RESUMEN
The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U(L)4, U(L)29, U(L)30, and U(L)31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.
Asunto(s)
Perfilación de la Expresión Génica , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética , Animales , Transporte Biológico/genética , Northern Blotting , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 2/crecimiento & desarrollo , Cinética , Ratones , Células 3T3 NIH , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , ARN Viral/análisis , Factores de Tiempo , Proteínas Virales/genéticaRESUMEN
We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Replicación Viral , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Ganglios Espinales/virología , Perfilación de la Expresión Génica , Genes Inmediatos-Precoces , Herpesvirus Humano 1/genética , Humanos , Ratones , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Transcripción Genética , Ensayo de Placa ViralRESUMEN
The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.
Asunto(s)
Herpesvirus Humano 1/fisiología , Queratitis Herpética/virología , Proteínas Virales/fisiología , Latencia del Virus , Animales , Córnea/virología , ADN Viral/análisis , Modelos Animales de Enfermedad , Genotipo , MicroARNs , Fenotipo , Conejos , Ganglio del Trigémino , Replicación ViralRESUMEN
BACKGROUND: Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication. METHODS: Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays. RESULTS: DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC50) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle. CONCLUSION: These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Dimetilsulfóxido/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Células Cultivadas , Chlorocebus aethiops , Dimetilsulfóxido/uso terapéutico , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Conejos , Células VeroRESUMEN
Neuronal immediate-early gene (IEG) expression is regulated by synaptic activity and plays an important role in the neuroplastic mechanisms critical to memory consolidation. IEGs can be divided into two functional classes: (1) regulatory transcription factors (RTFs), which can broadly influence cell function depending on the "downstream" genes they regulate, and (2) "effector" proteins, which may directly modulate specific cellular functions. The objective of the current study was to determine whether the expression of an effector IEG (Arc) was similar to, or different from, that of two well characterized RTF IEGs (c-fos and zif268) after learning. IEG RNA levels from rats trained in spatial and nonspatial water tasks were determined using RNase protection assays and in situ hybridization. Overall, the regulation of the three IEGs was similar in the hippocampus and the entorhinal and primary visual cortices. Consequently, IEG RNA levels were positively correlated within a structure. By contrast, Arc and zif268 RNA levels were not correlated or only weakly correlated across structures, although c-fos RNA levels were moderately correlated across structures. Arc RNA expression differed from that of zif268 and c-fos in two regards: (1) hippocampal Arc RNA levels were correlated with learning of the hippocampal-dependent spatial, but not hippocampal-independent cued response, water task, and (2) Arc RNA levels in the hippocampus and entorhinal cortex increased after spatial reversal learning relative to an asymptotic performance group. Thus, although the expression of Arc, zif268, and c-fos exhibited many similarities, Arc was most responsive to differences in behavioral task demands.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica/fisiología , Hipocampo/metabolismo , Proteínas Inmediatas-Precoces , Aprendizaje/fisiología , Aprendizaje por Laberinto/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Animales , Señales (Psicología) , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Giro Dentado/citología , Giro Dentado/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Corteza Entorrinal/metabolismo , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Hipocampo/citología , Hibridación Fluorescente in Situ , Masculino , Memoria/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-fos/genética , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genéticaRESUMEN
A class of strict late Herpes Simplex Virus Type 1 (HSV-1) promoters contains a conserved sequence element (termed the downstream activation sequence, DAS) located downstream of the transcription start site. These DAS-containing promoters also require both a TATA box and an initiator element for maximal levels of transcription. In this communication, we demonstrate that the downstream promoter element (DPE) found on a class of Drosophila TATA-less promoters and known to bind the homologue of human TAF(II)70 (a component of TFIID), can functionally substitute for DAS in the context of the strict late UL38 promoter in spite of no obvious sequence similarity. Although Drosophila DPE-containing promoters do not require a TATA box, the element does not remove the requirement for a TATA box when functioning in the HSV promoter. Next, we demonstrate that hTAF(II)70, interacts in a sequence specific manner with DAS as predicted from the fact that DPE binds Drosophila TBP. These results suggest that multiple TFIID/promoter interactions are important in the activation of HSV-1 late gene expression upon viral DNA replication. We propose that such interactions could be favored upon viral DNA replication since TFIID concentrates to viral transcription foci that form during the later stages of infection.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas , Factores de Transcripción TFII/química , Factores de Transcripción/metabolismo , Activación Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/metabolismo , Línea Celular , ADN Viral , Drosophila , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Microscopía Confocal , TATA Box , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII/metabolismo , Transcripción GenéticaRESUMEN
More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early alpha27 (U(L)54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the alpha27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.
