Alpha TIF Research Articles

Construction and characterization of a stably transformed HeLa cell line in which the expression of bovine herpesvirus 1 ICP0 (BICP0) is induced by tetracycline.

To explore the effects BICP0 (a principal transactivator of BHV-1 gene expression) on viral promoter elements, we established a cell line in which the expression of BICP0 is regulated by tetracycline. A hybrid promoter containing reiterated copies of the tet-operator (tet-O) and a minimal herpesviral alpha gene transinducing factor (alpha TIF) responsive element (minimal human cytomegalovirus immediate early promoter) was fused to the BICP0 gene and used to transform a HeLa cell line which expressed a fusion protein consisting of the repressor of the tet-O and the transactivating domain of alpha TIF. Simultaneously, the hygromycin resistance gene was transfected to select cells in media containing either hygromycin alone or both hygromycin and tetracycline. Immunofluorescent assays indicated that BICP0 was synthesised in the transformed cell lines solely upon induction of the gene by tetracycline removal. Only cells which had been kept constantly in medium containing tetracycline were able to synthesise BICP0 upon induction. Induced cell lines transactivated the native BICP0 promoter as well as the herpes simplex virus thymidine kinase promoter and the long terminal repeat sequences of human immunodeficiency virus in a dose dependent manner. These cell lines may help to further explore the functions of BICP0 as well as to investigate the molecular basis of interactions between herpes- and retroviruses.

The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines.

alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.

PREPs: herpes simplex virus type 1-specific particles produced by infected cells when viral DNA replication is blocked.

Herpes simplex virus (HSV)-infected cells produce not only infectious nucleocapsid-containing virions but also virion-related noninfectious light particles (L-particles) composed of the envelope and tegument components of the virus particle (J. F. Szilágyi and C. Cunningham, J. Gen. Virol. 62:661-668, 1991). We show that BHK and MeWO cells infected either with wild-type (WT) HSV type 1 (HSV-1) in the presence of viral DNA replication inhibitors (cytosine-beta-D-arabinofuranoside, phosphonoacetic acid, and acycloguanosine) or with a viral DNA replication-defective mutant of HSV-1 (ambUL8) synthesize a new type of virus-related particle that is morphologically similar to an L-particle but differs in its relative protein composition. These novel particles we term pre-viral DNA replication enveloped particles (PREPs). The numbers of PREPs released into the culture medium were of the same order as those of L-particles from control cultures. The particle/PFU ratios of different PREP stocks ranged from 6 x 10(5) to 3.8 x 10(8), compared with ratios of 3 x 10(3) to 1 x 10(4) for WT L-particle stocks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analyses revealed that true late proteins, such as 273K (VP1-2), 82/81K (VP13/14), and gC (VP8), were greatly reduced or absent in PREPs and that gD (VP17) and 40K proteins were also underrepresented. In contrast, the amounts of proteins 175K (VP4; IE3), 92/91K (VP11/12), 38K (VP22), and gE (with BHK cells) were increased. The actual protein composition of PREPs showed some cell line-dependent differences, particularly in the amount of gE. PREPs were biologically competent and delivered functional Vmw65 (VP16; alpha TIF) to target cells, but the efficiency of complementation of the HSV-1 (strain 17) mutant in1814 was 10 to 30% of that of WT L-particles.

The Cellular C1 Factor of the Herpes Simplex Virus Enhancer Complex Is a Family of Polypeptides

The alpha/immediate early genes of herpes simplex virus are regulated by the specific assembly of a multiprotein enhancer complex containing the Oct-1 POU domain protein, the viral alpha-transinduction factor alpha TIF, (VP16, ICP25), and the C1 cellular factor. The C1 factor from mammalian cells is a heterogeneous but related set of polypeptides that interact directly with the alpha-transinduction factor to form a heteromeric protein complex. The isolation of cDNAs encoding the polypeptides of the C1 factor suggests that these proteins are proteolytic products of a novel precursor. The sequence of the amino termini of these polypeptide products indicate that the proteins are generated by site-specific cleavages within a reiterated 20-amino acid sequence. Although the C1 factor appears to be ubiquitously expressed, it is localized to subnuclear structures in specific cell types.

