RESUMEN
In order to use herpes simplex virus (HSV) as a vector for the transmission and expression of foreign genes, it is obviously necessary to be able to prepare stocks of the virus and to propagate both HSV and the recombinant viruses derived from it. Similarly, the introduction of foreign genes into the virus will require the purification of viral DNA for use as a cloning vector, and preparation and analysis of viral DNA will also be necessary in order to characterize the recombinant viruses produced during the cloning procedures. This chapter will therefore discuss in turn the procedures used in our laboratory for the growth of HSV, and for the preparation of viral DNA.
RESUMEN
The non-permissivity of C1300 mouse neuroblastoma cells for herpes simplex virus (HSV) infection is due to a failure of such cells to transcribe the immediate-early (IE) genes following viral infection. We have transfected both C1300 cells and permissive cells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Proteínas Inmediatas-Precoces , Neuronas/fisiología , Regiones Promotoras Genéticas/genética , Simplexvirus/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Ratones , Datos de Secuencia Molecular , Neuroblastoma/genética , Células Tumorales CultivadasRESUMEN
The effect of herpes simplex virus type 1 infection in a series of immortalized dorsal root ganglion cell lines has been investigated. Following infection of one of these lines, the viral immediate-early genes are not transcribed and the lytic cycle is aborted at an early stage. In contrast these cells do support transcription of the gene encoding the latency-associated transcripts which are the only viral RNAs present in latently infected ganglia in vivo. These cell lines are therefore a suitable model system for studies of the processes regulating the interaction of HSV with neuronal cell types and the establishment of latent infections in vivo.
Asunto(s)
Ganglios Espinales/citología , Herpes Simple/genética , Neuronas Aferentes/citología , Simplexvirus/genética , Transcripción Genética/genética , Animales , Línea Celular , Ganglios Espinales/microbiología , Regulación Viral de la Expresión Génica , Genes Virales/genética , Neuronas Aferentes/microbiología , Ratas , Simplexvirus/aislamiento & purificaciónRESUMEN
Transcription of the herpes simplex virus (HSV) immediate-early (IE) genes in lytic infection is dependent upon the formation of a complex between the cellular transcription factor Oct-1 and the HSV virion protein Vmw65. This complex then binds to the TAATGARAT sequence in the IE promoters and trans-activates the IE genes. Following infection of neuronal cells such as the C1300 neuroblastoma cell line, however, the viral (IE) genes are not transcribed and the lytic cycle is aborted at an early stage. We show here that the cellular factors necessary to form a trans-activating complex with Vmw65 are present in C1300 cells and that trans-activation of both viral and cellular promoters by Vmw65 can be observed in these cells. In contrast with permissive cells, however, trans-activation is only observed in C1300 cells at a high concentration of the target viral promoter and not at a low concentration of the target promoter, regardless of the amount of Vmw65 transfected. The significance of these effects for the regulation of latent infection and cellular gene expression in neuronal cells is discussed.
Asunto(s)
Genes Virales/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , Simplexvirus/genética , Transactivadores/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , ADN Viral/genética , Regulación Viral de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Neuroblastoma , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The herpes simplex virus virion protein Vmw65 trans activates the viral immediate-early genes and some octamer-containing cellular genes, including that encoding histone H2B. We found, however, that a truncated form of this virion protein repressed H2B gene transcription and also dominantly inhibited induction of the gene by intact Vmw65. A cell line expressing this truncated protein expressed reduced levels of H2B and grew more slowly than the parental cell line or a similar line expressing the intact protein.
Asunto(s)
Regulación Viral de la Expresión Génica , Histonas/genética , Simplexvirus/genética , Transactivadores/genética , Transcripción Genética , Activación Transcripcional , Animales , División Celular , Línea Celular , Cinética , Células L/citología , Células L/metabolismo , Ratones , Plásmidos , Transactivadores/metabolismo , Transfección , Virión/genéticaRESUMEN
Undifferentiated U937 cells are non-permissive for herpes simplex virus (HSV) infection but can be rendered permissive by treatment with phorbol myristate acetate (PMA), which causes them to differentiate to a macrophage-like phenotype. Following infection with HSV, both PMA--treated and untreated cells correctly transcribe the viral immediate-early genes at levels comparable to those observed in fully permissive cell types, but immediate-early RNA and protein are detected only in the PMA-treated cells. Hence PMA acts by relieving an early block to HSV infection caused by the rapid turnover of immediate-early RNA. This block is not caused by the production of soluble inhibitors and can also be relieved by treatment with other agents that cause macrophage differentiation such as 1, 25 dihydroxycholecalciferol. These findings therefore indicate that the non-permissivity of undifferentiated U937 cells for HSV is mediated by post-transcriptional regulation of immediate-early gene expression.
