Human Cytomegalovirus Major Immediate-early Gene Research Articles

SAMHD1 Modulates Early Steps during Human Cytomegalovirus Infection by Limiting NF-κB Activation

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter.

Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements.

ABSTRACTTriggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-κB binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation.IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that costimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-κB family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-κB binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to repower the cycle of cytomegalovirus propagation.

Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state.

Alternative Splicing of the Human Cytomegalovirus Major Immediate-Early Genes Affects Infectious-Virus Replication and Control of Cellular Cyclin-Dependent Kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.

Phorbol ester-induced human cytomegalovirus major immediate-early (MIE) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells.

The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE(+)) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-kappaB (kappaB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and kappaB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-kappaB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and kappaB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-kappaB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

Cellular signals involved in cyclooxygenase-2 expression induced by human cytomegalovirus

Cyclooxygenase (COX)-2 plays an important role in inflammatory reaction and human cytomegalovirus (HCMV) is known to induce COX-2 gene expression. However, little is known for the mechanism of COX-2 gene expression by HCMV. In this study, three recently isolated HCMV strains including TB40/E and clinical isolates from Korean patients as well as highly laboratory adapted strain AD169 were used to stimulate COX-2 induction. Western blot analyses revealed that recently isolated HCMV strains induced COX-2 expression in human foreskin fibroblast (HFF) cells to higher levels than lab-adapted strain AD169. HCMV gene expression was not required for COX-2 induction since UV-inactivated virus was able to stimulate COX-2 gene expression during immediate early infection. Moreover, when HCMV-infected HFF cells were transfected with small interfering RNA for major immediate early (MIE) genes, HCMV-induced COX-2 expression was not abrogated, suggesting that HCMV MIE genes are not needed for COX-2 expression. In order to understand the signal pathways involved in COX-2 gene expression induced by HCMV, selective inhibitors were used. When HCMV-infected HFF cells were treated with epidermal growth factor receptor (EGFR) kinase inhibitors and c-Raf/mitogen-activated protein kinases (MAPK, MEK) 1/2 inhibitors, HCMV-induced COX-2 expression was diminished. However, COX-2 expression by HCMV infection was not inhibited if HCMV-infected HFF cells were treated with inhibitors of phosphatidylinositol-3-kinase, c-Jun N-terminal kinase, nuclear factor kappa B, p-38 MAPK and Rho-associated protein kinase. We concluded that EGFRK, Raf, MEK1/2 and extracellular signal-regulated kinases (ERK 1/2) pathway may participate in the COX-2 mediated inflammatory response to HCMV infection.

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.

Breaking human cytomegalovirus major immediate-early gene silence by vasoactive intestinal peptide stimulation of the protein kinase A-CREB-TORC2 signaling cascade in human pluripotent embryonal NTera2 cells.

The triggering mechanisms underlying reactivation of human cytomegalovirus (HCMV) in latently infected persons are unclear. During latency, HCMV major immediate-early (MIE) gene expression breaks silence to initiate viral reactivation. Using quiescently HCMV-infected human pluripotent embryonal NTera2 cells (NT2) to model HCMV reactivation, we show that vasoactive intestinal peptide (VIP), an immunomodulatory neuropeptide, immediately and dose-dependently (1 to 500 nM) activates HCMV MIE gene expression. This response requires the MIE enhancer cyclic AMP response elements (CRE). VIP quickly elevates CREB Ser133 and ATF-1 Ser63 phosphorylation levels, although the CREB Ser133 phosphorylation level is substantial at baseline. VIP does not change the level of HCMV genomes in nuclei, Oct4 (pluripotent cell marker), or hDaxx (cellular repressor of HCMV gene expression). VIP-activated MIE gene expression is mediated by cellular protein kinase A (PKA), CREB, and TORC2. VIP induces PKA-dependent TORC2 Ser171 dephosphorylation and nuclear entry, which likely enables MIE gene activation, as TORC2 S171A (devoid of Ser171 phosphorylation) exhibits enhanced nuclear entry and desilences the MIE genes in the absence of VIP stimulation. In conclusion, VIP stimulation of the PKA-CREB-TORC2 signaling cascade activates HCMV CRE-dependent MIE gene expression in quiescently infected NT2 cells. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.