Asunto(s)
Herpesvirus Humano 1/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética , Animales , Células Cultivadas , Eliminación de Gen , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , ConejosRESUMEN
The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (gamma1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at -48, a TATA box at -30, and an initiator (Inr) element at the cap site, here we show that elements upstream of -48 can functionally compensate for the mutational loss of the critical Sp1 site at -48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5' boundary of the VP16 promoter extends to ca. -90. This region includes an Sp1 binding site at -46, CAAT box homology at -77, and "E box" (CACGTG) at -85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity.
Asunto(s)
Cápside/genética , Regulación Viral de la Expresión Génica/genética , Proteína Vmw65 de Virus del Herpes Simple/genética , Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Animales , Unión Competitiva , Proteínas de la Cápside , Chlorocebus aethiops , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Fibroblastos/virología , Genes Virales/genética , Herpesvirus Humano 1/fisiología , Cinética , Caperuzas de ARN/genética , Conejos , Eliminación de Secuencia/genética , Factor de Transcripción Sp1/metabolismo , TATA Box/genética , Factores de Transcripción/metabolismo , Células Vero , Replicación Viral , Factor de Transcripción YY1RESUMEN
Previous analysis of two Herpes Simplex Virus Type-1 (HSV-1) promoters controlling expression of mRNA encoding early genes (U(L)37 and U(L)50) showed that the U(L)50 (dUTPase) promoter is at least 6-fold stronger both in its normal genomic location and in the non-essential gC locus. In the present report we demonstrate that the TATA element of either promoter is the major determinant of promoter strength. When the U(L)37 TATA element (CGTATAAC) was mutated with two base changes to the U(L)50 TATA sequence (CATAAAAC) in recombinant viruses, the activity of the U(L)37 promoter was increased to that of the U(L)50 promoter. Conversely, when the U(L)50 TATA element was changed to that of the U(L)37 promoter, U(L)50 promoter activity was reduced to the level of the U(L)37 promoter. In addition, we investigated the spacing of the TATA box with respect to upstream promoter elements. We found that re-positioning the U(L)37 TATA box to a location equivalent to that of the U(L)50 promoter relative to the transcript start site; i.e. three bases upstream of its cognate location, significantly diminished activity. Substitution of the U(L)50 TATA box at the new position could only partially restore promoter activity. Thus, we also conclude that the spacing of TATA elements vis-à-vis upstream promoter elements is also a critical determinant of promoter strength.
Asunto(s)
Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas/genética , Animales , Chlorocebus aethiops , Fibroblastos , Mutagénesis Sitio-Dirigida , ARN Mensajero/análisis , Conejos , TATA Box , Células VeroRESUMEN
We generated recombinant viruses in which the kinetics of expression of the leaky-late VP5 mRNA was altered. We then analyzed the effect of such alterations on viral replication in cultured cells. The VP5 promoter and leader sequences from positions -36 to +20, containing the TATA box and an initiator element, were deleted and replaced with a strong early (dUTPase), an equal-strength leaky-late (VP16), or a strict-late (U(L)38) promoter. We found that recombinant viruses containing the dUTPase promoter inserted in the VP5 locus expressed VP5-encoding mRNA with early kinetics, while virus with the U(L)38 promoter inserted expressed such mRNA with strict-late kinetics. Further, in spite of differences in its functional architecture, the VP16 promoter fully substituted for the VP5 promoter. Western blot analysis demonstrated that the amounts of VP5 capsid protein produced by the recombinant viruses differed somewhat; however, on complementing C32 and noncomplementing Vero cells, such viruses replicated to titers equivalent to those of the rescued wild-type virus controls. Multistep virus growth in mouse embryo fibroblasts, rabbit skin cells, and Vero cells also demonstrated equivalent replication efficiencies for both recombinant and wild-type viruses. Further, recombinant viruses did not show any impairment in their ability to replicate on serum-starved or quiescent human lung fibroblasts. We conclude that the kinetics of the essential VP5 mRNA expression is not critical for viral replication in cultured cells.