Asunto(s)
Herpes Simple/fisiopatología , Fagocitos/fisiología , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Dihidroxicolecalciferoles/farmacología , Regulación Viral de la Expresión Génica/fisiología , Genes Reguladores/fisiología , Genes Virales/fisiología , Herpes Simple/metabolismo , Humanos , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/microbiología , Leucemia Mielomonocítica Aguda/patología , Fagocitos/metabolismo , ARN Viral , Simplexvirus/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/fisiologíaRESUMEN
C1300 mouse neuroblastoma cells are nonpermissive for infection with herpes simplex virus owing to a failure of viral immediate-early gene transcription following infection. The weak activity of the immediate-early gene promoters in these cells is mediated by the binding of a repressor factor to the octamer-related TAATGARAT motifs in these promoters. This repressor activity is specific to cells of neuronal origin (being absent in a range of permissive nonneuronal cells) and is also able to repress the activity of cellular octamer-containing promoters introduced into C1300 cells. The role of this repressor in the regulation of octamer-containing cellular genes in neuronal cells and in the control of latent infections with herpes simplex virus is discussed.
Asunto(s)
Regulación Viral de la Expresión Génica , Genes Virales , Neuronas/metabolismo , Regiones Promotoras Genéticas , Simplexvirus/genética , Transcripción Genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/metabolismo , Herpes Simple/genética , Datos de Secuencia Molecular , Mutación , Transfección , Células Tumorales CultivadasRESUMEN
An HSV virion component, Vmw65, interacts with cellular transcription factors to transactivate TAATGARAT-containing viral genes and some cellular genes containing the related octamer element. We show that the octamer-containing histone H2B promoter can be trans-activated by transfection of Vmw65 but not by viral infection. The induction of H2B transcription by Vmw65 can be abolished by co-transfection of excess amounts of either a TAATGARAT element or a Vmw65 responsive octamer element. This effect cannot be overcome by addition of increasing amounts of Vmw65. The H2B promoter and TAATGARAT-containing viral promoters therefore compete for limiting cellular factors required for induction by Vmw65 resulting in repression of the H2B gene during lytic infection. The competitive effect of TAATGARAT elements on the H2B gene is not observed in the absence of Vmw65, but can be produced in the presence of a truncated form of Vmw65 lacking the acidic tail required for transcriptional activation. Hence a domain of Vmw65 distinct from that involved in transcriptional induction interacts with cellular octamer binding proteins favouring binding to the TAATGARAT motif.
Asunto(s)
Genes Virales , Histonas/genética , Simplexvirus/genética , Secuencia de Bases , Línea Celular , Regulación Viral de la Expresión Génica , Herpes Simple/genética , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Transactivadores , Transcripción Genética , TransfecciónRESUMEN
C1300 neuroblastoma cells are nonpermissive for infection with herpes simplex virus but can be rendered permissive by pretreatment with sodium butyrate. This increased permissivity which is specific for HSV is caused by increased transcription of the viral immediate-early genes following infection of butyrate-treated cells and can be observed for at least 24 hr following withdrawal of butyrate. The use of C1300 cells as a model system for studying the regulation of immediate-early gene expression in neuronal cells in vitro and its possible relevance to the study of the processes regulating latent infection in vivo is discussed.