Real-Time PCR Diagnostics Failure Caused by Nucleotide Variability within Exon 4 of the Human Cytomegalovirus Major Immediate-Early Gene

Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.

Circadian Regulation of a Viral Gene Promoter in Live Transgenic Mice Expressing Firefly Luciferase

This study was conducted to test for possible circadian control of viral infection in live animals using bioluminescence imaging of a firefly luciferase transgene. Transgenic mice expressing the firefly luciferase gene under the control of the promoter and enhancer of the human cytomegalovirus major immediate-early gene (CMV::luc) were examined through whole-animal imaging. Mice were crossed with HRS/J hairless albino mice to improve imaging of deep structures. Transgene expression in the extremities and head was elevated around dusk in mice maintained in cycles of light and dark. Signal was also elevated during the animal's night in mice maintained in extended darkness. The viral promoter was induced during the active phase of the circadian locomotor rhythm in several tissues. Both the acinar cells and islets expressed the transgene in dissociated pancreas cultures. These results suggest that viruses may exploit the circadian system for optimal timing of infection at particular phases in several tissue types.

Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells.

Differentiation-dependent expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 and IE2, may partly govern virus replication in monocytic THP-1 and embryonal carcinoma (Tera-2) cells. The modulator of the MIE promoter was shown previously in transient transfection assays to repress transcription from promoter segments in undifferentiated THP-1 and Tera-2 cells but not in differentiated cells. To determine the biological importance of these findings, we constructed a recombinant HCMV (r delta MSVgpt) without a modulator. In comparison to wild-type (WT) virus, r delta MSVgpt exhibits a slight delay in growth in human fibroblasts, but there is no appreciable change in IE1 and IE2 transcription. Moreover, there is no appreciable change in the early/late kinetics of transcription of RNAs colinear with the predicted UL128 coding region, which is adjacent to the modulator, although the size distribution and abundance of these RNAs are altered. In infected undifferentiated THP-1 and Tera-2 cells, WT and r alpha MSVgpt viruses produce minimal but comparable amounts of IE1 RNAs. The genomes of both viruses are detectable in similar amounts within these undifferentiated cells. Induction of cellular differentiation before infection overcomes the block in MIE gene transcription. WT and r alpha MSVgpt infections of differentiated THP-1 cells produce similar levels of IE1 and IE2 RNAs. Thus, differentiation-dependent control of MIE gene transcription involves regulatory mechanisms other than the modulator. Possible alternative functions of the modulator are discussed.

Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types.

Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction.

The human cytomegalovirus major immediate-early gene.

Immediate-early genes function to regulate viral and cellular gene expression during the course of virus replication. The major immediate-early gene region of human cytomegalovirus plays a key role in affecting activation and repression of viral and cellular genes. These proteins intimately associate and/or interact with viral and cellular proteins during this process. Herein, we will discuss the current understanding of this complex gene region.

Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

Site-specific inhibition of RNA polymerase II preinitiation complex assembly by human cytomegalovirus IE86 protein.

The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein (UL122, IE2), is a DNA-binding protein that represses the MIEP through its cognate recognition sequence (designated the cis repression signal [crs]) located between the TATA box and the initiation site of transcription. Purified recombinant IE86 protein was shown to repress MIEP transcription in vitro, in a cis-acting mediated pathway, with nuclear extracts from HeLa S3, U373-MG, and primary human foreskin fibroblast cells. Repression of the MIEP by IE86 was shown by two criteria to be dependent on the direct interaction of IE86 with the crs element. Core promoter constructs containing essentially the MIEP TATA box and crs element were also specifically repressed by IE86 but not by a mutant IE86 protein, indicating the general transcription machinery as the target for IE86 repression. Kinetic and template commitment experiments demonstrated that IE86 affects preinitiation complex formation but not the rate of reinitiation. Sarkosyl inhibition experiments further revealed that IE86 was unable to effect repression by either disassembling or preventing the elongation of a preexisting transcription complex. Further, the ability of IE86 to interact with the DNA-binding subunit of TFIID was shown not to be required for repression. These functional protein-DNA and protein-protein interaction experiments demonstrate that IE86 specifically interferes with the assembly of RNA polymerase II preinitiation complexes. The biological significance of these results and the precise mechanism by which IE86 represses transcription are discussed.

