and 8.5-kb series of L/STs was shown to initiate within the same sequences as less abundant 4.2-, 7.3-, and > 9.5-kb transcripts by Northern (RNA) blot analysis. S1 nuclease analysis revealed a single 5' terminus 28 bp downstream of a TATA box and 6 bp downstream of a consensus ICP4 binding site. The location of the transcriptional start site indicates that the promoter of the L/STs likely corresponds to the bidirectional promoter described by Bohenzky et al. (R. A. Bohenzky, A. G. Papavassiliou, I. H. Gelman, and S. Silverstein, J. Virol. 67:632-642, 1993). The L/STs accumulate with late kinetics in ICP4 mutant-infected cells and are polyadenylated. Mutant viruses encoding forms of ICP4 unable to bind the consensus site, ATCGTC, exhibited abundant expression of the L/STs, whereas mutants encoding forms of ICP4 able to bind this site expressed no detectable L/STs, suggesting that ICP4 plays a critical role in repressing L/ST expression. Their synthesis in ICP4 mutant-infected cells is inhibited by the protein synthesis inhibitor cycloheximide, indicating that they are induced either by an immediate-early viral protein other than ICP4 or by a virus-induced cellular protein. Preliminary evidence indicates that the L/STs are not present in latently infected ganglia. The abundant expression of the L/STs with late kinetics only in the absence of functional ICP4 and the sensitivity of their synthesis to cycloheximide indicate that they are not members of any of the recognized kinetic classes of herpes simplex virus type 1 transcripts but constitute a new class of viral transcript.

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

Herpes simplex virus infected cell polypeptide 4 preferentially represses Sp1-activated over basal transcription from its own promoter.

Herpes simplex virus type 1 infected cell polypeptide 4 (HSV-1 ICP4) is a multifunctional phosphoprotein that is essential for viral infection. It is both a repressor and an activator of viral gene expression depending upon the promoter. ICP4 represses transcription from its own promoter. In the present study, we used general transcription factors from HeLa cell nuclear extracts, recombinant TATA binding protein (TBP) and TFIIB, and the transcriptional activator Sp1 to reconstitute in vitro transcription for the ICP4 promoter and to examine the effects of purified ICP4 on transcription. ICP4 was able to effectively repress Sp1-induced transcription from ICP4 promoter templates that contain one or multiple Sp1 binding sites. The observed inhibition required the ICP4 binding site that spans the transcription initiation site. ICP4 did not inhibit basal transcription as inferred by its inability to inhibit transcription when (i) Sp1 was not included in transcription reactions, (ii) the templates contained no Sp1 binding sites, and (iii) TBP was used in place of TFIID in the reactions. The in vitro observations were consistent with the behavior of the same constructs expressed in cells from the herpes simplex virus type 1 genome. DNase I footprinting experiments revealed that ICP4 could co-occupy the ICP4 promoter region with TBP-TFIIB, indicating that ICP4 does not necessarily exclude these factors from binding to the TATA region. The data suggest that the repressive effects of ICP4 observed in this study result from ICP4 interfering with the interactions contributing to Sp1-induced transcription.

Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.

The ICP4 binding sites in the herpes simplex virus type 1 glycoprotein D (gD) promoter are not essential for efficient gD transcription during virus infection.

Activation of the early and late genes of herpes simplex virus type 1 during infection in tissue culture requires functional immediate-early regulatory protein ICP4. ICP4 is a specific DNA-binding protein which recognizes a variety of DNA sequences, many of which contain the consensus ATCGTC. In general, mutations which impair the ability of ICP4 to bind to DNA also eliminate its ability to activate viral early and late promoters both in transfection assays and in the infected cell. However, the role of ICP4 binding sites in the viral genome is unclear; many early and late promoters do not contain consensus binding sites in their vicinity. The glycoprotein D (gD) gene contains two well-characterized ICP4 binding sites upstream of its promoter and a third downstream of the transcription start site. Multimerization of one of these sites has been shown to increase the response of the gD promoter to ICP4 in transfection assays, while their removal reduces stimulation of the gD promoter by ICP4 in vitro. To assess the role of these binding sites during virus infection, we have constructed a recombinant viral genome which has mutations affecting all three. Comparison of the amounts of gD RNA synthesized by the recombinant and wild-type viruses indicated that the mutations had little or no effect on the activity of the gD promoter. Therefore, either the sites have no essential role in gD promoter regulation in the presence of all of the herpes simplex virus type 1 IE polypeptides during a normal infection or they can be functionally substituted by other ICP4 binding sites elsewhere in the genome.

