Immediate Early Gene Transcription Research Articles

Interferon-α Inhibits the Murine Cytomegalovirus Immediate-Early Gene Expression by Down-Regulating NF-κB Activity

Transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by the interaction of cellular transcription factors with a strong vital enhancer controlling promoters flanking both sides of the regulatory sequence. We have previously demonstrated that interferon-α (IFN-α) inhibits MCMV replication by impairing the transcription of IE genes. To define the cis-acting elements and trans-acting factors involved in this inhibition, permissive murine fibroblasts were transfected with DNA constructs containing the chloramphenicol acetyl transferase reporter gene and portions of the IE enhancer. The region spanning -1185 to -269 relative to the IE1-3 promoter was sufficient to allow IFN-α-induced inhibition. Since this segment contains several NF-κB sites, cells were transfected with a construct containing three copies of NF-κB element in front of the homologous minimal IE1-3 promoter. Upon IFN-α treatment the reporter gene activity was strongly reduced, indicating that NF-κB binding site is sufficient to confer inhibition. The specificity of this inhibition was demonstrated by the lack of a significant effect on the activity of DNA constructs containing either a mutated NF-κB trimer or an ATF/CRE trimer. Gel shift assays with NF-κB probes revealed that MCMV infection activated NF-κB proteins, whereas IFN-α treatment significantly reduced their ability to bind NF-κB sites. In cotransfection experiments using various NF-κB subunit expression vectors and a reporter driven by three copies of an NF-κB element, activation of NF-κB-dependent transcription was observed with expression of p65 or combinations of p50-p65. Taken as a whole, these results suggest that IFN-α inhibits MCMV IE gene enhancer activity by mechanisms that decrease the availability of virus-induced NF-κB transcriptionally active in the nuclei of infected cells.

Neuronal activity and neuropeptide gene transcription in the brains of immune-challenged rats.

The present study investigated the effect of the acute-phase response of a systemic immune activation on the transcription of various immediate early genes (IEGs) and neuropeptides in the brain of conscious rats. One, 3, 6, 9, and 12 h after a single intraperitoneal (i.p.) administration of either the immune activator lipopolysaccharide (LPS) or the vehicle solution, adult male rats were sacrificed and their brains cut in 30-microns coronal sections. mRNA encoding the IEGs c-fos and nerve growth factor inducible-B (NGFI-B), and neuropeptides corticotropin-releasing factor (CRF), oxytocin (OT), and vasopressin (AVP) were assayed by in situ hybridization histochemistry using a 35S-labeled riboprobes. The primary transcripts [heteronuclear (hn)RNA] for these neuropeptides were also detected using intronic probe technology, and colocalization of c-fos mRNA within CRF, AVP, and OT neurons was determined by means of a combination of immunocytochemistry and in situ hybridization techniques on same the brain sections. One h after LPS treatment, both c-fos and NGFI-B genes were expressed in the parvocellular division of the paraventricular nucleus (PVN) of the hypothalamus. The medial preoptic area/organum vasculosum of the lamina terminalis, the supraoptic nucleus (SON), the magnocellular division of the PVN, the arcurate nucleus/median eminence, the locus coeruleus, the nucleus of the solitary tract, and the area postrema also exhibited a strong signal for these two transcripts 3 h after endotoxin administration. A smaller but a significant c-fos expression was observed in various structures, including the dorsomedial hypothalamic area, the central nucleus of the amygdala, the ventral part of the tuberomammillary nucleus, the laterodorsal tegmental nucleus, the external lateral part of the parabrachial nucleus, the dorsal division of the ambiguus nucleus, and the lateral reticular nucleus of LPS-injected rats. The signal for c-fos and NGFI-B mRNA in most of these brain nuclei reached a maximum at 3 h postinjection, declined at 6 h, and vanished 9 to 12 h after LPS treatment. In the parvocellular nucleus of the PVN, c-fos was largely expressed in CRF-immunoreactive (ir) neurons, whereas in the magnocellular part of that nucleus and in the SON, this transcript was colocalized in numerous OT-ir and few AVP-ir neurons. Relative levels of CRF mRNA in the parvocellular PVN were also significantly increased 6 h following LPS, but endotoxin did not alter the genetic expression of this stress-related neuropeptide in other brain regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of Pseudorabies Virus Replication by a Chimeric trans-gene Product Repressing Transcription of the Immediate-Early Gene