Direct interaction of the human cytomegalovirus IE86 protein with the cis repression signal does not preclude TBP from binding to the TATA box.

The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein, represses the MIEP through a DNA sequence located between the TATA box and the transcription initiation site, designated the cis repression signal (crs). Through mutational analysis, amino acid domains within IE86 responsible for binding the crs element were located at the C terminus. Mutation of the putative zinc finger domain, which precluded IE86 from binding DNA, converted the protein from a repressor of MIEP transcription into an activator. DNase I protection analysis demonstrated that the IE86 footprint overlapped the sequence protected by the TATA-binding protein (TBP). Investigation of whether IE86 was able to displace TBP from DNA revealed that both proteins could bind DNA simultaneously. However, higher concentrations of IE86 were required to obtain protection of the crs element in the presence of prebound TBP. Similarly, higher concentrations of TBP were required to obtain protection in the presence of prebound IE86. These observations indicate that steric hinderance impairs but does not prevent both proteins from binding DNA synchronously.

Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector

Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system. We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3. This vector, denoted Cgal/13, contains the E, coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome. We studied the efficiency of Cgal/13 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer. Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS. Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia. Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expresssion of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression. Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.

Thyroid-Specific Enhancer-Binding Protein (T/EBP): cDNA Cloning, Functional Characterization, and Structural Identity with Thyroid Transcription Factor TTF-1

A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP) was isolated from a rat thyroid-derived FRTL-5 cell lambda gt 11 expression library, using a double-stranded oligonucleotide probe. This oligonucleotide was previously demonstrated to have the strongest binding affinity among three cis-acting DNA elements within the thyroid-specific enhancer region located 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. Nucleotide and deduced amino acid sequences of the cDNA revealed that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter conferred thyroid-specific enhancer activity of as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown, by gel mobility shift experiments, to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity.

Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1.

A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP) was isolated from a rat thyroid-derived FRTL-5 cell lambda gt 11 expression library, using a double-stranded oligonucleotide probe. This oligonucleotide was previously demonstrated to have the strongest binding affinity among three cis-acting DNA elements within the thyroid-specific enhancer region located 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. Nucleotide and deduced amino acid sequences of the cDNA revealed that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter conferred thyroid-specific enhancer activity of as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown, by gel mobility shift experiments, to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity.

Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system

Herpes simplex virus type 1 glycoprotein H (HSV-1 gH) was synthesized in an inducible mammalian cell expression system, and its properties were examined. The gH coding sequence, together with the stable 5' untranslated leader sequence from xenopus beta-globin, was placed under control of the strong promoter from the human cytomegalovirus major immediate-early gene in an amplifiable plasmid which contains the simian virus 40 (SV40) virus origin for replication (ori). This expression vector was transfected into ts COS cells constitutively expressing a temperature-sensitive SV40 T antigen which allows utilization of the SV40 ori at permissive temperatures. The results of transient expression assays at the permissive temperature showed that HSV-1 gH could be synthesized in greater amounts than those produced by a high-multiplicity virus infection. The proteins produced were detected in Western blots (immunoblots) with a HSV-1 gH-specific polyclonal serum raised against a TrpE-gH fusion protein. The transfected gH had an apparent molecular weight of approximately 105,000, intermediate in size to those of the precursor (100,000) and fully processed forms (110,000) of HSV-1 gH from infections. Antigenicity was investigated by reactions with three virus-neutralizing monoclonal antibodies specific for conformational epitopes on gH. Only one of these monoclonal antibodies could immunoprecipitate the synthesized gH. However, equal recognition of the transfected gH was achieved by superinfection with virus. In addition, detectable amounts of gH were not expressed on the cell surface unless the cells were superinfected with virus. Studies with a temperature-sensitive mutant, ts1201, defective in encapsidation showed that the changes in antigenic structure and cell surface expression caused by superinfection with virus were not due simply to incorporation of gH into virions. These results suggest that gH requires additional virus gene products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity.