Ie1 Promoter Research Articles

Altering central nervous system physiology with a defective herpes simplex virus vector expressing the glucose transporter gene.

Because of their postmitotic nature, neurons are difficult subjects for gene transfer. To circumvent this, we have used a defective herpes simplex virus vector to overexpress the rat brain glucose transporter (GT) gene under the control of the human cytomegalovirus ie1 promoter. This vector, designated vIE1GT, was propagated using a herpes simplex virus type 1 temperature-sensitive mutant, ts756. GT expressed from vIE1GT was readily immunoprecipitated from membrane fractions of vIE1GT-infected Vero cells. By using indirect double immunofluorescence techniques, vIE1GT was shown to be capable of enhancing GT expression in cultured hippocampal neurons and glia. Glucose transport in such vIE1GT-infected cultures was increased approximately 2-fold relative to controls. The efficacy of this system in vivo was then tested by microinjection of vIE1GT into adult rat hippocampus. When examined 2 days later, GT expression from vIE1GT was demonstrated in hippocampal neurons by in situ hybridization; a small but significant increase in glucose transport was detected in tissue immediately surrounding the injection site by 2-deoxy[14C]glucose uptake and autoradiography. Such injections did not cause marked cytopathology. Thus, this approach can be used to alter central nervous system physiology in vitro and in vivo.

Human beta 2-adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP-binding protein to adenylyl cyclase.

Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human beta 2-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350,000 human receptors/cell. The number of receptors did not change with cell density or age of culture. The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. Coupling of the receptors to endogenous GTP-binding proteins is demonstrated by hormone-dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells. Western-blot analysis revealed that the endogenous GTP-binding protein appears to be of the heterotrimeric type. Antibodies raised against the mammalian alpha subunit of stimulatory GTP-binding proteins cross-react with the insect alpha subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The beta subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the C-terminal amino acids of the mammalian beta subunit of GTP-binding proteins, but is about approximately 2 kDa larger than that of the beta subunit of GTP-binding proteins from bovine brain. Exposure of the transformed insect cells to L-isoproterenol rapidly induces uncoupling and internalization of 30% of the heterologously expressed receptors. In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remaining 70% of the receptors.

Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences.

We have developed a sensitive genetic assay to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination. Recombination substrates containing mu and gamma 3 chain switch (S)-region sequences, S mu and S gamma 3, are transiently introduced into primary murine B cells cultured with lipopolysaccharide to induce isotype switching. Recombination involving S-region sequences deletes a conditionally lethal marker, the leftward promoter of phage lambda (lambda PL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S mu-lambda PL-S gamma 3, about 2% of replicated molecules undergo deletion of lambda PL during transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of S mu increases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.

Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus

A second immediate early (IE) regulatory gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) has been identified. The IE-2 gene which is homologous to the IE-N gene of Autographa californica MNPV was mapped to the HindIII A fragment of OpMNPV between 0.41 to 1.37 map units. The IE-2 gene codes for a predicted protein of 45,640 Da and analysis of the amino acid sequence shows that the protein has a highly basic amino terminal domain and a cysteine-rich domain that is similar to a zinc finger motif that is also found in the baculovirus proteins GC30 and PE-38. The I E-2 gene is expressed as a 1.3-kb transcript that was detectable by 0.5 hr postinfection (hr p.i.), reached maximum steady state levels by 6 hr p.i., and declined slightly by 48 hr p.i. Cis-acting 5′ regulatory sequences were analyzed by deletion analysis of the IE-2 promoter linked to the reporter gene chloramphenicol acetyl transferase. Maximum expression was obtained when the IE-2 promoter contained sequences 275 by upstream from the transcriptional start site, trans-Activation analysis revealed that IE-2 trans-activated the IE-1 promoter and in addition appeared to be autoregulatory.

Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.

Sodium butyrate-inducible replication of human cytomegalovirus in a human epithelial cell line

Replication of human cytomegalovirus (HCMV) in a human epithelial thyroid papillary carcinoma cell line (TPC-1) was restricted. However, pretreatment of these cells with 5 m M sodium butyrate (NaB) for 24 hr before infection enhanced both HCMV yield and infectious center titer to a similar level of that seen in human embryonic lung fibroblast cells. Immunofluorescence staining, gel electrophoresis, and Northern blot analysis revealed that TPC-1 cells are nonpermissive for expression of HCMV major immediate early (IE1) functions, but many of the cells become permissive after being treated with NaB. The presence of cycloheximide during NaB pretreatment of the cells efficiently diminished the stimulatory effect of NaB on expression of the IE1 gene. Therefore, it appeared that NaB induces the synthesis of a cellular protein(s) which apparently plays an important role in the conversion of nonpermissive cells to a permissive state for expression of this critical viral gene. Transient chloramphenicol acetyltransferase (CAT) assay experiments indicated that in TPC-1 cells the HCMV-CAT construct which contains the complete IE1 promoter regulatory region was expressed poorly, whereas a high level of CAT activity was detectable in the NaB-treated cells. Therefore, these results suggest that the enhancing effect of NaB on HCMV replication is expressed through some host cellular factor(s), and the HCMV IE1 promoter regulatory region is most likely to be the primary target of NaB action.

Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus

The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus expresses two immediate-early genes from the HindIII-G region (map units 90.4 to 96.8) of the genome. During the early phase of infection, nonspliced 1.9-kb and spliced 2.1-kb transcripts are expressed which encode the IE1 and IE0 (spliced IE1) gene products, respectively. These two gene products differ only in that IE0 contains an additional 54 amino acids at the amino terminus. RNA analysis of these two genes during infection revealed that they were differentially expressed. IE1 was expressed early and late, whereas IE0 was expressed only early in infection. The regulation of these two immediate-early genes was analyzed by transient expression assays. The IE1 gene product stimulated expression of IE1 promoter-directed expression but down-regulated expression from the IE0 promoter. The IE0 gene product also transactivated the IE1 promoter but did not affect expression from its own promoter. Unlike IE1, which transactivates the delayed early 39K gene in the presence and absence of the homologous region (hr) enhancers, IE0 transactivated the 39K promoter only in the presence of cis-linked hr5 enhancer. The results of this study in conjunction with previous results suggest that the IE1 gene encodes a multifunctional gene product that may be involved in (i) repression of immediate-early gene expression, (ii) continued expression of its own gene product during infection, and (iii) transactivation of the delayed early and late classes of genes.

The Vmw175 binding site in the IE-1 promoter has no apparent role in the expression of Vmw110 during herpes simplex virus type 1 infection

The immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1) are the first to be expressed during infection in tissue culture. Since they are transcribed at abnormally high levels in the absence of IE protein synthesis they appear to be subject to repression during normal infection. One of the major HSV-1 regulatory proteins, Vmw175 (the product of IE gene 3), is required for normal IE gene regulation since mutations which inactivate it lead to abnormally high levels of IE gene expression. The mechanism of repression of the IE-3 promoter requires both the ability of Vmw175 to bind to DNA and the presence of a Vmwl75 recognition DNA binding sequence at the cap site of the IE-3 promoter. A similar Vmw175 DNA binding sequence has been defined within the IE-1 promoter. This paper describes the construction of a variant of HSV-1 with a mutation within the IE-1 Vmwl 75 DNA binding site. Although the mutation destroyed the ability of Vmwl75 to bind to the site, and greatly reduced the ability of Vmwl75 to repress the IE-1 promoter in transfection assays, the mutation had no effect on the levels of Vmwl 10 expression during normal HSV-1 infection.

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

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