Biomedical subjects

R D Everett

Publications and source records attributed to R D Everett.

At least 73 records · Page 4Linked to original sources

PubMed1988

Promoter sequence and cell type can dramatically affect the efficiency of transcriptional activation induced by herpes simplex virus type 1 and its immediate-early gene products Vmw175 and Vmw110.

The activation of transcription of the early and late classes of viral genes during infection by herpes simplex virus type 1 (HSV-1) requires the prior expression of immediate-early (IE) gene products. The IE gene products can also activate certain cellular and heterologous viral promoters. This paper presents a thorough analysis of transactivation of the HSV-1 glycoprotein gD and simian virus 40 early promoters, and two other promoters that are hybrids of both, under a variety of experimental conditions. Two methods of transactivation (superinfection with virus and co-transfection with isolated IE genes) have been used with all four target promoters in a variety of cell types. The conclusions are: (1) promoter sequence affects the efficiency of promoter activation by infectious HSV-1 virus, but this activation is not restricted to HSV promoters; (2) cell type affects the efficiency of promoter activation by HSV-1, and this can lead to a failure to activate a promoter in one cell type but not in others in which activation is generally more efficient; (3) a promoter can be activated to different extents in co-transfection experiments using plasmids carrying isolated IE genes that express Vmw110 or Vmw175 or when both are used together; (4) the pattern of activation of a promoter by the IE gene products in cotransfection experiments varies in different cell types; (5) changes in promoter sequence can alter the pattern of activation by the different IE polypeptides, and this pattern can again differ in different cell types; (6) other apparently minor experimental variables, as might exist between the standard methods used in different laboratories, can also affect the patterns of activation observed. The results are discussed in terms of the mechanism of action of the HSV-1 IE gene products and the limitations of the co-transfection assay.

Animals↗

PubMed1988

Analysis of the functional domains of herpes simplex virus type 1 immediate-early polypeptide Vmw110.

At the onset of infection by herpes simplex virus type 1 (HSV-1) in tissue culture, five immediate-early (IE) genes are expressed. At least four of the IE gene products have some role in the transcriptional regulation of later classes of viral genes. This study concerns the product of IE gene 1, Vmw110. Several laboratories have shown that a cloned copy of IE gene 1 can stimulate gene expression in trans in transfection experiments; this transactivation can occur either with Vmw110 by itself or in a strongly synergistic fashion with the product of IE gene 3, Vmw175. As an approach to study the mechanism of this effect, a large number of in-frame insertion mutations in the Vmw110 coding region have been constructed. The effect of the insertions on the ability of Vmw110 to activate transcription in the presence of Vmw175 has been described. This report concerns the effect of the insertions on the ability of Vmw110 to activate gene expression in the absence of Vmw175. In addition, a family of in-frame deletion mutations has been constructed and tested in transfection and immunofluorescence assays. The results show (1) that mutations in different domains of the Vmw110 polypeptide can have different effects on function in the presence or absence of Vmw175; (2) that mutations can affect the pattern of immunofluorescent intranuclear staining of Vmw110; and (3) that deletions in the region of a particular highly basic region of the polypeptide result in failure of Vmw110 to be transported into the nucleus.

DNA Transposable Elements↗

PubMed1988

Mutational dissection of the HSV-1 immediate-early protein Vmw175 involved in transcriptional transactivation and repression.

Vmw175 is one of five immediate-early (IE) proteins encoded by herpes simplex virus type-1 (HSV-1). It is required for the transcription of later classes of genes and for the accompanying repression of IE expression. Vmw175 has been shown to be a transactivator of transcription and also to autoregulate its own synthesis. We have made a large number of small, in-frame, insertion and deletion mutants of a plasmid-borne copy of the gene encoding Vmw175. Study of the activity of the resultant mutant polypeptides in transient transfection assays has defined the regions of the protein important for the repression of its own promoter, and for the transactivation of an HSV early promoter in synergy with another HSV IE protein, Vmw110. Large stretches of the protein are relatively unimportant for either function, while the regions most sensitive to disruption correlate to sequences conserved between Vmw175 and VZV 140K, the corresponding transactivating protein of Varicella-Zoster virus. The region from amino acids 275 to 490 is particularly important for both repression and transactivation, whereas that from around 840 to 1100 seems to be more important for transactivation than repression. The nuclear localization signal has been mapped to within residues 682-774.