Asunto(s)
Cápside/genética , Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/fisiología , ARN Mensajero , ARN Viral , Replicación Viral , Animales , Cápside/biosíntesis , Proteínas de la Cápside , Línea Celular , Chlorocebus aethiops , Genoma Viral , Proteína Vmw65 de Virus del Herpes Simple/genética , Herpesvirus Humano 1/genética , Humanos , Cinética , Ratones , Mutagénesis , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Ácido Fosfonoacético/farmacología , Regiones Promotoras Genéticas , Pirofosfatasas/genética , Conejos , Recombinación Genética , Células Tumorales Cultivadas , Células VeroRESUMEN
Previous studies using cell culture systems to evaluate LAT expression demonstrated that the LAT promoter expresses at much higher levels in neuroblastoma cell lines than fibroblast lines. The high level of LAT expression in neuronal-derived cell lines correlates with the high level of LAT accumulation observed in sensory ganglia neurons during a latent infection. We have found that using LAT promoters to express reporter genes from recombinant viruses in vivo produces high levels of LAT promoter activity in the epithelium of the mouse foot. An analysis of LAT promoter activity during an acute infection in the mouse clearly demonstrates that in contrast to studies performed with selected cell lines, the LAT promoter expresses similar levels of reporter gene product in peripheral cells and in neurons. In addition, the amount of reporter gene product is higher when the LAT promoter is located within the R(L) as compared to the U(L) region, and when expression is adjusted for copy number of the reporter construct, expression is roughly the same. These results suggest the activity of the LAT promoter varies greatly according to cell type and that high levels of expression is not limited solely to neurons, especially in the in vivo setting.
Asunto(s)
Regulación Viral de la Expresión Génica , Genes Virales/genética , Herpes Simple/virología , Herpesvirus Humano 1/genética , Latencia del Virus/genética , Enfermedad Aguda , Animales , Línea Celular , ADN Recombinante/genética , Epitelio/metabolismo , Epitelio/virología , Femenino , Pie/virología , Ganglios Espinales/metabolismo , Ganglios Espinales/virología , Dosificación de Gen , Genes Reporteros/genética , Herpesvirus Humano 1/fisiología , Cinética , Ratones , Neuronas/metabolismo , Neuronas/virología , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Pirofosfatasas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Replicación Viral/genéticaRESUMEN
Several polysulfonate compounds have been shown to have the potential to inhibit the replication of herpesviruses by blocking binding and penetration of the host cell. We analyzed the actions of the polysulfonate compound suramin on the replication of herpes simplex virus type 1 (HSV-1) and compared them with the actions of heparin. We used the expression of a reporter gene (beta-galactosidase) recombined into the latency-associated transcript region of the 17syn+ strain of HSV-1 to quickly evaluate productive cycle activity and have shown that it can be directly correlated with virus replication under the conditions used. We find that suramin, like heparin, blocks the binding of HSV-1 to the cell membrane. Also, suramin efficiently blocks the cell-to-cell spread of the virus; this effect has not been previously reported. Our control experiments demonstrate that heparin also has some effect on intercellular spread of HSV-1 but to a significantly lesser degree than does suramin. We suggest that suramin and related polysulfonate compounds have potential for developing of antiherpes treatments.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Suramina/farmacología , Animales , Chlorocebus aethiops , Replicación del ADN , ADN Viral/biosíntesis , Expresión Génica , Genes Reporteros , Heparina/farmacología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Fusión de Membrana/fisiología , Factores de Tiempo , Células Vero , Ensayo de Placa Viral , Virión/fisiología , Ensamble de Virus , Replicación Viral , beta-Galactosidasa/genéticaRESUMEN
The herpes simplex virus type 1 (HSV-1) transcription program is a regulated cascade in which early and late phases of gene expression are separated by viral DNA replication. While promoters controlling expression of transcripts encoding immediate-early proteins contain virus-specific cis-acting elements, these are in the context of cellular promoter elements, and the promoters controlling expression of other viral transcripts contain only cellular cis-acting elements. We had developed and continue to refine a general method for the production of recombinant viruses in which modified promoters can be inserted into nonessential loci within the viral genome through homologous recombination. This approach has been especially useful in defining the features of model promoters of the various kinetic classes. Our work suggests that class-specific differences in promoter architecture are critical factors in the ability of the cellular transcription machinery to form stable preinitiation complexes at various phases of infection and, thus, mediate kinetic class-specific transcription. Early (beta) promoters contain a TATA box and upstream activation elements while sequences downstream of the TATA homology are dispensible for transcription. Late transcripts can be catagorized as either leaky-late (beta gamma) or strict late (gamma) depending on whether they are readily detectable prior to viral DNA replication. Promoters controlling both types are clearly distinct from early ones in that sequences near the transcription start site which resemble consensus mammalian initiator elements are required along with the TATA box and activator elements. Strict late promoters do not contain elements upstream of the TATA box but include what appears to be a class specific element downstream of the transcription start site.