Asunto(s)
Neuronas/microbiología , Simplexvirus/genética , Animales , Butiratos/farmacología , Ácido Butírico , Regulación de la Expresión Génica/efectos de los fármacos , Genes Virales , Técnicas In Vitro , Ratones , Neuroblastoma , ARN Viral/genética , Factores de Tiempo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Lytic infection with herpes virus type 1 (HSV-1) causes the accumulation of a 40-kDa cellular protein (p40) which is also overexpressed in cultured cells transformed by HSV or other agents and in human cervical tumors. Accumulation of p40 is dependent upon viral protein synthesis but not viral DNA replication in the infected cell and occurs in the HSV-1 mutants tsK and tsLB2 in which only a defective ICP4 protein and the four other immediate-early proteins are synthesized. By using a panel of HSV-1 strains, each defective in one of these four proteins, we show that only a mutation in the gene encoding ICP27 abolishes p40 accumulation. The defect in this mutant virus can be rescued by a plasmid encoding ICP27 alone indicating that ICP27 is obligately required for p40 accumulation. The significance of this effect as one aspect of the interaction of viral control proteins with cellular genes is discussed.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces , Biosíntesis de Proteínas , Simplexvirus/fisiología , Proteínas Virales/fisiología , Animales , Western Blotting , Línea Celular , Cicloheximida/farmacología , Replicación del ADN , Mutación , Proteínas/genética , Simplexvirus/genética , Transfección , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Replicación ViralRESUMEN
The gene encoding the U3 SnRNA is transcriptionally induced early in infection with herpes simplex virus (HSV). This effect is due to the action of the HSV virion protein Vmw65 and is dependent upon the octamer motif in the U3 promoter. In contrast the U1 SnRNA gene which also contains an octamer is not activated by Vmw65 in infections or transfections. We show that this is due to sequence differences between the octamers in the U1 and U3 genes. Thus the U3 octamer can confer responsiveness to Vmw65 to truncated U1 or U3 promoters whereas the U1 octamer cannot. The use of Vmw65 as a tool to analyse the role of the octamer sequence in mediating a variety of expression patterns in different genes is discussed.
Asunto(s)
Genes , Simplexvirus/genética , Animales , Secuencia de Bases , ADN/genética , Regulación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Transcripción Genética , TransfecciónRESUMEN
Lytic infection with herpes simplex virus causes increased ubiquitin gene transcription. This effect is reproduced in cells expressing the viral immediate early protein ICP4 in the absence of other viral proteins but is not seen at the nonpermissive temperature in cells expressing the temperature-sensitive ICP4 protein of HSV-1 tsK. Studies with probes specific to the three human ubiquitin genes indicate that only the Ubi B gene is sensitive to ICP4-mediated induction whereas the Ubi A and C genes are unaffected. The significance of these effects for the mechanism by which ICP4 transcriptionally activates viral and a few cellular genes is discussed.
Asunto(s)
Proteínas Inmediatas-Precoces , Simplexvirus/genética , Factores de Transcripción/genética , Ubiquitinas/genética , Proteínas Virales/genética , Regulación de la Expresión Génica , Humanos , Transcripción GenéticaRESUMEN
Nuclear run-off and pulse-labelling techniques have been used to study the changes in transcription rates of a number of cellular genes during infection with Herpes simplex virus. The majority of these genes show a decrease in transcription rate to about 60% of that observed prior to infection. In contrast, a small number of genes are transcriptionally activated during infection. These effects, which occur at a point in infection after the synthesis of viral proteins but prior to the onset of viral DNA synthesis, are mediated by different immediate-early proteins of the virus. Thus we show that, whilst transcriptional activation requires a functional ICP4 protein, repression is dependent upon the presence of another immediate early protein--ICP22.
Asunto(s)
Herpes Simple/metabolismo , Supresión Genética , Proteínas Virales/farmacología , Aciclovir/farmacología , Animales , Cricetinae , Cicloheximida/farmacología , ADN Viral/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Riñón , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , Simplexvirus/efectos de los fármacos , Simplexvirus/genética , Simplexvirus/patogenicidad , Transcripción Genética/efectos de los fármacosRESUMEN
Lytic infection with herpes simplex virus results in transcriptional induction of a cellular gene encoding ubiquitin, causing increased accumulation of ubiquitin RNA and protein in the infected cell. This induction, which is dependent upon viral protein synthesis, does not occur in the HSV-1 mutant tsK which is defective in the gene encoding the viral protein ICP4. Transfected cells expressing the viral ICP4 protein exhibit higher levels of ubiquitin gene transcription than untransfected controls indicating that transcriptional induction can be mediated by the ICP4 protein alone.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces , Simplexvirus/fisiología , Ubiquitinas/biosíntesis , Proteínas Virales/fisiología , Animales , Línea Celular , Cricetinae , Fibroblastos/metabolismo , Simplexvirus/genética , Transcripción Genética , Ubiquitinas/genética , Proteínas Virales/genética , Replicación ViralRESUMEN
Infection of cultured cells with herpes simplex virus (HSV) results in the transcriptional induction of a small number of cellular genes. Although the majority of such genes are dependent upon viral protein synthesis for their induction, a small minority are not. These genes are induced by events occurring prior to the onset of viral protein synthesis, in particular by binding of the virus to the cell surface and cellular entry of the virion. The significance of such cellular gene induction early in viral infection is discussed in terms of virus-cell interaction in general and the mechanism of transformation by HSV in particular.