Genomic Organization of the Human VP16 Accessory Protein, a Housekeeping Gene (HCFC1) Mapping to Xq28

The region between DXS52 and Factor VIII gene in the human Xq28 chromosomal band contains a G+C-rich isochore to which many genes have been mapped. We report here the isolation and characterization of a transcript mapping about 50 kb telomeric from the vasopressin type 2 receptor gene in a 180-kb YACs/cosmid contig containing the L1CAM gene at its centromeric end. The determined transcribed sequence from a human fetal brain library is identical to that of the recently identified accessory protein HCFC1 (host cell factor, also called C1) that activates herpes simplex virus VP16 (alpha TIF) transactivator protein for association with the octamer motif-binding protein Oct-1 (Cell 74: 115, 1993). The gene is expressed in a ubiquitous pattern and a larger transcript of approximately 10 kb is present in all the tissues tested, while an alternatively spliced RNA of approximately 8.0 kb is present in muscle and heart tissues. Genomic sequencing allowed us to determine that the sequenced transcript is assembled from 26 exons spread over a relatively small genomic region of approximately 24 kb. This alllowed us to determine that a previously reported cDNA clone arises from the splicing out of an internal portion of exon 8 which does not change the reading frame. All together these results raise the possibility that alternative mRNA processing could partly contribute to the diversity of the polypeptide HCFC1 family in a subset of tissues.

Protein and DNA elements involved in transactivation of the promoter of the bovine herpesvirus (BHV) 1 IE-1 transcription unit by the BHV alpha gene trans-inducing factor.

In herpes simplex virus (HSV)-infected cells, the transcription of immediate-early (alpha) genes is regulated by a virion component, the alpha gene trans-inducing factor (alpha TIF). This protein forms a complex with cellular factors and TAATGARAT motifs present in one or more copies in the promoters of all alpha genes. We have characterized the bovine herpesvirus 1 (BHV-1) homolog of this protein. Like its HSV counterpart, the BHV alpha TIF was synthesized in the later stages of infection and could be demonstrated to be a component of purified virions. In transient expression assays, BHV alpha TIF was a strong transactivator and stimulated the activity of IE-1, the major BHV-1 alpha gene promoter, with an efficiency comparable to that of HSV alpha TIF. This stimulation was largely dependent on a TAATGAGCT sequence present in a single copy in IE-1, and BHV alpha TIF, in conjunction with cellular factors, formed a complex with oligonucleotides containing this sequence. Despite these similarities between the two alpha TIFs, our preliminary observations suggest that the proteins may activate transcription by different mechanisms. Although BHV alpha TIF strongly transactivated IE-1, it differed from its HSV counterpart in that the carboxyl terminus of BHV alpha TIF, when fused to the DNA-binding domain of GAL4, was a relatively poor stimulator of a promoter containing GAL4-binding sites. Also unlike HSV alpha TIF, removal of the carboxyl terminus of BHV alpha TIF reduced but did not eliminate the ability of the protein to transactivate IE-1. These results are discussed in view of the structural similarities and differences among the alpha TIFs of alphaherpes-viruses.

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.

An 85-kilodalton herpes simplex virus type 1 alpha trans-induction factor (VP16)-VP13/14 fusion protein retains the transactivation and structural properties of the wild-type molecule during virus infection.

The 65-kDa herpes simplex virus type 1 encoded alpha trans-induction factor (alpha TIF or VP16) has two important functions: it is required for the efficient transcriptional induction of the alpha or immediate-early genes, and it acts as an essential structural component of the virion. The transcription properties of alpha TIF have been well studied in vitro. The protein is a powerful inducer of RNA polymerase II-directed transcription and, similar to the cellular transcriptional transactivators GAL4 and CGN4, contains separable DNA binding and transactivation domains. In contrast, little is known about the structural function of alpha TIF because this function can be studied only during virus replication and structural mutants are lethal. The in vivo analysis of alpha TIF is further complicated by the likelihood that the transcription and structural functions are not entirely separable. In this study, we take an alternate approach toward the development of alpha TIF mutants and their subsequent characterization. Rather than analyzing the effects of intragenic mutations, we have examined the properties of a mutant virus which expresses an alpha TIF fusion protein containing 61 amino acids of another herpes simplex virus type 1 virion protein, VP13/14, fused to its C terminus. This is the first report which demonstrates that the C-terminus of alpha TIF can tolerate the addition of an adjacent protein domain without compromising its transactivation function in vivo. Moreover, the VP13/14 sequences do not interfere with the protein-protein interactions required for virion targeting and assembly.

