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1.
PLoS Pathog ; 15(3): e1007667, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30901352

RESUMO

Host innate immune defences play a critical role in restricting the intracellular propagation and pathogenesis of invading viral pathogens. Here we show that the histone H3.3 chaperone HIRA (histone cell cycle regulator) associates with promyelocytic leukaemia nuclear bodies (PML-NBs) to stimulate the induction of innate immune defences against herpes simplex virus 1 (HSV-1) infection. Following the activation of innate immune signalling, HIRA localized at PML-NBs in a Janus-Associated Kinase (JAK), Cyclin Dependent Kinase (CDK), and Sp100-dependent manner. RNA-seq analysis revealed that HIRA promoted the transcriptional upregulation of a broad repertoire of host genes that regulate innate immunity to HSV-1 infection, including those involved in MHC-I antigen presentation, cytokine signalling, and interferon stimulated gene (ISG) expression. ChIP-seq analysis revealed that PML, the principle scaffolding protein of PML-NBs, was required for the enrichment of HIRA onto ISGs, identifying a role for PML in the HIRA-dependent regulation of innate immunity to virus infection. Our data identifies independent roles for HIRA in the intrinsic silencing of viral gene expression and the induction of innate immune defences to restrict the initiation and propagation of HSV-1 infection, respectively. These intracellular host defences are antagonized by the HSV-1 ubiquitin ligase ICP0, which disrupts the stable recruitment of HIRA to infecting viral genomes and PML-NBs at spatiotemporally distinct phases of infection. Our study highlights the importance of histone chaperones to regulate multiple phases of intracellular immunity to virus infection, findings that are likely to be highly pertinent in the cellular restriction of many clinically important viral pathogens.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fibroblastos/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 1/patogenicidade , Chaperonas de Histonas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Chaperonas de Histonas/genética , Humanos , Fatores de Transcrição/genética , Replicação Viral
2.
PLoS Pathog ; 14(1): e1006769, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29309427

RESUMO

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Corpos de Inclusão Viral/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Química Click , Deleção de Genes , Herpes Simples/tratamento farmacológico , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Corpos de Inclusão Viral/efeitos dos fármacos , Corpos de Inclusão Viral/patologia , Corpos de Inclusão Viral/virologia , Cinética , Lisogenia/efeitos dos fármacos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína da Leucemia Promielocítica/antagonistas & inibidores , Proteína da Leucemia Promielocítica/genética , Interferência de RNA , Inibidores da Transcriptase Reversa/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
PLoS Pathog ; 12(8): e1005842, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27575707

RESUMO

Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes.


Assuntos
Cromatina/metabolismo , Regulação Viral da Expressão Gênica/genética , Herpes Simples/virologia , Proteínas Imediatamente Precoces/metabolismo , Replicação Viral/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Imunofluorescência , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Histonas/metabolismo , Humanos , Transcrição Gênica , Transfecção , Células Vero
5.
J Virol ; 90(9): 4807-4826, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26937035

RESUMO

UNLABELLED: Small ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE: Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.


Assuntos
Herpes Simples/genética , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Proteínas Inibidoras de STAT Ativados/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Replicação do DNA , DNA Viral , Progressão da Doença , Expressão Gênica , Genoma Viral , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/química , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão , Proteína SUMO-1/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral
6.
J Virol ; 90(13): 5939-5952, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27099310

RESUMO

UNLABELLED: Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE: Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.


Assuntos
Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Proteína da Leucemia Promielocítica/química , Proteínas Inibidoras de STAT Ativados/metabolismo , Fibroblastos/virologia , Prepúcio do Pênis , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Masculino , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/metabolismo , Proteína SUMO-1/antagonistas & inibidores , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Replicação Viral
7.
PLoS Pathog ; 9(10): e1003695, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24130491

RESUMO

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.


Assuntos
Cromatina/metabolismo , DNA Viral/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Histonas/metabolismo , Modelos Biológicos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/genética , Cromatina/virologia , DNA Viral/genética , Herpes Simples/genética , Herpesvirus Humano 1/genética , Histonas/genética , Humanos , Células Vero
8.
bioRxiv ; 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39185184

RESUMO

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

10.
J Virol ; 85(24): 13234-52, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21994445

RESUMO

The infecting genomes of herpes simplex virus 1 (HSV-1) are assembled into unstable nucleosomes soon after nuclear entry. The source of the histones that bind to these genomes has yet to be addressed. However, infection inhibits histone synthesis. The histones that bind to HSV-1 genomes are therefore most likely those previously bound in cellular chromatin. In order for preexisting cellular histones to associate with HSV-1 genomes, however, they must first disassociate from cellular chromatin. Consistently, we have shown that linker histones are mobilized during HSV-1 infection. Chromatinization of HSV-1 genomes would also require the association of core histones. We therefore evaluated the mobility of the core histones H2B and H4 as measures of the mobilization of H2A-H2B dimers and the more stable H3-H4 core tetramer. H2B and H4 were mobilized during infection. Their mobilization increased the levels of H2B and H4 in the free pools and decreased the rate of H2B fast chromatin exchange. The histones in the free pools would then be available to bind to HSV-1 genomes. The mobilization of H2B occurred independently from HSV-1 protein expression or DNA replication although expression of HSV-1 immediate-early (IE) or early (E) proteins enhanced it. The mobilization of core histones H2B and H4 supports a model in which the histones that associate with HSV-1 genomes are those that were previously bound in cellular chromatin. Moreover, this mobilization is consistent with the assembly of H2A-H2B and H3-H4 dimers into unstable nucleosomes with HSV-1 genomes.


Assuntos
Herpesvirus Humano 1/patogenicidade , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Animais , Linhagem Celular , Chlorocebus aethiops , DNA Viral/metabolismo , Humanos , Ligação Proteica
11.
J Virol ; 82(17): 8629-46, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18579611

RESUMO

Histones interact with herpes simplex virus type 1 (HSV-1) genomes and localize to replication compartments early during infections. However, HSV-1 genomes do not interact with histones in virions and are deposited in nuclear domains devoid of histones. Moreover, late viral replication compartments are also devoid of histones. The processes whereby histones come to interact with HSV-1 genomes, to be later displaced, remain unknown. However, they would involve the early movement of histones to the domains containing HSV-1 genomes and the later movement away from them. Histones unbind from chromatin, diffuse through the nucleoplasm, and rebind at different sites. Such mobility is upregulated by, for example, phosphorylation or acetylation. We evaluated whether HSV-1 infection modulates histone mobility, using fluorescence recovery after photobleaching. All somatic H1 variants were mobilized to different degrees. H1.2, the most mobilized, was mobilized at 4 h and further so at 7 h after infection, resulting in increases in its "free" pools. H1.2 was mobilized to a "basal" degree under conditions of little to no HSV-1 protein expression. This basal mobilization required nuclear native HSV-1 genomes but was independent of HSV-1 proteins and most likely due to cellular responses. Mobilization above this basal degree, and increases in H1.2 free pools, however, depended on immediate-early or early HSV-1 proteins, but not on HSV-1 genome replication or late proteins. Linker histone mobilization is a novel consequence of cell-virus interactions, which is consistent with the dynamic interactions between histones and HSV-1 genomes during lytic infection; it may also participate in the regulation of viral gene expression.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Histonas/metabolismo , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Progressão da Doença , Recuperação de Fluorescência Após Fotodegradação , Técnica Direta de Fluorescência para Anticorpo , Variação Genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 1/genética , Histonas/genética , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mutação , Plasmídeos , Transfecção , Células Vero
12.
Viruses ; 5(7): 1758-86, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23863878

RESUMO

Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells.


Assuntos
Cromatina/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Liberação de Vírus , Replicação Viral , Humanos
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