Asunto(s)
Herpes Simple/genética , Transcripción Genética , Proteínas Virales/biosíntesis , Células Cultivadas , Cicloheximida/farmacología , Herpes Simple/metabolismo , Hibridación de Ácido Nucleico , ARN Viral/análisis , Receptores Virales/análisisRESUMEN
The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the xrs strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronied populations of xrs cells, estimated to have been irradiated in late S and G2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.
Asunto(s)
Aberraciones Cromosómicas , Fibroblastos/efectos de la radiación , Animales , Ciclo Celular , Línea Celular , Cromátides/efectos de la radiación , Cromosomas/efectos de la radiación , Cricetinae , Cricetulus/genética , Reparación del ADN , Femenino , Fibroblastos/ultraestructura , Ovario , Rayos XRESUMEN
Herpes simplex virus Type 2 causes a severe repression of host cell biosynthesis at a number of levels. We show that despite this, non-viral cDNA clones derived from cellular RNA species which accumulate to high levels after infection can be isolated using differential screening techniques. By using nuclear run-off assays, we have shown that this RNA accumulation is mediated by transcriptional induction of the corresponding cellular genes.
Asunto(s)
Regulación de la Expresión Génica , Poli A/biosíntesis , ARN Mensajero/biosíntesis , Simplexvirus/fisiología , Transcripción Genética , Animales , Línea Celular , Cricetinae , ADN/análisis , Fibroblastos , Genes , Mesocricetus/genética , Poli A/genética , ARN Mensajero/genéticaRESUMEN
A small number of cellular proteins accumulate to high levels in cells infected with Herpes Simplex Virus (HSV) despite a generalised repression of most host cell bio-synthesis. An antibody to one such protein has been used to screen a lambda gt11 library and for polysome immunoprecipitation in order to isolate cDNA clones derived from the corresponding gene. The cDNA clones have been used in dot blot and nuclear run-off assays to show that HSV, like other DNA tumour viruses can transcriptionally induce a cellular gene. The mechanism of this effect which is dependent on viral protein synthesis and its possible significance in transformation by HSV are discussed.
Asunto(s)
Transformación Celular Neoplásica , Clonación Molecular , ADN/aislamiento & purificación , Genes , Simplexvirus/genética , Transcripción Genética , Animales , Línea Celular , Núcleo Celular/metabolismo , Cricetinae , Riñón , Hibridación de Ácido Nucleico , Homología de Secuencia de Ácido NucleicoRESUMEN
Six CHO mutants have previously been described as being sensitive to ionizing radiation and bleomycin treatment, with little or no cross sensitivity to UV-radiation (Jeggo and Kemp, 1983). Their ability to rejoin single- and double-strand breaks has been examined here. Using two techniques, gradient sedimentation and alkaline elution, no difference could be observed between wild-type and mutant strains in the initial number of single-strand breaks induced, the rate of rejoining, or the final level of single-strand breaks rejoined. Thus, a major inability to rejoin single-strand breaks is not the basis for sensitivity in these mutants. In contrast, all 6 mutants showed a decreased ability to rejoin the double-strand breaks induced by gamma-irradiation as measured by neutral elution. Rejoining of half of the breaks occurred in 37 min in wild-type cells and reached a maximum level of 72% after 2 h. All the mutants showed a decreased rate of rejoining, and the final level was 17% of that observed in the wild-type in the most defective mutant, and ranged from 35 to 69% in the other 5 mutants. These are the first mammalian cell mutants to be described with a defect in double-strand break rejoining.