Asunto(s)
Regiones Promotoras Genéticas/genética , Recombinación Genética/genética , Transcripción Genética/genética , Replicación del ADN/genética , ADN Viral/genética , Regulación Viral de la Expresión Génica/genética , Genes Reporteros/genética , Genes Virales/genética , Herpesvirus Humano 1/metabolismo , Cinética , Mutagénesis/genética , ARN Mensajero/metabolismo , TATA Box/genética , Proteínas Virales/metabolismoRESUMEN
Previous work on the strict late (gamma) UL38 promoter of herpes simplex virus type 1 identified three cis-acting elements required for wild-type levels of transcription: a TATA box at -31, a consensus mammalian initiator element at the transcription start site, and a downstream activation sequence (DAS) at +20 to +33. DAS is found in similar locations on several other late promoters, suggesting an important regulatory role in late gene expression. In this communication, we further characterize the interaction between DAS and a cellular protein which is found in both uninfected and infected nuclear extracts. This protein was purified from HeLa nuclear extracts and identified as the DNA binding component (Ku heterodimer) of DNA-dependent protein kinase (DNA-PK) by peptide mapping. Highly purified DNA-PK was able to stimulate UL38 transcription in vitro approximately 10-fold. DAS is similar in sequence to another element, nuclear regulatory element 1 (NRE1) of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat. NRE1 is known to specifically bind Ku in the absence of DNA ends. We demonstrated that NRE1 is able to substitute for DAS in the UL38 promoter to activate transcription as measured by in vitro transcription and in vivo during infection of tissue culture cells with recombinant virus. Also, we found that the binding of DNA-PK to DAS involves the bases demonstrated to be important in UL38 transcription and that the 70-kDa subunit of Ku binds to DAS.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Proteínas de Unión al ADN , Proteínas Nucleares/aislamiento & purificación , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN , Proteína Quinasa Activada por ADN , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción GenéticaRESUMEN
Functional analysis of two promoters controlling early herpes simplex virus type 1 (HSV-1) transcripts encoding the UL37 and UL50 (dUTPase) proteins are described in this report. Transcripts expressed under the control of these promoters were found to be expressed early regardless of the position of the transcription unit within the viral genome. Despite this, wt dUTPase mRNA was 6-10 times more abundant than the UL37 transcript both in wt and recombinant viruses. This same difference in transcript abundance was seen when a reporter gene (beta-galactosidase) was controlled by the two promoters in recombinant viruses in the heterologous glycoprotein C (gC) locus. Thus, both the kinetics and relative abundance of UL50 and UL37 transcripts are a direct function of their respective promoter regulatory elements. Characterization of mutated UL37 and UL50 promoters in recombinant viruses showed that the functional modules important for expression from these promoters are concentrated upstream of the transcription start site; however the extent and composition of these modules in terms of the cis-acting elements they contain was different for each. For the UL37 promoter, both a HiNF-P factor binding site (-53 to -58 bp) and the TATA homology (-22 to -27) were required for any detectable expression, while an Sp1 binding site at -123 augmented this but was not absolutely required. In contrast, the only functional elements crucial for expression from the UL50 promoter were the TATA box (-25 to -31) and an Sp1 binding site at -117 bp relative to the cap site. Despite differences in detail, when the functional architecture of these two early promoters were compared to the extensively characterized HSV-1 thymidine kinase (UL23) promoter, class-specific similarities are clearly apparent.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Genes Inmediatos-Precoces/genética , Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , ADN Recombinante , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Pirofosfatasas/genética , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Conejos , Eliminación de Secuencia , TATA Box/genética , Activación Transcripcional/genética , Proteínas del Envoltorio Viral/genética , Proteínas Estructurales Virales/genéticaRESUMEN