Animals↗

PubMed1987

A detailed mutational analysis of Vmw110, a trans-acting transcriptional activator encoded by herpes simplex virus type 1.

Viral genes expressed during infection of tissue culture cells by herpes simplex virus type 1 (HSV-1) can be divided into three broad temporal groups called immediate-early (IE), early and late. Prior expression of IE gene products is required before later classes of genes can be transcribed. Using cloned copies of viral genes in plasmid vectors, it has been shown that the products of IE genes 1 and 3 (Vmw110 and Vmw175) can activate transcription from both viral and cellular promoters in short-term transfection assays. The effects of these two IE gene products are markedly synergistic in HeLa cells; the activation observed with both genes present is about 20 times that observed with Vmw175 alone. The mechanism of this activation and the properties of the IE proteins that are required are not well understood. This paper describes the construction of a large number of in-frame insertion and deletion mutations in a plasmid-encoded copy of IE gene 1. The ability of the mutant Vmw110 polypeptides to activate gene expression (in the presence of Vmw175) from the HSV-1 glycoprotein gD promoter linked to the chloramphenicol acetyl transferase gene was studied. The results show that the structural integrity of at least five regions of the polypeptide are important for its function in the presence of Vmw175.

Chromosome Deletion↗

PubMed1987

Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts.

We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications.

Animals↗

PubMed1986

The control of herpes simplex virus type-1 late gene transcription: a 'TATA-box'/cap site region is sufficient for fully efficient regulated activity.

The transcriptional programme of herpes simplex virus type 1 (HSV-1) is organised into three principle phases; immediate-early (IE), early (E) and late. The appearance of IE gene products provides the switch for E transcription. Abundant expression of late genes requires viral DNA replication. There is some overlap between E and late genes according to their degree of dependence on DNA replication. The pattern of expression of gene US11 is regulated with 'true-late' kinetics (Johnson et al., 1986). In a transient assay system, regulation of a plasmid-borne US11 promoter mimics its viral counterpart, and has a similar dependence on DNA replication for abundant expression. Using plasmids which contain a functional HSV-1 origin of replication (ORIS), we have identified the sequence requirements for the expression of late genes. All DNA sequence elements necessary for fully efficient regulated expression of US11 lie within 31 bp of the RNA cap sites; therefore it appears that a late gene promoter consists only of a proximal 'TATA-box' and cap-site region. We tested this hypothesis by removing the distal upstream region of the gD promoter (which is required for its normal regulation as an early promoter) and linking this truncated promoter to ORIS. This resulted in the conversion of gD promoter regulation to late gene kinetics during virus superinfection. The implications of these results for the mechanisms of HSV gene regulation are discussed.

Base Sequence↗

PubMed1986

DNA replication is required for abundant expression of a plasmid-borne late US11 gene of herpes simplex virus type 1.

During herpes simplex virus type 1 (HSV-1) infection, the appearance of true-late gene products is severely reduced under conditions of DNA synthesis inhibition. This report describes the use of a plasmid-borne promoter of a true-late HSV-1 gene (US11), linked to the rabbit beta-globin gene, to study the requirement of DNA replication for late gene expression. The activity of the plasmid-borne US11 promoter in constructs containing or lacking an HSV-1 origin of replication (ORIS) was analysed by quantitative S1 mapping of correctly initiated hybrid transcripts. Following HSV-1 superinfection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to the viral US11 promoter. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE-5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression.

Cloning, Molecular↗

PubMed1986

Characterization of the IE110 gene of herpes simplex virus type 1.