A predictive model for DNA recognition by the herpes simplex virus protein ICP4

The herpes simplex virus (HSV) type 1 immediate early protein ICP4 is an essential regulatory enzyme that binds DNA directly in order to stimulate or repress gene expression. The degree of transaction is related to the locations and affinities of the ICP4 binding sites. A number of binding sites have been identified; some sites showed obvious homology to one another, and these were called consensus ICP4 binding sites. Other binding sites did not appear to be related, and these were termed non-consensus sites. We hypothesized, however, that a single model could describe all ICP4 binding sites, given the appropriate characterizations of sites. We performed statistical analyses on a set of ICP4 binding sites and found that the bases important for defining binding were located within a 13 base region. Missing contact analyses on several high-affinity binding sites revealed the same 13 base region as important for critical protein-DNA contacts. From these data we derived the consensus sequence RTCGTCNNYNYSG, where R is purine, Y is pyrimidine, S is C or G, and N is any base. In addition, we found that a better profile for ICP4 binding sites involves use of a matrix of base proportions from the binding site data; sites are analyzed by calculating the Matrix Mean score. We show that this Matrix Mean model could accurately predict the locations of novel ICP4 binding sites. Finally, we analyzed the entire HSV-1 genome for potential ICP4 binding sites and speculate about what these results suggest for the role of ICP4 in viral gene regulation.

The interaction of ICP4 with cell/infected-cell factors and its state of phosphorylation modulate differential recognition of leader sequences in herpes simplex virus DNA.

Regulation of herpes simplex virus (HSV) gene expression requires the synthesis of functional ICP4, a phosphoprotein that binds to several specific sites in virus DNA and acts in trans either to activate or to repress transcription of the three major kinetic classes of virus genes. Binding of ICP4 to specific sites in alpha genes (which are the first to be transcribed) causes repression of alpha-gene expression. ICP4 also indirectly participates in the formation of DNA-protein complexes with sequences present in the promoter/regulatory and leader regions of the beta and gamma genes that are sequentially activated later in infection. Here we demonstrate that the extent of phosphorylation of ICP4 contributes to its ability to participate differentially in complex formation with cis-acting elements present in beta and gamma genes. Dephosphorylated ICP4 retains its binding properties for the high affinity sites present in alpha promoters, whereas only phosphorylated forms of the protein are able to participate in complex formation with model beta and gamma sequences. These studies also reveal a requirement for cell and infected-cell factors to recognize the beta and gamma sequences. Our data suggest that the state of phosphorylation and concentration of ICP4 within the nucleus of infected cells determine the extent to which ICP4 interacts with these other factors.

DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element.

We report the results of fine mapping the sequences responsible for negative regulation of immediate-early (IE) gene 3 by its own gene product, ICP4. Affinity-purified ICP4 binds the transcriptional start site of IE gene 3 and protein-protein interactions induce a secondary mobility shift that footprints exactly as the primary complex. Since these DNA-protein complexes contain ICP4, it is likely that the two differ only in stoichiometry of protein. Additional data show that the DNA-binding domain recognized by ICP4 can be embedded as a cassette in foreign DNA and that native ICP4 will recognize and bind the resulting DNA. In two different immediate-early promoters, the ICP4 binding site can be located either 3' or 5' of the TATA box; however, the ICP4 site is rotationally displaced from the transcription factor IID (TFIID) site by a roughly one-half helical turn, suggesting that ICP4 and TFIID are on the opposite helical face when bound at their respective sites. In the IE1 and IE3 promoters, binding of ICP4 causes an alteration in the helical geometry of the minor groove of the TATA region as visualized by copper footprinting. In contrast, TATA hypersensitivity was not detected in the glycoprotein D promoter (an early gene promoter containing the ICP4 site separated from TATA by eight helical turns) or in an artificial IE3 promoter construct in which the TATA-A4 separation was increased from 2.5 to roughly 5 helical turns. Such stereospecific and distance-dependent conformational alterations in the TATA box under the influence of ICP4 binding may be important in the repression of immediate-early genes.