RESUMEN
Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Infecciones por Virus ADN/inmunología , Virus ADN/fisiología , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , Animales , AMP Cíclico/metabolismo , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Células THP-1 , Replicación ViralRESUMEN
In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-ß mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.
Asunto(s)
Apoptosis , Herpesvirus Humano 1 , Viroterapia Oncolítica , Virus Oncolíticos , Replicación Viral , Herpesvirus Humano 1/fisiología , Humanos , Viroterapia Oncolítica/métodos , Línea Celular Tumoral , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Caspasas/metabolismo , Animales , Melanoma/terapia , Melanoma/inmunologíaRESUMEN
The dynamic nature of interactions between invading viral pathogens and their hosts has fascinated scientists for several decades. The well-known capacity of herpes simplex virus (HSV) to establish life-long infections in humans reflects a dynamic balance between maintaining a latent state in which viral genomes are silenced and re-entry into the lytic phase during reactivation. Silencing of the viral genome has been shown to be a function of innate immune signalling, intrinsic cellular antiviral mechanisms and epigenetic repression. Thus, although many important observations have been made identifying cellular processes that contribute to the repression of the viral genome and latency, the field has lacked an understanding of how these factors work together. In this issue of EMBO Reports, Suzich et al (2021) present convincing evidence that brings together individual observations into a cohesive model that explains many of these outstanding mysteries. Here, we will review the background data that lead to this outstanding piece of work.
Asunto(s)
Herpesvirus Humano 1 , Represión Epigenética , Genoma Viral , Herpesvirus Humano 1/genética , Humanos , Latencia del Virus/genéticaRESUMEN
In herpes simplex virus type 1 (HSV-1) infection, the coupling of genome replication and transcription regulation has been known for many years; however, the underlying mechanism has not been elucidated. We performed a comprehensive transcriptomic assessment and factor-binding analysis for Pol II, TBP, TAF1, and Sp1 to assess the effect genome replication has on viral transcription initiation and elongation. The onset of genome replication resulted in the binding of TBP, TAF1, and Pol II to previously silent late promoters. The viral transcription factor, ICP4, was continuously needed in addition to DNA replication for activation of late gene transcription initiation. Furthermore, late promoters contain a motif that closely matches the consensus initiator element (Inr), which robustly bound TAF1 postreplication. Continued DNA replication resulted in reduced binding of Sp1, TBP, and Pol II to early promoters. Therefore, the initiation of early gene transcription is attenuated following DNA replication. Herein, we propose a model for how viral DNA replication results in the differential utilization of cellular factors that function in transcription initiation, leading to the delineation of kinetic class in HSV-productive infection.
Asunto(s)
Proteínas Inmediatas-Precoces/genética , ARN Polimerasa II/genética , Simplexvirus/genética , Transcripción Genética , Animales , Chlorocebus aethiops , Replicación del ADN/genética , Genoma Viral/genética , Humanos , Regiones Promotoras Genéticas , Unión Proteica , TATA Box/genética , Factores de Transcripción/genética , Células Vero , Replicación Viral/genéticaRESUMEN
Lysine-specific demethylase 1 (LSD1) targets cellular proteins, including histone H3, p53, E2F, and Dnmt1, and is involved in the regulation of gene expression, DNA replication, the cell cycle, and the DNA damage response. LSD1 catalyzes demethylation of histone H3K9 associated with herpes simplex virus 1 (HSV-1) immediate early (IE) promoters and is necessary for IE gene expression, viral DNA replication, and reactivation from latency. We previously found that LSD1 associates with HSV-1 replication forks and replicating viral DNA, suggesting that it may play a direct role in viral replication or coupled processes. We investigated the effects of the LSD1 inhibitor SP-2509 on the HSV-1 life cycle. Unlike previously investigated LSD1 inhibitors tranylcypromine (TCP) and OG-L002, which covalently attach to the LSD1 cofactor flavin adenine dinucleotide (FAD) to inhibit demethylase activity, SP-2509 has previously been shown to inhibit LSD1 protein-protein interactions. We found that SP-2509 does not inhibit HSV-1 IE gene expression or transcription factor and RNA polymerase II (Pol II) association with viral DNA prior to the onset of replication. However, SP-2509 does inhibit viral DNA replication, late gene expression, and virus production. We used EdC labeling of nascent viral DNA to image aberrant viral replication compartments that form in the presence of SP-2509. Treatment resulted in the formation of small replication foci that colocalize with replication proteins but are defective for Pol II recruitment. Taken together, these data highlight a potential new role for LSD1 in the regulation of HSV-1 DNA replication and gene expression after the onset of DNA replication.IMPORTANCE Treatment of HSV-1-infected cells with SP-2509 blocked viral DNA replication, gene expression after the onset of DNA replication, and virus production. These data support a potential new role for LSD1 in the regulation of viral DNA replication and successive steps in the virus life cycle, and further highlight the promising potential to utilize LSD1 inhibition as an antiviral approach.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Histona Demetilasas/efectos de los fármacos , Hidrazinas/farmacología , Sulfonamidas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , ADN Viral , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces , Herpes Simple/tratamiento farmacológico , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , Células VeroRESUMEN
Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4-6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks. Pulse chase analyses and imaging studies reveal temporal and spatial dynamics between viral replication forks and associated proteins and demonstrate that several DNA repair complexes and key transcription factors are recruited to or near replication forks. Consistent with these observations we show that the initiation of viral DNA replication is sufficient to license late gene transcription. These data provide insight into mechanisms that couple HSV-1 DNA replication with transcription and repair for the coordinated expression and maintenance of the viral genome.
Asunto(s)
Replicación del ADN/genética , Herpes Simple/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Interacciones Huésped-Patógeno/genética , Replicación Viral/genética , Animales , Línea Celular , Chlorocebus aethiops , ADN Viral/análisis , ADN Viral/genética , Técnica del Anticuerpo Fluorescente , Genes Virales/genética , Genoma Viral/genética , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Espectrometría de Masas , Células Vero , Replicación Viral/fisiologíaRESUMEN
Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.
Asunto(s)
Regulación Viral de la Expresión Génica , Genoma Viral , Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , Pulmón/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Animales , Western Blotting , Núcleo Celular/genética , Células Cultivadas , Chlorocebus aethiops , Ensamble y Desensamble de Cromatina , Replicación del ADN , Técnica del Anticuerpo Fluorescente , Herpes Simple/genética , Herpes Simple/virología , Humanos , Pulmón/citología , Pulmón/embriología , Proteínas Nucleares/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero , Proteínas Virales/genética , Replicación ViralRESUMEN
Malignant melanoma is an aggressive tumour of the skin with increasing incidence, frequent metastasis and poor prognosis. At the same time, it is an immunogenic type of cancer with spontaneous regressions. Most recently, the tumoricidal effect of plasmacytoid dendritic cells (pDC) and their capacity to overcome the immunosuppressive tumour microenvironment are being investigated. In this respect, we studied the effect of the infectious, but replication-deficient, herpes simplex virus 1 (HSV-1) d106S vaccine strain, which lacks essential immediate early genes, in pDC co-cultures with 11 melanoma cell lines. We observed a strong cytotoxic activity, inducing apoptotic and necrotic cell death in most melanoma cell lines. The cytotoxic activity of HSV-1 d106S plus pDC was comparable to the levels of cytotoxicity induced by natural killer cells, but required only a fraction of cells with effector : target ratios of 1 : 20 (P < 0·05). The suppressive activity of cell-free supernatants derived from virus-stimulated pDC was significantly neutralized using antibodies against the interferon-α receptor (P < 0·05). In addition to type I interferons, TRAIL and granzyme B contributed to the inhibitory effect of HSV-1 d106S plus pDC to a minor extent. UV-irradiated viral stocks were significantly less active than infectious particles, both in the absence and presence of pDC (P < 0·05), indicating that residual activity of HSV-1 d106S is a major component and sensitizes the tumour cells to interferon-producing pDC. Three leukaemic cell lines were also susceptible to this treatment, suggesting a general anti-tumour effect. In conclusion, the potential of HSV-1 d106S for therapeutic vaccination should be further evaluated in patients suffering from different malignancies.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/trasplante , Herpesvirus Humano 1/inmunología , Inmunoterapia Adoptiva/métodos , Leucemia/terapia , Melanoma/terapia , Neoplasias Cutáneas/terapia , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Leucemia/inmunología , Leucemia/metabolismo , Leucemia/patología , Leucemia/virología , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/patología , Melanoma/virología , Necrosis , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Factores de Tiempo , Transfección , Microambiente TumoralRESUMEN
UNLABELLED: Herpes simplex virus 1 (HSV-1) can undergo a productive infection in nonneuronal and neuronal cells such that the genes of the virus are transcribed in an ordered cascade. HSV-1 can also establish a more quiescent or latent infection in peripheral neurons, where gene expression is substantially reduced relative to that in productive infection. HSV mutants defective in multiple immediate early (IE) gene functions are highly defective for later gene expression and model some aspects of latency in vivo. We compared the expression of wild-type (wt) virus and IE gene mutants in nonneuronal cells (MRC5) and adult murine trigeminal ganglion (TG) neurons using the Illumina platform for cDNA sequencing (RNA-seq). RNA-seq analysis of wild-type virus revealed that expression of the genome mostly followed the previously established kinetics, validating the method, while highlighting variations in gene expression within individual kinetic classes. The accumulation of immediate early transcripts differed between MRC5 cells and neurons, with a greater abundance in neurons. Analysis of a mutant defective in all five IE genes (d109) showed dysregulated genome-wide low-level transcription that was more highly attenuated in MRC5 cells than in TG neurons. Furthermore, a subset of genes in d109 was more abundantly expressed over time in neurons. While the majority of the viral genome became relatively quiescent, the latency-associated transcript was specifically upregulated. Unexpectedly, other genes within repeat regions of the genome, as well as the unique genes just adjacent the repeat regions, also remained relatively active in neurons. The relative permissiveness of TG neurons to viral gene expression near the joint region is likely significant during the establishment and reactivation of latency. IMPORTANCE: During productive infection, the genes of HSV-1 are transcribed in an ordered cascade. HSV can also establish a more quiescent or latent infection in peripheral neurons. HSV mutants defective in multiple immediate early (IE) genes establish a quiescent infection that models aspects of latency in vivo. We simultaneously quantified the expression of all the HSV genes in nonneuronal and neuronal cells by RNA-seq analysis. The results for productive infection shed further light on the nature of genes and promoters of different kinetic classes. In quiescent infection, there was greater transcription across the genome in neurons than in nonneuronal cells. In particular, the transcription of the latency-associated transcript (LAT), IE genes, and genes in the unique regions adjacent to the repeats persisted in neurons. The relative activity of this region of the genome in the absence of viral activators suggests a more dynamic state for quiescent genomes persisting in neurons.
Asunto(s)
Fibroblastos/virología , Regulación Viral de la Expresión Génica , Genoma Viral , Herpes Simple/virología , Herpesvirus Humano 1/genética , Neuronas/virología , Ganglio del Trigémino/virología , Replicación Viral , Animales , Herpesvirus Humano 1/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Activación ViralRESUMEN
Innate sensing of microbial components is well documented to occur at many cellular sites, including at the cell surface, in the cytosol, and in intracellular vesicles, but there is limited evidence of nuclear innate signaling. In this study we have defined the mechanisms of interferon regulatory factor-3 (IRF-3) signaling in primary human foreskin fibroblasts (HFF) infected with herpes simplex virus 1 (HSV-1) in the absence of viral gene expression. We found that the interferon inducible protein 16 (IFI16) DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d109 infection and induction of IRF-3 signaling. Consistent with the IFI16 sensor being nuclear, conditions that block viral DNA release from incoming capsids inhibit IRF-3 signaling. An unknown factor must be exported from the nucleus to activate IRF-3 through cytoplasmic STING, which is required for IRF-3 activation and signaling. However, when the viral ICP0 protein is expressed in the nucleus, it causes the nuclear relocalization and degradation of IFI16, inhibiting IRF-3 signaling. Therefore, HSV-1 infection is sensed in HFF by nuclear IFI16 upon release of encapsidated viral DNA into the nucleus, and the viral nuclear ICP0 protein can inhibit the process by targeting IFI16 for degradation. Together these results define a pathway for nuclear innate sensing of HSV DNA by IFI16 in infected HFF and document a mechanism by which a virus can block this nuclear innate response.
Asunto(s)
Herpes Labial/metabolismo , Proteínas Inmediatas-Precoces/fisiología , Factor 3 Regulador del Interferón/metabolismo , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Células Cultivadas , ADN Viral/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces/metabolismo , Simplexvirus/genética , Animales , Línea Celular , Análisis Mutacional de ADN , Eliminación de Gen , Prueba de Complementación Genética , Proteínas Inmediatas-Precoces/genética , Mapeo de Interacción de Proteínas , Eliminación de Secuencia , Factor de Transcripción TFIID/metabolismo , Transcripción GenéticaRESUMEN
Infected cell polypeptide 4 (ICP4) activates transcription from most viral promoters. Two transactivation domains, one N-terminal and one C terminal, are largely responsible for the activation functions of ICP4. A mutant ICP4 molecule lacking the C-terminal activation domain (n208) efficiently activates many early genes, whereas late genes are poorly activated, and virus growth is severely impaired. The regions within the N terminus of ICP4 (amino acids 1 to 210) that contribute to activation were investigated by analysis of deletion mutants in the presence or absence of the C-terminal activation domain. The mutants were assessed for their abilities to support viral replication and to regulate gene expression. Several deletions in regions conserved in other alphaherpesviruses resulted in impaired activation and viral growth, without affecting DNA binding. The single small deletion that had the greatest effect on activation in the absence of the C terminus corresponded to a highly conserved stretch of amino acids between 81 and 96, rendering the molecule nonfunctional. However, when the C terminus was present, the same deletion had a minimal effect on activity. The amino terminus of ICP4 was predicted to be relatively disordered compared to the DNA-binding domain and the C-terminal 500 amino acids. Moreover, the amino terminus appears to be in a relatively extended conformation as determined by the hydrodynamic properties of several mutants. The data support a model where the amino terminus is an extended and possibly flexible region of the protein, allowing it to efficiently interact with multiple transcription factors at a distance from where it is bound to DNA, thereby enabling ICP4 to function as a general activator of polymerase II transcription. The C terminus of ICP4 can compensate for some of the mutations in the N terminus, suggesting that it either specifies redundant interactions or enables the amino terminus to function more efficiently.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de Secuencia , Activación Transcripcional , Células VeroRESUMEN
ICP0 is a transcriptional activating protein required for the efficient replication and reactivation of latent herpes simplex virus 1 (HSV-1). Multiple regions of ICP0 contribute its activity, the most prominent of which appears to be the RING finger, which confers E3 ubiquitin ligase activity. A region in the C terminus of ICP0 has also been implicated in several activities, including the disruption of a cellular repressor complex, REST/CoREST/HDAC1/2/LSD1. We used quiescent infection of MRC-5 cells with a virus that does not express immediate-early proteins, followed by superinfection with various viral mutants to quantify the ability of ICP0 variants to reactivate gene expression and alter chromatin structure. Superinfection with wild-type virus resulted in a 400-fold increase in expression from the previously quiescent d109 genome, the removal of heterochromatin and histones from the viral genome, and an increase in histone marks associated with activated transcription. RING finger mutants were unable to reactivate transcription or remove heterochromatin from d109, while mutants that are unable to bind CoREST activate gene expression from quiescent d109, albeit to a lesser degree than the wild-type virus. One such mutant, R8507, resulted in the partial removal of heterochromatin. Infection with R8507 did not result in the hyperacetylation of H3 and H4. The results demonstrate that (i) consistent with previous findings, the RING finger domain of ICP0 is required for the activation of quiescent genomes, (ii) the RF domain is also crucial for the ultimate removal of repressive chromatin, (iii) activities or interactions specified by the carboxy-terminal region of ICP0 significantly contribute to activation, and (iv) while the effects of the R8507 on chromatin are consistent with a role for REST/CoREST/HDAC1/2/LSD1 in the repression of quiescent genomes, the mutation may also affect other activities involved in derepression.
Asunto(s)
Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Activación Viral , Línea Celular , Expresión Génica , Heterocromatina/metabolismo , Humanos , Transcripción GenéticaRESUMEN
The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. Since viral genes are transcribed by cellular RNA polymerase II (RNA pol II), ICP4 must interact with components of the pol II machinery to regulate viral gene expression. It has been shown previously that ICP4 interacts with TATA box-binding protein (TBP), TFIIB, and the TBP-associated factor 1 (TAF1) in vitro. In this study, ICP4-containing complexes were isolated from infected cells by tandem affinity purification (TAP). Forty-six proteins that copurified with ICP4 were identified by mass spectrometry. Additional copurifying proteins were identified by Western blot analysis. These included 11 components of TFIID and 4 components of the Mediator complex. The significance of the ICP4-Mediator interaction was further investigated using immunofluorescence and chromatin immunoprecipitation. Mediator was found to colocalize with ICP4 starting at early and continuing into late times of infection. In addition, Mediator was recruited to viral promoters in an ICP4-dependent manner. Taken together, the data suggest that ICP4 interacts with components of TFIID and Mediator in the context of viral infection, and this may explain the broad transactivation properties of ICP4.
Asunto(s)
Herpesvirus Humano 1/patogenicidad , Proteínas Inmediatas-Precoces/metabolismo , Complejo Mediador/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Western Blotting , Línea Celular , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Cromatografía de Afinidad/métodos , Técnica del Anticuerpo Fluorescente , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/aislamiento & purificación , Espectrometría de Masas , Complejo Mediador/genética , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/aislamiento & purificación , Activación Transcripcional , Células VeroRESUMEN
Persisting latent herpes simplex virus genomes are to some degree found in a heterochromatic state, and this contributes to reduced gene expression resulting in quiescence. We used a relatively long-term quiescent infection model in human fibroblasts, followed by provision of ICP0 in trans, to determine the effects of ICP0 on the viral chromatin state as gene expression is reactivated. Expression of ICP0, even at low levels, results in a reduction of higher-order chromatin structure and heterochromatin on quiescent viral genomes, and this effect precedes an increase in transcription. Concurrent with transcriptional activation, high levels of ICP0 expression result in the reduction of the heterochromatin mark trimethylated H3K9, removal of histones H3 and H4 from the quiescent genome, and hyperacetylation of the remaining histones. In contrast, low levels of ICP0 did not appreciably change the levels of histones on the viral genome. These results indicate that ICP0 activity ultimately affects chromatin structure of quiescent genomes at multiple levels, including higher-order chromatin structure, histone modifications, and histone association. Additionally, the level of ICP0 expression affected its ability to change chromatin structure but not to reactivate gene expression. While these observations suggest that some of the effects on chromatin structure are possibly not direct, they also suggest that ICP0 exerts its effects through multiple mechanisms.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Activación Viral , Células Cultivadas , Fibroblastos/virología , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Transcripción GenéticaRESUMEN
RNA polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and is commonly targeted during cancer and viral infection. We find that Herpes Simplex Virus-1 (HSV-1) stimulates tRNA expression 10-fold. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts. tRNA with a specific codon bias were not targeted-rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection results in depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the viral genome, which suggests a previously unrecognized role in HSV-1 gene expression. These findings provide insight into mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.
Asunto(s)
Herpesvirus Humano 1/fisiología , ARN Polimerasa III/metabolismo , Factores de Transcripción , Genoma Viral , Herpesvirus Humano 1/genética , Humanos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Mensajero/metabolismo , ARN de Transferencia , ARN no Traducido , Transcripción Genética , Activación Transcripcional , Replicación ViralRESUMEN
The ability of herpes simplex virus to persist in cells depends on the extent of viral-gene expression, which may be controlled by epigenetic mechanisms. We used quiescent infection with the viral mutants d109 and d106 to explore the effects of cell type and the presence of the viral protein ICP0 on the expression and chromatin structure of the human cytomegalovirus (HCMV) tk and gC promoters on the viral genome. Expression from the HCMV promoter on the d109 genome decreased with time and was considerably less in HEL cells than in Vero cells. Expression from the HCMV promoter in d106 was considerably more abundant than in d109, and this increased with time in both cell types. The same pattern of expression was seen on the tk and gC genes on the viral genomes, although the levels of tk and gC RNA were approximately 10(2)- and 10(5)-fold lower than those of wild-type virus in d106 and d109, respectively. In micrococcal-nuclease digestion experiments, nucleosomes were evident on the d109 genome, and the amount of total H3 as determined by chromatin immunoprecipitation was considerably greater on d109 than d106 genomes. The acetylation of histone H3 on the d106 genomes was evident at early and late times postinfection in Vero cells, but only at late times in HEL cells. The same pattern was observed for H3 acetylated on lysine 9. Trimethylation of H3K9 on d109 genomes was evident only at late times postinfection in Vero cells, while it was observed both early and late in HEL cells. Heterochromatin protein 1gamma (HP1gamma) was generally present only on d109 genomes at late times postinfection of HEL cells. The observations of chromatin structure correlate with the expression patterns of the three analyzed genes on the quiescent genomes. Therefore, several mechanisms generally affect the expression and contribute to the silencing of persisting genomes. These are the abundance of nucleosomes, the acetylation state of the histones, and heterochromatin. The extents to which these different mechanisms contribute to repression vary in different cell types and are counteracted by the presence of ICP0.
Asunto(s)
Regulación Viral de la Expresión Génica , Histonas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Simplexvirus/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Animales , Línea Celular , Chlorocebus aethiops , Citomegalovirus/genética , Perfilación de la Expresión Génica , Heterocromatina/metabolismo , Humanos , Metilación , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Simplexvirus/genética , Timidina Quinasa/genética , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
The binding of herpes simplex virus type 1 ICP4, TATA-binding protein (TBP), and RNA polymerase II (polII) to the promoter regions of representative immediate-early (IE) (ICP0), early (E) (thymidine kinase [tk]), and late (L) (glycoprotein C [gC]) genes on the viral genome was examined as a function of time postinfection, viral DNA replication, cis-acting sites for TFIID in the tk and gC promoters, and genetic background of ICP4. The binding of TBP and polII to the IE ICP0 promoter was independent of the presence of ICP4, whereas the binding of TBP and polII to the tk and gC promoters occurred only when ICP4 also bound to the promoters, suggesting that the presence of ICP4 at the promoters of E and L genes in virus-infected cells is crucial for the formation of transcription complexes on these promoters. When the TATA box of the tk promoter or the initiator element (INR) of the gC promoter was mutated, a reduction in the amount of TBP and polII binding was observed. However, a reduction in the amount of ICP4 binding to the promoters was also observed, suggesting that the binding of TBP-containing complexes and ICP4 is cooperative. The binding of ICP4, TBP, and polII was also observed on the gC promoter at early times postinfection or when DNA synthesis was inhibited, suggesting that transcription complexes may be formed early on L promoters and that additional events or proteins are required for expression. The ability to form these early complexes on the gC promoter required the DNA-binding domain but in addition required the carboxyl-terminal 524 amino acids of ICP4, which is missing the virus n208. This region was not required to form TBP- and polII-containing complexes on the tk promoter. n208 activates E but not L genes during viral infection. These data suggest that a region of ICP4 may differentiate between forming TBP- and polII-containing complexes on E and L promoters.
Asunto(s)
Herpesvirus Humano 1/metabolismo , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Proteína de Unión a TATA-Box/genética , Animales , Chlorocebus aethiops , Replicación del ADN , Herpesvirus Humano 1/enzimología , Proteínas Inmediatas-Precoces/metabolismo , Unión Proteica , ARN Polimerasa II/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Células VeroRESUMEN
We have recently shown that ICP4 has a differential requirement for the general transcription factor TFIIA in vitro (S. Zabierowski and N. DeLuca, J. Virol. 78:6162-6170, 2004). TFIIA was dispensable for ICP4 activation of a late promoter (gC) but was required for the efficient activation of an early promoter (tk). An intact INR element was required for proficient ICP4 activation of the late promoter in the absence of TFIIA. Because TFIIA is known to stabilize the binding of both TATA binding protein (TBP) and TFIID to the TATA box of core promoters and ICP4 has been shown to interact with TFIID, we tested the ability of ICP4 to stabilize the binding of either TBP or TFIID to the TATA box of representative early, late, and INR-mutated late promoters (tk, gC, and gC8, respectively). Utilizing DNase I footprinting analysis, we found that ICP4 was able to facilitate TFIIA stabilized binding of TBP to the TATA box of the early tk promoter. Using mutant ICP4 proteins, the ability to stabilize the binding of TBP to both the wild-type and the INR-mutated gC promoters was located in the amino-terminal region of ICP4. When TFIID was substituted for TBP, ICP4 could stabilize the binding of TFIID to the TATA box of the wild-type gC promoter. ICP4, however, could not effectively stabilize TFIID binding to the TATA box of the INR-mutated late promoter. The additional activities of TFIIA were required to stabilize the binding of TFIID to the INR-mutated late promoter. Collectively, these data suggest that TFIIA may be dispensable for ICP4 activation of the wild-type late promoter because ICP4 can substitute for TFIIA's ability to stabilize the binding of TFIID to the TATA box. In the absence of a functional INR, ICP4 can no longer stabilize TFIID binding to the TATA box of the late promoter and requires the additional activities of TFIIA. The stabilized binding of TFIID by TFIIA may in turn allow ICP4 to more efficiently activate transcription from non-INR containing promoters.
Asunto(s)
ADN Viral/metabolismo , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , TATA Box , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/metabolismo , Animales , Chlorocebus aethiops , Huella de ADN , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/genética , Eliminación de Secuencia , Factor de Transcripción TFIID/metabolismo , Células VeroRESUMEN
Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.