We have determined the DNA sequence of the herpes simplex virus type 1 (HSV-1) gene encoding the immediate early protein IE110, which is involved in transcriptional activation of later virus genes. The locations of the 5' and 3' termini of IE110 mRNA together with the positions of two introns, were identified. Examination of the DNA sequence suggested that translation starts at the first ATG after the 5' terminus of the mRNA, and that both introns occur in protein-coding sequence. The predicted IE110 polypeptide contains 775 amino acids, and has a molecular weight of 78452. It contains a cysteine-rich region resembling regions found in several proteins which interact functionally with DNA. An antiserum was raised to the predicted C terminal amino acid sequence of the IE110 polypeptide and was shown to immunoprecipitate the native protein from HSV-1-infected cell extracts. The functional importance of regions of the protein was evaluated by construction of frameshift and deletion mutants of a plasmid-borne IE110 gene. The mutants were tested for IE110 function by short-term transfection assays, and the results were correlated with the DNA sequence and RNA mapping studies.

Amino Acid Sequence↗

PubMed1986

The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans.

Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.

Gene Expression Regulation↗

PubMed1986

The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene.

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

Animals↗

PubMed1985

Activation of cellular promoters during herpes virus infection of biochemically transformed cells.

At least two of the immediate-early (IE) products of herpes simplex virus-1 (HSV-1) are responsible for the activation of transcription from viral early promoters. This process appears not to be promoter specific since several unrelated viral and cellular plasmid-borne promoters can also be activated in short-term transfection assays. This paper describes experiments that show that cellular promoters integrated into the host genome can also be activated during viral infection, and that this process is brought about by IE gene products. Biochemically transformed cell lines were isolated following transfection of plasmids containing the human epsilon-globin promoter linked to the herpes thymidine kinase coding region (as selectable marker), and an unselected rabbit beta-globin gene. Infection of some, but not all, such cell lines with HSV-1 resulted in a rapid and considerable stimulation of the integrated epsilon- and beta-globin promoters. Both promoters could also be activated (albeit less efficiently) during pseudorabies virus infection, and after introduction by transfection of plasmids containing HSV IE genes. The implications of these results for viral-host interactions and the mechanism of viral-induced promoter activation are discussed.

Animals↗

PubMed1984

Trans activation of transcription by herpes virus products: requirement for two HSV-1 immediate-early polypeptides for maximum activity.

The transcriptional programme of the herpes viruses is organised into three principal phases. The immediate-early (IE) genes are the first to be transcribed, by the pre-existing host RNA polymerase II, and their promoters are strongly stimulated by a polypeptide component of the virus particle. The E and L gene promoters become active only after the appearance of IE gene products. Genetic and biochemical evidence has shown that the HSV-1 IE polypeptide Vmw175 (ICP 4) is essential for the trans activation of HSV early promoters, but the role of none of the other four IE gene products was known. This paper describes functional tests that show, by co-transfection of recombinant plasmids into HeLa cells, that (i) Vmw175 alone can activate an HSV-1 E gene promoter, (ii) the four other HSV-1 IE gene products by themselves are unable to activate transcription, (iii) the combination of Vmw175 plus the product of IE gene 1, Vmw110 (ICP 0), is a much better activator than Vmw175 alone, (iv) cloned IE gene products of human cytomegalovirus (CMV), varicella-zoseter virus (VZV) and pseudorabies virus (PRV) can also activate transcription from an HSV-1 early promoter, and (v) this activation also occurs with cellular promoters.

Cloning, Molecular↗

PubMed1984

Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses.

This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA isolated after infection. The results showed that Herpes simplex viruses 1 and 2, Pseudorabies virus, Variella Zoster virus, Human Cytomegalovirus, Equine herpes virus-1 and Adenovirus-2 activate transcription from both promoters tested. In contrast, SV40 did not activate transcription in trans in this assay. The possible mechanisms of this activation are discussed.

Adenoviruses, Human↗

PubMed1984

A detailed analysis of an HSV-1 early promoter: sequences involved in trans-activation by viral immediate-early gene products are not early-gene specific.

