RESUMO
Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells.
Assuntos
Inativação Gênica , Genoma Viral , Herpesvirus Humano 6/genética , Proteínas Nucleares/genética , Replicação Viral , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Replicação do DNA , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Herpesvirus Humano 6/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Proteína da Leucemia Promielocítica/genética , Fatores de Transcrição/metabolismo , Latência ViralRESUMO
Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.
Assuntos
Infecções por Herpesviridae/genética , Muromegalovirus/genética , Proteínas do Envelope Viral/genética , Animais , Infecções por Herpesviridae/virologia , Camundongos , Muromegalovirus/patogenicidade , Deleção de Sequência/genética , Vírion/genética , Vírion/crescimento & desenvolvimentoRESUMO
The HIRA histone chaperone complex deposits histone H3.3 into nucleosomes in a DNA replication- and sequence-independent manner. As herpesvirus genomes enter the nucleus as naked DNA, we asked whether the HIRA chaperone complex affects herpesvirus infection. After infection of primary cells with HSV or CMV, or transient transfection with naked plasmid DNA, HIRA re-localizes to PML bodies, sites of cellular anti-viral activity. HIRA co-localizes with viral genomes, binds to incoming viral and plasmid DNAs and deposits histone H3.3 onto these. Anti-viral interferons (IFN) specifically induce HIRA/PML co-localization at PML nuclear bodies and HIRA recruitment to IFN target genes, although HIRA is not required for IFN-inducible expression of these genes. HIRA is, however, required for suppression of viral gene expression, virus replication and lytic infection and restricts murine CMV replication in vivo. We propose that the HIRA chaperone complex represses incoming naked viral DNAs through chromatinization as part of intrinsic cellular immunity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Viral/metabolismo , Herpesvirus Humano 1/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/virologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Células HEK293 , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Chaperonas de Histonas/genética , Chaperonas de Histonas/imunologia , Humanos , Corpos de Inclusão/imunologia , Corpos de Inclusão/metabolismo , Corpos de Inclusão/virologia , Camundongos Endogâmicos C57BL , Muromegalovirus/genética , Muromegalovirus/fisiologia , Proteína da Leucemia Promielocítica/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-ß neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-ß pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-ß in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-ß.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Antígenos Nucleares/biossíntese , Autoantígenos/biossíntese , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interferon beta/imunologia , Proteínas Nucleares/biossíntese , Proteína da Leucemia Promielocítica/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Antígenos Nucleares/genética , Antígenos Nucleares/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Linhagem Celular , Proteínas Correpressoras , Infecções por Citomegalovirus/virologia , Regulação Viral da Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunidade Inata/imunologia , Interferon beta/genética , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/imunologia , Interferência de RNA , RNA Interferente Pequeno/genética , Regulação para Cima/imunologiaRESUMO
Covalent linkage to members of the small ubiquitin-like (SUMO) family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1) ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection.
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 1 , Proteoma/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Proteoma/genética , Proteômica/métodos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/imunologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Virais/metabolismoRESUMO
Death domain-associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein-protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenovírus Humanos/genética , Cromatina/metabolismo , DNA Helicases/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Linhagem Celular , Cromatina/química , Proteínas Correpressoras , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Proteína Nuclear Ligada ao XRESUMO
Upon the entry of the viral genome into the nucleus, herpes simplex virus type 1 (HSV-1) gene expression is rapidly repressed by constitutively expressed cellular proteins. This intrinsic antiviral defense is normally counteracted by ICP0, which allows virus infection to proceed efficiently. Replication of ICP0-null mutant HSV-1, however, is severely repressed by mechanisms that are conferred, at least in part, by nuclear domain 10 (ND10) components, including hDaxx, the promyelocytic leukemia (PML) protein, and Sp100. To investigate if these ND10 components repress viral gene expression in a cooperative manner, we simultaneously depleted host cells for hDaxx, PML, and Sp100 by multiple short hairpin RNA (shRNA) knockdown from a single lentivirus vector. We found that replication and gene expression of ICP0-null mutant HSV-1 were cooperatively repressed by hDaxx, PML, and Sp100 immediately upon infection, and all stages of virus replication were inhibited. Plaque-forming efficiency was enhanced at least 50-fold in the triple-depleted cells, a much larger increase than achieved by depletion of any single ND10 protein. Similar effects were also observed during infection of triple-depleted cells with human cytomegalovirus (HCMV). Moreover, using a cell culture model of quiescent infection, we found that triple depletion resulted in a much larger number of viral genomes escaping repression. However, triple depletion was unable to fully overcome the ICP0-null phenotype, implying the presence of additional repressive host factors, possibly components of the SUMO modification or DNA repair pathways. We conclude that several ND10 components cooperate in an additive manner to regulate HSV-1 and HCMV infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Citomegalovirus/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 1/imunologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Replicação Viral , Linhagem Celular , Proteínas Correpressoras , Citomegalovirus/fisiologia , Deleção de Genes , Herpesvirus Humano 1/fisiologia , Humanos , Chaperonas Moleculares , Proteína da Leucemia Promielocítica , Ensaio de Placa Viral , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.
Assuntos
Herpesvirus Humano 1/enzimologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Regulação Viral da Expressão Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.
Assuntos
Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Proteína SUMO-1/genética , Especificidade por Substrato , Transativadores , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Transcriptional targeting of viral genes is a promising strategy to achieve tumor-specific replication of oncolytic viruses. Due to its natural tropism, herpes simplex virus type 1 (HSV-1) may be an ideal tool for oncolytic therapy of brain tumors such as malignant glioblastoma. To study whether glioma-specific gene expression can be accomplished within the HSV-1 genome, four cellular regulatory elements were exemplarily studied. Whereas the human telomerase reverse transcriptase (hTERT) and survivin promoters and the nestin and vascular endothelial growth factor A (VEGF-A) enhancers displayed pronounced glioma specificity after plasmid transfection, only the nestin enhancer conferred a certain selectivity for glioma cells and notable activity when transferred into the viral genome. The nestin enhancer was also found to be highly useful for tumor cell-specific expression of a therapeutically relevant gene (interleukin-2) when tested in combination with the hTERT or simian virus 40 (SV40) early promoter in the HSV-1 genome. Because activity of the chosen promoter in a tumor is a prerequisite for the successful application of an oncolytic virus, we examined whether the activity of a promoter can be deduced from the amounts of cellular mRNA or protein expressed under its control. We found little correlation between promoter activity and mRNA levels of the corresponding gene, whereas protein expression was more closely related to promoter activity. We conclude that the cellular elements are differently regulated in the viral and cellular genomes. Mechanistic insight into the differential regulation is required to improve and refine the design of transcriptionally targeted HSV vectors.