PCR analysis of herpes simplex virus (HSV) genome replication and productive-cycle transcription was used to examine the role of the cornea in the latency-associated transcript (LAT)-mediated reactivation of HSV type 1 (HSV-1) in the rabbit eye model. The reduced relative reactivation frequency of 17 delta Pst (a LAT- virus) compared to those of wild-type and LAT+ rescuants correlated with reduced levels of viral DNA and transcription in the cornea following epinephrine induction. The timing of virus appearance in the cornea was most consistent with tissue peripheral to the cornea itself mediating a LAT-sensitive step in the reactivation process. Specific results include the following. (i) While viral DNA was found in the corneas of rabbits latently infected with either the LAT+ or LAT- virus prior to and during the first 16 to 24 h following induction, more was found in animals infected with the LAT+ virus. (ii) A significant increase in levels of viral DNA occurred 20 to 168 h following induction. (iii) The average relative amount of viral DNA was lower at all time points following reactivation of animals infected with the LAT- virus. (iv) Expression of productive-cycle transcripts could be detected in corneas of some rabbits latently infected with either the LAT+ or LAT- virus, and the amount recovered and the timing of appearance differed during the reactivation of rabbits latently infected with the LAT+ or LAT- virus. (v) Despite the reduced recoveries of LAT- virus DNA and productive-cycle transcripts in reactivating corneas in vivo compared to those of their LAT+ counterparts, such differences were not detected in cultured keratinocytes or in experiments in which relatively high titers of virus were superinfected into the eyes of latently infected rabbits. (vi) A number of LAT(+)-virus-infected rabbits expressed LAT in corneas isolated from uninduced rabbits. When seen, its amount was significantly higher than that of a productive-cycle (VP5) transcript.
Asunto(s)
Córnea/virología , Herpesvirus Humano 1/genética , Transcripción Genética , Activación Viral , Replicación Viral , Agonistas Adrenérgicos/farmacología , Animales , Cápside/genética , Proteínas de la Cápside , Células Cultivadas , Chlorocebus aethiops , Replicación del ADN , ADN Viral/biosíntesis , Modelos Animales de Enfermedad , Epinefrina/farmacología , Genoma Viral , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 1/fisiología , Humanos , Queratinocitos/citología , Queratinocitos/virología , Reacción en Cadena de la Polimerasa , ARN Viral/biosíntesis , Conejos , Ganglio del Trigémino/virología , Células Vero , Latencia del VirusRESUMEN
The clinical manifestations of herpes simplex virus infection generally involve a mild and localized primary infection followed by asymptomatic (latent) infection interrupted sporadically by periods of recrudescence (reactivation) where virus replication and associated cytopathologic findings are manifest at the site of initial infection. During the latent phase of infection, viral genomes, but not infectious virus itself, can be detected in sensory and autonomic neurons. The process of latent infection and reactivation has been subject to continuing investigation in animal models and, more recently, in cultured cells. The initiation and maintenance of latent infection in neurons are apparently passive phenomena in that no virus gene products need be expressed or are required. Despite this, a single latency-associated transcript (LAT) encoded by DNA encompassing about 6% of the viral genome is expressed during latent infection in a minority of neurons containing viral DNA. This transcript is spliced, and the intron derived from this splicing is stably maintained in the nucleus of neurons expressing it. Reactivation, which can be induced by stress and assayed in several animal models, is facilitated by the expression of LAT. Although the mechanism of action of LAT-mediated facilitation of reactivation is not clear, all available evidence argues against its involving the expression of a protein. Rather, the most consistent models of action involve LAT expression playing a cis-acting role in a very early stage of the reactivation process.
Asunto(s)
Herpes Simple/complicaciones , Simplexvirus/fisiología , Latencia del Virus/genética , Animales , Modelos Animales de Enfermedad , Encefalitis , Regulación Viral de la Expresión Génica , Herpes Simple/inmunología , Herpesvirus Humano 3/patogenicidad , Herpesvirus Humano 3/fisiología , Humanos , Hibridación Fluorescente in Situ , Ratones , Neuronas/virología , Regiones Promotoras Genéticas , Conejos , Simplexvirus/genética , Simplexvirus/crecimiento & desarrollo , Simplexvirus/inmunología , Activación Viral , Latencia del Virus/inmunologíaRESUMEN
Latency-associated transcript (LAT) promoter deletion mutants of herpes simplex virus type 1 have a reduced capacity to reactivate following adrenergic induction in the rabbit eye model. We have mapped a reactivation phenotype within LAT and describe the construction of recombinants in which poly(A) addition sites have been placed at intervals within the LAT region to form truncated LAT transcripts. These mutants localize the induced reactivation phenotype to the 5' end of LAT. To further define this region, we constructed a recombinant containing a 348-bp deletion located 217 bp downstream of the transcription start site of the 8.5-kb LAT. This virus, 17delta348, expresses LAT but exhibits a significantly reduced ability to reactivate following epinephrine iontophoresis into the cornea. Quantitative DNA PCR analysis reveals that 17delta 348 establishes a latent infection within rabbit trigeminal ganglia with the same efficiency as does either the rescuant or wild-type virus. The region deleted in 17delta348 encodes three potential translational initiators (ATGs) which we have mutated and demonstrated to be dispensable for epinephrine-induced reactivation. In addition, three smaller deletions within this region have been constructed and were shown to reactivate at wild-type (parent) frequencies. These studies indicate that an undefined portion of the 348-bp region is required to facilitate induced reactivation. Sequence analysis of this 348-bp region revealed a CpG island which extends into the LAT promoter and which possesses homology to conserved elements within the mouse and human XIST transcript encoded on the X chromosome. Possible implications of these elements in the regulation of LAT expression are discussed.
Asunto(s)
Herpesvirus Humano 1/genética , ARN Viral/fisiología , Activación Viral/genética , Animales , Secuencia de Bases , Secuencia Conservada , Epinefrina/farmacología , Herpesvirus Humano 1/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Poli A/metabolismo , Conejos , Eliminación de Secuencia , Repeticiones de Trinucleótidos , Activación Viral/efectos de los fármacosRESUMEN
The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter.
Asunto(s)
Cápside/genética , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Proteínas de la Cápside , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , ADN Viral/genética , ADN Viral/metabolismo , Células HeLa , Humanos , Metilación , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares/metabolismo , Caperuzas de ARN , Conejos , Piel/citología , Piel/metabolismo , Células Vero , Proteínas ViralesRESUMEN
A region of the herpes simplex virus type 1 genome located upstream of the alpha 0 promoter contains a promoter which regulates transcription in the opposite orientation to that driven by alpha 0. Analyses of mutants from which this promoter, alpha X, was deleted and a mutant in which a fragment that serves as a transcription terminator and polyadenylation signal was inserted upstream of this promoter demonstrate that two distinct transcription units overlap this region of the genome and are transcribed in a direction antisense to the neurovirulence gene gamma (1)34.5. One unit, dependent on the alpha X promoter, is active when cells are infected in the presence of the protein synthesis inhibitor cycloheximide. The second unit, independent of alpha X, is active during the course of productive infection. This transcription unit originates from a promoter upstream of alpha X which is distinct from the latency-associated promoter (LAP). Two polyadenylated transcripts of 0.9 and 4.9 kb accumulate from this region of the genome during productive infection, but no mature transcripts accumulate in infected cells maintained in the presence of cycloheximide. Kinetic analyses demonstrate that the transcripts that accumulate during productive infection fall into the beta class of herpes simplex virus type 1 genes.