RESUMO
The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Receptor Notch1 , Transativadores , Ativação Viral , Latência Viral , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/genética , Humanos , Animais , Transativadores/metabolismo , Transativadores/genética , Receptor Notch1/metabolismo , Receptor Notch1/genética , Células Vero , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Chlorocebus aethiops , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação Viral da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a DNARESUMO
Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein Jκ recombination signal-binding protein (RBP-Jκ or CSL). RBP-Jκ normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-Jκ DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-Jκ motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-Jκ binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-Jκ bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-Jκ DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-Jκ-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta- and RBP-Jκ-bound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-Jκ DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-Jκ during KSHV reactivation.
Assuntos
DNA/metabolismo , Herpesvirus Humano 8/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fatores do Domínio POU/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Herpesvirus Humano 8/genética , HumanosRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi's Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman's Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis.
RESUMO
Reactivation of Kaposi's sarcoma-associated herpesvirus (KHSV; also known as Human herpesvirus (HHV)-8) from latency is associated with progression to disease. The primary experimental models for studying KSHV reactivation are B lymphocyte cell lines derived from patients with primary effusion lymphoma (PEL). PEL models have remained essential tools for understanding molecular details of latency and reactivation, yet they have shortcomings. In particular, PEL cells are difficult to transfect with plasmid DNA, which limits their routine use in studies that require introduction of exogenous DNA. Moreover, PELs produce poorly infectious virus, which limits functional quantitation of the ultimate step in KSHV reactivation. In this study, we show that a recently published reporter virus system overcomes inherent difficulties of using PELs for studying viral reactivation. Vero rKSHV.294 cells harbor a recombinant reporter KSHV clone and produce infectious virus whose quantitation is strictly dependent on passage to naïve 293 cells. We show that the cells are easily transfectable, and produce significant amount of infectious virus in response to ectopically-expressed lytic switch protein Rta. In thus study, we derive optimal conditions to measure fold reactivation by varying experimental time periods and media volumes in infections and reporter enzyme reactions, and by eliminating background cellular and media activities. By measuring production of infectious virus, we demonstrate that Rta, but not the cellular transactivator Notch Intracellular Domain (NICD)-1, is sufficient to reactivate KSHV from latency. These data confirm previous studies that were limited to measuring viral gene expression in PELs as indicators of reactivation.
Assuntos
Herpesvirus Humano 8/fisiologia , Transfecção/métodos , Virologia/métodos , Ativação Viral , Latência Viral , Linhagem Celular , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismoRESUMO
In Kaposi's sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth. The KSHV Rta protein is the lytic switch for reactivation. Rta transactivates essential genes via interactions with cofactors such as the cellular RBP-Jk and Oct-1 proteins, and the viral Mta protein. Given that robust viral reactivation would facilitate antiviral responses and culminate in host cell lysis, regulation of Rta's expression and function is a major determinant of the latent-lytic balance and the fate of infected cells. Our lab recently showed that Rta transactivation requires the cellular peptidyl-prolyl cis/trans isomerase Pin1. Our data suggest that prolinedirected phosphorylation regulates Rta by licensing binding to Pin1. Despite Pin1's ability to stimulate Rta transactivation, unchecked Pin1 activity inhibited virus production. Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function. We propose that Pin1 is a molecular timer that can regulate the balance between viral lytic gene expression and host cell lysis. Intriguing scenarios for Pin1's underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/metabolismo , Peptidilprolil Isomerase/metabolismo , Transativadores/metabolismo , Ativação Viral , Latência Viral , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Transcrição GênicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1's effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.
Assuntos
Herpesvirus Humano 8/fisiologia , Peptidilprolil Isomerase/metabolismo , Sarcoma de Kaposi/virologia , Ativação Viral , Latência Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Replicação do DNA , DNA Viral/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Camundongos , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Regiões Promotoras Genéticas , Homologia de Sequência de AminoácidosRESUMO
In primary effusion lymphoma (PEL) cells infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta's promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.
Assuntos
Infecções por Herpesviridae/enzimologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/fisiologia , Histona Desacetilases/metabolismo , Ativação Viral , Linhagem Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por Herpesviridae/genética , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/genética , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Regiões Promotoras Genéticas , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
RNA polymerase II (RNAP-II) synthesizes the m(7)G-capped Spliced Leader (SL) RNA and most protein-coding mRNAs in trypanosomes. RNAP-II recruitment to DNA usually requires a set of transcription factors that make sequence-specific contacts near transcriptional start sites within chromosomes. In trypanosomes, the transcription factor TFIIB is necessary for RNAP-II-dependent SL RNA transcription. However, the trypanosomal TFIIB (tTFIIB) lacks the highly basic DNA binding region normally found in the C-terminal region of TFIIB proteins. To assess the precise pattern of tTFIIB binding within the SL RNA gene locus, as well as within several other loci, we performed chromatin immunoprecipitation/microarray analysis using a tiled gene array with a probe spacing of 10 nucleotides. We found that tTFIIB binds non-randomly within the SL RNA gene locus mainly within a 220-nt long region that straddles the transcription start site. tTFIIB does not bind within the small subunit (SSU) rRNA locus, indicating that trypanosomal TFIIB is not a component of an RNAP-I transcriptional complex. Interestingly, discrete binding sites were observed within the putative promoter regions of two loci on different chromosomes. These data suggest that although trypanosomal TFIIB lacks a highly basic DNA binding region, it nevertheless localizes to discrete regions of chromatin that include the SL RNA gene promoter.