A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the immediate-early (IE) protein of pseudorabies virus (PRV) and a tail-truncated Vmw65 of herpes simplex virus 1, lacking the transcription activation domain, was constructed The chimeric gene product inhibited transcription from the PRV IE promoter in a transient expression assay. A HeLa cell line stably transformed with the chimeric gene showed remarkable resistance to PRV infection In the transformed cells infected with PRV, transcription of the PRV IE gene was repressed, indicating that the resistance of the cells to PRV infection was due to interference with IE gene transcription by the fusion protein.

Adenomatous polyposis coli, protein kinases, protein tyrosine phosphatase: The effect of sulindac

A putative explanation of the effect of sulindac on adenomatous colon and duodenal polyps from clinical observations and related in vitro experiments is presented. In cells with mutant APC genes, persistent high prostaglandin content of polyps leads to desensitization, downregulation of adenylate cyclase, uncoupling of cAMP synthesis from prostaglandin, and inactivation of protein kinase A (PKA). It is suggested that in normal cells, (APC) protein binds to catenins and microtubules to maintain structure and contribute to cell-cell communication, adherence, and the dephosphorylated state, a necessary condition for such functions. Cells with mutant APC product become isolated, deprived of communication and adhesion to other epithelial cells, overphosphorylated, and without corrective capability. The latter is largely due to downregulation of cAMP synthesis and protein kinase A activity secondary to high prostaglandin. Three main biochemical defects ensue: (1) the restrictive influence of PKA catalyzed phosphorylation of Raf-1 kinase and resultant effects on the MAP kinase cascade and transcription is lost, (2) the transcription of immediate early genes, including cyclooxygenase is stimulated, and (3) the stimulation of protein tyrosine phosphatase (PTPase) by PKA is in abeyance. These putative abnormalities are reversed by inhibition of cyclooxygenase-1 by sulindac. cAMP synthesis and PKA activity return to normal. PKA catalyzed phosphorylations block Raf-1 kinase at the confluence of the Ras and protein kinase C pathways. The MAP kinase cascade is inhibited as is transcription of immediate early genes. At the same time PKA stimulates PTPase, which dephosphorylates the cytoskeleton and restores cell-cell communication, adherence, and structure. The transformed phenotype is circumvented by adjustment of the phosphorylation state and mutant cells rejoin the epithelial community. The redox state of cytoplasm in mutant cells may be shifted toward reduction.

Human cytomegalovirus (HCMV) immediate-early enhancer/promoter specificity during embryogenesis defines target tissues of congenital HCMV infection

Congenital human cytomegalovirus (HCMV) infection is a common cause of deafness and neurological disabilities. Many aspects of this prenatal infection, including which cell types are infected and how infection proceeds, are poorly understood. Transcription of HCMV immediate-early (IE) genes is required for expression of all other HCMV genes and is dependent on host cell transcription factors. Cell type-specific differences in levels of IE transcription are believed to underlie differences in infection permissivity. However, DNA transfection experiments have paradoxically suggested that the HCMV major IE enhancer/promoter is a broadly active transcriptional element with little cell type specificity. In contrast, we show here that expression of a lacZ gene driven by the HCMV major IE enhancer/promoter -524 to +13 segment is restricted in transgenic mouse embryos to sites that correlate with known sites of congenital HCMV infection in human fetuses. This finding suggests that the IE enhancer/promoter is a major determinant of HCMV infection sites in humans and that transcription factors responsible for its regulation are cell type-specifically conserved between humans and mice. The lacZ expression patterns of these transgenic embryos yield insight into congenital HCMV pathogenesis by providing a spatiotemporal map of the sets of vascular, neural, and epithelial cells that are likely targets of infection. These transgenic mice may constitute a useful model system for investigating IE enhancer/promoter regulation in vivo and for identifying factors that modulate active and latent HCMV infections in humans.