This report describes a detailed analysis of the functional DNA sequences within the HSV-1 glycoprotein D gene promoter. The transcriptional activity of deletion and insertion promoter mutants was studied after both trans activation, mediated by viral products, and cis activation by a linked SV40 enhancer. Two G-rich areas (upstream of a TATA signal) were identified as important regions of the promoter. These "upstream" signals were active in both orientations. A functional TATA-box region was detected. A second region, not homologous to the concensus TATA sequence, also appeared to have a role in the positioning of the RNA cap-sites, which included both purine and pyrimidine 5' ends. Deletion of the cap-site region resulted in a moderate reduction in transcription. All the promoter elements were important for both cis and trans activated transcription. No sequence specific for viral (trans) regulation was detected, implying that Early promoters are not distinguished by specific sequences. Since HSV-1 and some other animal viruses can activate transcription from unrelated promoters, this process is probably non-specific and applicable to many, particularly extra-chromosomal, genes. The possible mechanisms of this activation are discussed.

Base Sequence↗

PubMed1984

Mutational dissection of the 21 bp repeat region of the SV40 early promoter reveals that it contains overlapping elements of the early-early and late-early promoters.

Using quantitative S1 nuclease analysis and recombinants which contain the SV40 early promoter region linked to the rabbit beta-globin gene coding sequence, we have studied the effect of deletion, inversion and point mutations, located within the 21 bp repeat region, on initiation of transcription from the early-early and late-early startsites in the absence of T-antigen. Our data establish unequivocally that the six GC-rich repeats present in the 21 bp repeat region are essential elements of both the early-early and late-early promoters and that they are not redundant, since mutations, which affect only the GC-rich repeat most proximal to the TATA box, decrease drastically the activity of the early-early promoter, but increase that of the late-early promoter. On the other hand, the four GC-rich repeats most proximal to the 72 bp repeat are common elements of the two overlapping early-early and late-early promoters. Our results, which confirm that the early-early promoter is stronger than the late-early one, also support our previous suggestion that they are in competition for the transcriptional machinery. The general organization of the SV40 early promoter region is discussed.

Base Composition↗

PubMed1983

DNA sequence elements required for regulated expression of the HSV-1 glycoprotein D gene lie within 83 bp of the RNA capsites.

The genes of Herpes simplex virus type 1 (HSV-1) are classified into three temporally regulated groups. The Immediate-Early (IE) genes are transcribed first by the pre-existing transcription apparatus of the cell. The Early genes are transcribed only after IE-gene expression, and finally the Late genes are activated. The control of transcription of the HSV-1 glycoprotein D (gD) gene (an Early function) was studied by quantitative S1 mapping of RNA produced in HSV-1 infected HeLa cells after short-term transfection experiments using plasmids containing the gD promoter linked to the rabbit beta-globin gene. The viral promoter in the plasmid was activated in the same way as that in the virus itself; the RNA showed a similar time-course of appearance, dependence on prior IE-gene expression and pattern of RNA cap-sites. Deletion analysis showed that the DNA sequences necessary for Early promoter activation lie within 83 bp of the RNA cap-sites in this instance. Surprisingly, a plasmid-borne beta-globin promoter was also activated by HSV-1 infection. The mechanism of this activation, and DNA sequence similarities between the promoters of HSV-1 Early and rabbit beta-globin genes are discussed.

Base Sequence↗

PubMed1983

The repeated GC-rich motifs upstream from the TATA box are important elements of the SV40 early promoter.

We have constructed deletion and point mutations within the Simian virus 40 (SV40) early promoter region which contains two tandemly repeated 21 bp sequences and a related 22 bp sequence (the "upstream" 21 bp repeat region). After transfection into permissive CV-1 cells and non-permissive mouse 3T3-4E cells, the effect of the mutations on early gene expression was studied by measuring T-antigen production, using indirect immunofluoresence. Our results demonstrate that the 21 bp repeat region, and in particular the six GC-rich motifs 5'-CCGCCC-3' which are repeated in this region constitute an important element of the SV40 early promoter. Surprisingly, we found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.

Animals↗

PubMed1982

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.

Base Sequence↗