Nervous system inflammatory lesions and viral nucleic acids in rabbits with herpes simplex virus encephalitis-induced rotational behaviour.

Rabbits with herpes simplex virus (HSV) encephalitis induced by corneal virus challenge exhibit rotational behaviour linked with altered brain dopamine functions. The neuropathology and the distribution of the HSV-specific nucleic acids were studied, using probes for the viral trans-inducing factor alpha TIF and for the latency-associated transcript LAT-1 RNA to detect productive and latent infections, respectively. The rotational behaviour began 4 days after inoculation, and at that time the inflammatory process was observed only in the brain stem and the productive infection, revealed by in situ hybridisation, was seen in the trigeminal entry and nuclei. No HSV-specific nucleic acids or neural destruction were observed in the regions of the serotoninergic raphe or dopaminergic substantia nigra. At 8 days after inoculation, when the rotational behaviour was beginning to attenuate, the inflammatory lesions spread into the hemispheres, involving particularly the ventral parts of the limbic system including the olfactory system. In no cases were HSV-specific nucleic acids detected in the olfactory system. The inflammation in the limbic system was also detectable in animals without inflammatory lesions in the olfactory bulbs or tracts, suggesting that the infection had spread from the brain stem. The present study shows that in this model the altered neurotransmitter functions observed previously, appearing as rotational behaviour, occur without productive infection or necrosis, suggesting specific interaction of HSV with monoaminergic neurons. Additionally, the results suggest that HSV could reach the limbic system via ascending serotoninergic projections from the raphe neurons.

A minimal transcription activation domain consisting of a specific array of aspartic acid and leucine residues.

Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, alpha TIF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus alpha-trans-induction factor (VP16).

The assembly of specific multiprotein complexes on the herpes simplex virus alpha/IE (immediate early) enhancer elements requires the interactions of the Oct-1 POU homeodomain, the viral alpha TIF (alpha-trans-induction factor) (VP16), and at least one additional cellular factor, the C1 factor. The C1 factor interacts directly with alpha TIF, likely forming an intermediate protein complex that recognizes the Oct-1 homeodomain-DNA complex. The biochemical purification of the mammalian C1 factor suggests that it is composed of multiple subunits of related, but heterogeneous, polypeptides. The interaction of a subset of these polypeptides with alpha TIF is stimulated by post-translational modifications of the C1 proteins, suggesting that this factor may be a critical target for the regulation of the herpes simplex virus alpha/IE transcription.

Herpes simplex virus type 1 UL46 and UL47 deletion mutants lack VP11 and VP12 or VP13 and VP14, respectively, and exhibit altered viral thymidine kinase expression.

The gene products of herpes simplex virus type 1 UL46 and UL47 enhance the efficiency of alpha TIF (VP16)-mediated alpha gene expression through an unknown mechanism of action. To further characterize the function of the UL46- and UL47-encoded proteins during virus infection, a series of isogenic herpes simplex virus type 1 strain F-derived UL46 and UL47 single-deletion mutants and a UL46/47 double-deletion mutant were constructed and compared with the wild type. Analysis of purified virions obtained from the UL46 deletion mutant showed for the first time that UL46 encoded the viron tegument phosphoproteins VP11 and VP12 (VP11/12). Similar analyses of the UL47 deletion mutants confirmed an earlier report by McLean et al. that UL47 also encoded two virion tegument phosphoproteins, VP13 and VP14 (VP13/14) (G. McLean, F. Rixon, N. Langeland, L. Haarr, and H. Marsden, J. Gen. Virol. 71:2953-2960, 1990). Kinetic analysis demonstrated a delay of approximately 2 h in the appearance of thymidine kinase (TK) activity in all of the UL46 and UL47 single-deletion mutants. In the UL46/47 double-deletion mutant, the delay in TK activity increased twofold, suggesting that the proteins encoded by UL46 and UL47 may act at the same level. Since the delay in TK expression occurred within the first 4 h of infection, the actions of VP11/12 and VP13/14 resulted from their virion association and not from their de novo synthesis as late (beta gamma and gamma) genes. Densitometric analysis of purified virions showed that the levels of VP11/12 and VP13/14 in the virion tegument were near the molar ratios of alpha TIF. On the basis of these observations, we predict that the abilities of UL46 and UL47 to enhance alpha TIF-mediated transcription could result from a stoichiometric association of VP11/12 and VP13/14 with alpha TIF within the infecting virion.