Intracellular signaling and genetic reprogramming during agonist-induced hypertrophy of cardiomyocytes.

Cardiac myocytes stop dividing shortly after birth and subsequently the heart grows by enlargement of individual cells, a process that can be observed in vitro as well.' However, growth of individual cardiomyocytes can also be observed in the adult heart, a process called hypertrophy. Chronic hernodynamic overload of the heart by, e.g. , infarction, hypertension or volume or pressure overload ultimately leads to hypertrophy of the left ventricular wall. The hypertrophy process involves induction of transcription of specific contractile as well as noncontractile protein genes resulting in growth as well as change in contractile function of the c a r d i o r n y o ~ y t e . ~ ~ ~ This allows the heart to maintain normal beat-to-beat volume under the circumstances mentioned above. Although only limited evidence was presented for their importance during in vivo development of hypertr~phy,~-' hormonal and mechanical stimuli are thought to be the initial signals that trigger the onset of cell growth. The evidence for the involvement of hormonal and mechanical signals in the induction of hypertrophy mainly comes from in vitro models employing cultured neonatal cardiomyocytes. Here it was shown that induction of hypertrophy is accompanied by activation of the phosphoinositide (PI) cycle and by rapid stimulation of transcription of so-called immediate early genes, transcription factors, ultimately leading to rearrangement of gene expression of structural proteins in the cardiomyocyte. Recently, studies of hypertrophy were also performed on cultured adult cardiomyocytes,8-'1 confirming results from the neonatal cardiomyocyte model. In this review we focus on the influence of these hormonal and mechanical signals that activate the PI cycle during development of hypertrophy in the model of cultured cardiomyocytes and the ultimate outcome

Angiotensin as neuromodulator/neurotransmitter in central control of body fluid and electrolyte homeostasis.

Stimulation of central angiotensin receptors promotes, among others, drinking behaviour, stimulation of natriuresis and increased release of vasopressin. Angiotensin (ANG II)-containing pathways in the lamina terminalis and the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, brain areas involved in the regulation of body fluid homeostasis, have been described. All these areas express predominantly AT1 receptors. The drinking response and the vasopressin release to centrally administered ANG II are mediated by AT1 receptors, while AT2 receptors exert inhibitory effects. Evidence for the involvement of the catecholaminergic and angiotensinergic pathways in the PVN and SON in mediating the ANG II-induced release of vasopressin is presented. ANG II is released in the PVN upon local osmotic stimulation and water deprivation. Finally, we present evidence that activation of central angiotensinergic receptors, water deprivation, or hypertonicity induce transcription of immediate-early genes and expression of the respective proteins in the lamina terminalis and in the PVN and SON. The summarized data implicate ANG II as a neuromodulator/neurotransmitter in central control of body fluid and electrolyte homeostasis.

Serum response factor has functional roles both in indirect binding to the CArG box and in the transcriptional activation function of human T-cell leukemia virus type I Tax.

We previously reported that Tax1 of human T-cell leukemia virus type I interacts directly with serum response factor (SRF) and that Tax1 activates the transcription of several cellular immediate-early genes through the SRF binding site (CArG box). This activation required the transcriptional activation function of Tax1, identified as an activity of GALTax (a chimeric Tax1 with the yeast transcription factor GAL4) at the GAL4-binding site. In this study, we examined whether SRF plays a role in the transcriptional activation function of Tax1. Expression of Tax1 suppressed the GALTax activity at the GAL4 site as a result of squelching, and the suppressed activity was recovered by the overexpression of SRF, suggesting that SRF is a factor that is required for GALTax activity and that is inhibited by competition with Tax1. The expression of antisense SRF RNA specifically inhibited GALTax activity to less than 20%. Deletion of the Tax1 interaction domain of SRF at its C terminus converted SRF from an activator of GALTax to an inhibitor. These results suggest that SRF is an essential component of the transcriptional activation of Tax1 in addition to a mediator of CArG box binding.

Presence of human cytomegalovirus (HCMV) immediate early mRNA but not ppUL83 (lower matrix protein pp65) mRNA in polymorphonuclear and mononuclear leukocytes during active HCMV infection

During an active human cytomegalovirus (HCMV) infection, leukocytes, harbouring the HCMV lower matrix protein pp65 (ppUL83) are present in the peripheral blood and can be detected with the HCMV antigenaemia assay. In the present study, it was investigated whether the presence of pp65 in these cells was due to transcription of the virus genome or might be the result of uptake of this viral protein. Peripheral blood leukocytes of transplant recipients and AIDS patients with an active HCMV infection were investigated for the presence of HCMV immediate early (IE) antigen and pp65 using well characterized monoclonal antibodies, and for the presence of the corresponding mRNAs using non-radioactive in situ hybridization. Both mononuclear and polymorphonuclear cells were found to contain IE antigen and pp65. However, only mRNAs encoding IE antigen were found in these cells, whereas mRNAs encoding pp65 were not detected. In contrast, both IE antigen and pp65, as well as their corresponding mRNAs, were detected in the circulating late-stage HCMV-infected endothelial cells that were also present in the leukocyte fractions. These findings demonstrate that a restricted viral gene expression (transcription of IE genes) does occur in mononuclear and polymorphonuclear leukocytes. However, the abundant presence of the early antigen pp65 without detectable presence of the corresponding mRNA in these cells strongly indicates uptake of this protein by the phagocytic leukocytes, rather than de novo synthesis.

Roles of calmodulin-dependent protein kinases and phosphatase in calcium-dependent transcription of immediate early genes.

Recent studies indicate multiple mechanisms are involved in Ca2+ stimulation of gene expression. We have used cell-permeable, specific inhibitors of calmodulin-dependent protein kinases (CaM kinases) and phosphatase (calcineurin) to investigate the involvement of these enzymes in transcriptional regulation of three immediate early genes in PC12 cells stimulated with A23187 or KCl. Preincubation of PC12 cells with the CaM kinase inhibitor KN-62 blocked autophosphorylation of CaM kinase II in response to stimulation by the Ca2+ ionophore A23187. KN-62 treatment also resulted in a 60-70% inhibition of Ca(2+)-dependent transcription of c-fos, NGFI-A (zif 268), and NGFI-B (nur 77) as assessed by either Northern or nuclear run-on analyses. Preincubation with the calcineurin inhibitors FK-506 or cyclosporin A strongly enhanced expression of NGFI-A and blocked transcription of NGFI-B, but it had no significant effect on Ca(2+)-stimulated transcription of c-fos. Both FK-506 and KN-62 were specific for Ca(2+)-stimulated transcription as neither effected transcription in response to forskolin or phorbol ester (12-O-tetradecanoylphorbol-13-acetate) treatment. This is the first report of CaM kinase and calcineurin involvement in transcriptional regulation of NGFI-A and NGFI-B. Activation of CaM kinases and calcineurin, in response to elevated intracellular Ca2+, would exert antagonistic effects on transcription of NGFI-A. Since inhibition of either the kinase or phosphatase decreased transcription of NGFI-B by 60-90%, this suggests that each enzyme is necessary but not sufficient for Ca2+ stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca(2+)-stimulated gene expression.

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.

MK-801 Does Not Attenuate Immediate-Early Gene Expression Following an Amygdala Afterdischarge

It has been suggested that the increased transient expression of immediate-early gene transcription factors seen in nerve cells following an afterdischarge may initiate longer lasting or permanent changes in gene expression which underly the development of kindling. Since the development of kindling is sensitive to pharmacological blockade of the N -methyl-d-aspartate receptor, we tested whether the increased expression of the immediate-early genes c-fos, jun-B, c-jun, krox-20, and krox-24 following a kindling afterdischarge was also sensitive to N -methyl-d-aspartate receptor blockade by MK-801. In this report we demonstrate that all five immediate-early genes are induced by an amygdala afterdischarge. N-methyl-d-aspartate receptor blockade by a dose of MK-801 that significantly retards the development of amygdala kindling failed to attenuate immediate-early gene expression. These results suggest that although expression of these five immediate-early genes occurs after an amygdala afterdischarge their expression is not involved in the N -methyl-d-aspartate receptor-mediated component of amygdala kindling.

Trans-activation of the mouse cytomegalovirus immediate early gene enhancer by ras oncogenes

The ras gene family encodes 21K proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-ras (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltransferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NF kappa B, AP1, ATF and SP1. Activation of the p21ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.

Activation of pirenzepine-sensitive muscarinic receptors induces a specific pattern of immediate-early gene expression in rat brain neurons

Accumulating evidence suggests that immediate-early gene transcription factors such as c-Fos, form part of an intracellular signalling pathway linking the activation of neuronal receptors by neurotransmitters to changes in neuronal gene expression. Recently it has been demonstrated that the centrally active muscarinic receptor agonist pilocarpine induces both c- fos mRNA and protein in rat brain. In this report using immunocytochemical and in situ hybridization techniques we demonstrate for the first time that in addition to c- fos, pilocarpine administration increases the neuronal expression of jun-B, krox-20 and krox-24 ( zif-268) but not related c- jun and jun-D genes in rat cortex and hippocampus. Pretreatment of animals with atropine or pirenzepine significantly reduced induction of c- fos, jun-B, krox-20 and krox-24 genes in both hippocampus and cortex. These results show that activation of pirenzepine-sensitive muscarinic receptors results in a specific pattern of immediate-early gene expression in rat brain neurons. We suggest that the combinatorial complexity of immediate-early gene induction may allow receptor-specific control of gene expression in vivo.

Localization of a 34-amino-acid segment implicated in dimerization of the herpes simplex virus type 1 ICP4 polypeptide by a dimerization trap.

The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form. The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution. In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif. Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.

Rapid response genes as markers of cellular signaling during cortical histogenesis: Their potential in understanding mental retardation

Aberrations of dendritic morphology are seen in most forms of mental retardation (MR). Normal cortical development is dependent on neural activity that modulates developmental processes such as dendritic differentiation. Indeed, many of the classical histological correlates of MR are reproduced in models that alter activity during development. To explore the hypothesis that MR results from aberrant activity signals during development, it would be useful to have histochemical markers that are sensitive to neural activity. Recent studies indicate that certain immediate early genes (IEGs) are normally expressed at relatively high levels in cortical neurons during postnatal development and are rapidly regulated by natural activity. We have begun to assess the possible use of IEG markers to study MR by examining the pre- and postnatal developmental time course of a panel of known IEG transcription factors as well as a set of novel IEGs identified in our laboratory. One of these recently characterized clones encodes a novel, mitogen-inducible cyclo-oxygenase that is expressed during a critical period for dendritic formation and is regulated by N-methyl-d-aspartate-dependent synaptic activity and by environmental stimuli. These data suggest a role for prostaglandin signaling in postnatal cortical development. Other partially characterized novel IEGs are expressed in a cell-specific fashion in the cortical plate. Application of these histochemical markers to the study of MR pathogenesis in animal models is discussed.

1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels.

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus VP16 forms a complex with the virion host shutoff protein vhs.

Herpes simplex virus (HSV) virions contain at least two regulatory proteins that modulate gene expression in infected cells: the transcriptional activator VP16 and the virion host shutoff protein vhs. VP16 stimulates transcription of the HSV immediate-early genes, and vhs suppresses host protein synthesis and induces accelerated turnover of cellular and viral mRNAs. We report here that vhs binds directly to VP16: vhs and VP16 were coprecipitated from infected cells by an anti-vhs antiserum, and vhs and VP16 protein A fusions each bound intact versions of the other protein in a solid-phase capture assay. In addition, vhs and VP16 interacted in the two-hybrid activator system when coexpressed in Saccharomyces cerevisiae. vhs residues 238 to 344 were sufficient for the interaction, and the VP16 acidic transcriptional activation domain was not required. vhs blocked the ability of VP16 to enter a multiprotein complex on an immediate-early TAATGARATTC consensus sequence, indicating that vhs interacts with one or more regions of VP16 required for promoter recognition. We suggest that this interaction may play a structural role in the assembly of HSV virions and modulate the activity of vhs during infection.

Interferons Inhibit Onset of Murine Cytomegalovirus Immediate-Early Gene Transcription

Treatment of NIH 3T3 fibroblasts with interferon-α or interferon-γ (IFN-α or IFN-γ) significantly reduced murine cytomegalovirus (MCMV) replication. Determination of viral DNA in the nuclei of the infected cells before onset of DNA replication demonstrated that virus uptake, transport to the nucleus, and DNA stability were not decreased. Analysis of the virus specified mRNAs soon after infection revealed that in the cells exposed to IFNs expression of the immediate early (IE) genes was strongly reduced. Nuclear run-off transcription analysis showed that this inhibition is due to significant reduction of IE gene transcription rates following IFN treatment. Since transcription of the MCMV IE region is regulated by a strong enhancer element, a construct containing the chloramphenicol acetyltransferase (CAT) reporter gene, driven by an 1.2 kb segment spanning the enhancer and IE 1/3 promoter region of the IE transcription unit, was transfected into NIH 3T3 cells. Treatment with IFN-α or IFN-γ after transfection strongly reduced CAT activity compared to untreated controls. In an attempt to define a negative IFN-responsive element in the IE enhancer, a series of deletion mutants driving the CAT reporter gene were transfected into NIH 3T3 cells that were then treated with IFN-α With the sole exception of the construct containing the minimal MCMV IE 1/3 promoter (-102 to the cap site) all other deletion mutants were strongly down-regulated by IFN-α-treatment. Taken as a whole, these results suggest that IFNs inhibit MCMV replication by impairing the transcription of the IE transcription units, and that this negative regulation is carried out by sequences scattered throughout the IE enhancer region.

Differential Transcription and Translation of Immediate Early Genes in the Gerbil Hippocampus after Transient Global Ischemia

Excitotoxic activation of glutamate receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA-1 neurons in the gerbil hippocampus. Glutamate receptor stimulation also causes induction of transcription factors that belong to the class of immediate early genes. We examined the expression of six different immediate early genes in the gerbil hippocampus after transient global ischemia. Comparative analysis of c-fos and Krox-24 expression was carried out in the same animals at the transcriptional and translational level by in situ hybridization and immunocytochemistry. Postischemic synthesis of four additional immediate early gene (IEG)-encoded proteins (FOS-B, c-JUN, JUN-B, and JUN-D) was investigated by immunocytochemistry at recirculation intervals between 1 and 48 h. After 5 min of ischemia, transcription of c-fos and Krox-24 mRNA was induced in all hippocampal subpopulations with peak expression at 1 h after recirculation. In vulnerable CA-1 neurons, increased transcription of c-fos and Krox-24 was not followed by translation into protein. Induction of immediate early gene-encoded proteins was restricted to neuronal populations less vulnerable to brief ischemia and identified neurons that are targets of glutamate receptor-mediated neurotoxicity but that are destined to survive. Our data indicate an asynchronous synthesis and persistence of individual IEG-encoded proteins in these neurons. The staggered induction implies that combinatorial changes of transcription factors allow a differential postischemic regulation of target gene expression both spatially and over time.