Assuntos
Cromossomos/genética , Cromossomos/metabolismo , Regiões Promotoras Genéticas , RNA Líder para Processamento , Fator de Transcrição TFIIB/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Análise em Microsséries , Ligação ProteicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several neoplastic diseases: Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV replicates actively, via a controlled gene expression program, but can also remain latent. It had been thought that the transition from latent to lytic replication was controlled exclusively by the replication and transcription activator protein RTA (open reading frame 50 [ORF50] gene product). A dominant-negative (DN) ORF50 mutant, ORF50ΔSTAD, blocks gene expression and replication. We produced a PEL cell line derivative containing both latent KSHV genomes and an inducible ORF50ΔSTAD. We unexpectedly found that induction of apoptosis triggered high-level viral replication, even when DN ORF50ΔSTAD was present, suggesting that apoptosis triggers KSHV replication through a distinct RTA-independent pathway. We verified that apoptosis triggers KSHV replication independent of RTA using ORF50 small interfering RNA (siRNA) and also showed that caspase activity is required to trigger KSHV replication. We showed that when apoptosis triggers KSHV replication, the kinetics of late gene expression is accelerated by 12 to 24 h and that virus produced following apoptosis has reduced infectivity. KSHV therefore appears to replicate via two distinct pathways, a conventional pathway requiring RTA, with slower replication kinetics, producing virus with higher infectivity, and an alternative apoptosis-triggered pathway that does not require RTA, has faster replication kinetics, and produces virus with lower infectivity. The existence of a distinct apoptosis-triggered, accelerated replication pathway may have evolutionary advantages for the virus and clinical significance for the treatment of KSHV-associated neoplasms. It also provides further evidence that KSHV can sense and react to its environment.
Assuntos
Apoptose/genética , Herpesvirus Humano 8/genética , Replicação Viral/genética , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Expressão Gênica , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Cinética , Mutação , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Vírion/efeitos dos fármacos , Vírion/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi's sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic "CANT DNA repeats" in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta's role as the switch is inefficient. Many factors modulate K-Rta's function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.
RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is the etiologic agent of Kaposi's sarcoma (KS) and lymphoproliferative diseases. We previously demonstrated that the KSHV lytic switch protein Rta stimulates DNA binding of the cellular RBP-Jk/CSL protein, the nuclear component of the Notch pathway, on Rta target promoters. In the current study, we define the promoter requirements for formation of transcriptionally productive Rta/RBP-Jk/DNA complexes. We show that highly pure Rta footprints 7 copies of a previously undescribed repetitive element in the promoter of the essential KSHV Mta gene. We have termed this element the "CANT repeat." CANT repeats are found on both strands of DNA and have a consensus sequence of ANTGTAACANT(A/T)(A/T)T. We demonstrate that Rta tetramers make high-affinity interactions (i.e., nM) with 64 bp of the Mta promoter but not single CANT units. The number of CANT repeats, their presence in palindromes, and their positions relative to the RBP-Jk binding site determine the optimal target for Rta stimulation of RBP-Jk DNA binding and formation of ternary Rta/RBP-Jk/DNA complexes. DNA binding and tetramerization mutants of Rta fail to stimulate RBP-Jk DNA binding. Our chromatin immunoprecipitation assays show that RBP-Jk DNA binding is broadly, but selectively, stimulated across the entire KSHV genome during reactivation. We propose a model in which tetramerization of Rta allows it to straddle RBP-Jk and contact repeat units on both sides of RBP-Jk. Our study integrates high-affinity Rta DNA binding with the requirement for a cellular transcription factor in Rta transactivation.
Assuntos
Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Regiões Promotoras Genéticas , Transativadores/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Pegada de DNA , DNA Viral/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Transativadores/genética , Ativação ViralRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are coinfected with Epstein-Barr virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA-binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen 2 (EBNA-2) to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV(+)/EBV(+) PEL cells but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA-2 growth deficiency in an autocrine/paracrine manner. Complementation of EBNA-2 deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV(+)/EBV(+) PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA-2 and Rta induce distinct alterations in the cellular proteomes that contribute to the growth of infected cells.
Assuntos
Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/fisiologia , Receptores Notch/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Proliferação de Células , Meios de Cultivo Condicionados , Primers do DNA/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Expressão Gênica , Genes Virais , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas , Transdução de Sinais , Transativadores/genética , Transativadores/fisiologia , Transfecção , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia , Ativação Viral/fisiologiaAssuntos
Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Macrófagos/virologia , MicroRNAs/fisiologia , RNA Viral/fisiologia , Fatores de Transcrição E2F/antagonistas & inibidores , Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno , Humanos , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-6/biossíntese , Macrófagos/metabolismo , Mimetismo Molecular , Biossíntese de Proteínas , Isoformas de Proteínas/antagonistas & inibidores , RNA Viral/genética , Proteína do Retinoblastoma/antagonistas & inibidores , Ativação Transcricional , Proteínas Virais/genética , Proteínas Virais/fisiologia , Replicação ViralRESUMO
Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.
Assuntos
Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismo , Proteínas Virais/biossíntese , Ativação Viral , Núcleo Celular/química , Proteínas Imediatamente Precoces/genética , Sinais de Localização Nuclear , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Deleção de Sequência , Tamoxifeno/farmacologia , Transativadores/genéticaRESUMO
The Kaposi's sarcoma-associated herpesvirus (KSHV) Mta protein, encoded by open reading frame 57, is a transactivator of gene expression that is essential for productive viral replication. Previous studies have suggested both transcriptional and posttranscriptional roles for Mta, but little is known regarding Mta's transcriptional function. In this study, we demonstrate that Mta cooperates with the KSHV lytic switch protein, Rta, to reactivate KSHV from latency, but Mta has little effect on reactivation when expressed alone. We demonstrate that the Mta and Rta proteins are expressed with similar but distinct kinetics during KSHV reactivation. In single-cell analyses, Mta expression coincides tightly with progression to full viral reactivation. We demonstrate with promoter reporter assays that while Rta activates transcription in all cell lines tested, Mta's ability to transactivate promoters, either alone or synergistically with Rta, is cell and promoter specific. In particular, Mta robustly transactivates the nut-1/PAN promoter independently of Rta in 293 and Akata-31 cells. Using nuclear run-on assays, we demonstrate that Mta stimulates transcriptional initiation in 293 cells. Rta and Mta physically interact in infected cell extracts, and this interaction requires the intact leucine repeat and central region of Rta in vitro. We demonstrate that Mta also binds to the nut-1/PAN promoter DNA in vitro and in infected cells. An Mta mutant with a lesion in a putative A/T hook domain is altered in DNA binding and debilitated in transactivation. We propose that one molecular mechanism of Mta-mediated transactivation is a direct effect on transcription by direct and indirect promoter association.
Assuntos
Herpesvirus Humano 8/fisiologia , Regiões Promotoras Genéticas , Transativadores/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Ativação Viral , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Humanos , Proteínas Imediatamente Precoces/metabolismo , Regiões Promotoras Genéticas/genética , Transativadores/genética , Ativação Transcricional , Proteínas Virais/genética , Latência Viral , Replicação ViralRESUMO
The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Proteínas Repressoras/genética , Transativadores/metabolismo , Ativação Transcricional , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Células Cultivadas , DNA/metabolismo , Fibroblastos/metabolismo , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/análise , Proteínas Imediatamente Precoces/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Fator 1 de Transcrição de Octâmero/análise , Fator 1 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Elementos de Resposta , Transativadores/análise , Transativadores/genética , Transfecção , Proteínas Virais/análiseRESUMO
The Kaposi's sarcoma-associated herpesvirus open reading frame 50 (ORF50) protein (called Rta), is necessary and sufficient for reactivation of the virus from latency. We previously demonstrated that a truncated mutant of ORF50 lacking its C-terminal transcriptional activation domain, called ORF50DeltaSTAD, formed mixed multimers with wild-type (WT) ORF50 and functioned as a dominant negative inhibitor of reactivation. For this report, we investigated the requirements for multimerization of ORF50/Rta in transactivation and viral reactivation. We analyzed multimerization of WT, mutant, and chimeric ORF50 proteins, using Blue Native polyacrylamide gel electrophoresis and size exclusion chromatography. WT and mutant ORF50 proteins form tetramers and higher-order multimers, but not monomers, in solution. The proline-rich, N-terminal leucine heptapeptide repeat (LR) of ORF50 (amino acids [aa] 244 to 275) is necessary but not sufficient for oligomer formation and functions in concert with the central portion of ORF50/Rta (aa 245 to 414). The dominant negative mutant ORF50DeltaSTAD requires the LR to form mixed multimers with WT ORF50 and inhibit its function. In the context of the WT ORF50/Rta protein, mutagenesis of the LR, or replacement of the LR by heterologous multimerization domains from the GCN4 or p53 proteins, demonstrates that tetramers of Rta are sufficient for transactivation and viral reactivation. Mutants of Rta that are unable to form tetramers but retain the ability to form higher-order multimers are reduced in function or are nonfunctional. We concluded that the proline content, but not the leucine content, of the LR is critical for determining the oligomeric state of Rta.
Assuntos
Herpesvirus Humano 8/química , Proteínas Imediatamente Precoces/química , Estrutura Quaternária de Proteína , Sarcoma de Kaposi/virologia , Transativadores/química , Proteínas Virais/química , Ativação Viral , Sequência de Aminoácidos , Linhagem Celular , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Leucina/química , Leucina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Prolina/química , Prolina/genética , Transativadores/genética , Transativadores/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia , Ativação Viral/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.