Deletion of the VP16 open reading frame of herpes simplex virus type 1.

VP16 (also called Vmw65 and alpha TIF) is a structural protein of herpes simplex virus type 1 (HSV-1) that trans-induces HSV-1 immediate-early gene transcription. This report describes an HSV-1 VP16 deletion mutant that was constructed and propagated in a cell line transformed with a VP16 expression vector. The VP16 deletion mutant replicated like wild-type HSV-1 during infection of the VP16-expressing cell line. Deletion mutant virions propagated in this cell line contained wild-type, cell-derived VP16 protein that was recruited during virion assembly and was functional for immediate-early gene trans-induction. The mutant failed to replicate during subsequent infection of cells that do not express VP16, as determined in plaque assays and single-step replication assays. The deletion mutant induced nearly normal levels of viral DNA synthesis and capsid production during these infections, but it induced slightly lower levels of viral DNA encapsidation and appeared by transmission electron microscopy to be defective in further steps of virion maturation. A genetic revertant of the deletion mutant that was restored for VP16-coding sequences exhibited fully wild-type replication properties in both VP16-expressing and nonexpressing cells. The absence of VP16 protein synthesis at late times of HSV-1 infection prevents the production of infectious progeny virus and correlates with a profound defect in HSV-1 particle assembly.

Role of alpha-transinducing factor (VP16) in the induction of alpha genes within the context of viral genomes

In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.

Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.

Role of herpes simplex virus type 1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: characterization of three viral deletion mutants

The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.

Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome.

In herpes simplex virus 1-infected cells, the first set of genes to be expressed (alpha genes) is induced by the alpha gene trans-inducing factor (alpha TIF), a virion structural protein. The cis-acting site in the 5' untranscribed domain of alpha genes was previously reported to be the sequence 5'-GYATGNTAATGARATTCYTTGNGGG noncoding (where Y is a pyrimidine, R is a purine, N is any base), which binds a host protein designated alpha H1 (also termed the octamer binding protein, OTF-1, Oct-1, etc.) and which, together with this and possibly other proteins, forms complexes with alpha TIF. To determine the role of the various components of this cis-acting site and of other sequences shared by the alpha genes, we constructed 17 mutants spanning 110 nucleotides of the promoter domain of the alpha 27 gene and made a series of chimeric genes. Each chimeric gene embodying one set of these mutations was inserted into the viral genome and measurements were made of (i) accumulated mRNA under conditions in which only alpha genes were expressed and (ii) the capacity of the mutated sequence to form complexes containing alpha TIF and alpha H1 proteins. We report that (i) transversions in the "TAAT" sequence abolished both complex formation and induction of the chimeric alpha gene, (ii) mutations in the octamer binding site sequence upstream from TAAT or of the downstream GARAT abolished alpha TIF complex formation and also reduced alpha mRNA accumulation, (iii) mutations in a "CAAT" box also reduced expression of mRNA without affecting the formation of DNA-protein complexes containing alpha TIF, and (iv) mutations in sequences immediately downstream from TAATGARAT and in a pair of GA-rich elements reduced alpha mRNA expression whereas mutations between these elements had no effect on the accumulation of the mRNA. The results are consistent with the conclusion that both the alpha H1 octamer binding site ATGNTAAT and the GARAT sequence play a significant role in the induction of alpha genes. For optimal gene expression, however, additional elements downstream from the GARAT sequence and in other regions of the alpha promoter must be present.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.