RESUMO
IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas E1A de Adenovirus , Proteínas de Transporte , DNA Helicases , Imunidade Inata , Complexo de Endopeptidases do Proteassoma , Estresse Fisiológico , Humanos , Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/enzimologia , Adenovírus Humanos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , DNA Helicases/metabolismo , Interferons/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Quaternária de Proteína , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação , Replicação ViralRESUMO
Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.
Assuntos
Proteínas E1A de Adenovirus , Adenovírus Humanos , Proteínas Reguladoras de Apoptose , Replicação Viral , Humanos , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , Antivirais/farmacologia , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.
Assuntos
Adenoviridae , Ciclo Celular , Replicação do DNA , Replicação Viral , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células/efeitos dos fármacos , Células/virologia , Replicação do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Mutação , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Replicação Viral/fisiologia , HumanosRESUMO
E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.
Assuntos
Proteína de Ligação a CREB , Ensaios de Triagem em Larga Escala , Prolina , Histona Acetiltransferases , Proteínas E1A de Adenovirus/metabolismo , Fatores de Transcrição de p300-CBP , DNA , TecnologiaRESUMO
Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Domínios Proteicos , Proteína do Retinoblastoma/metabolismoRESUMO
Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein-protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein-protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.
Assuntos
Proteínas E1A de Adenovirus , Evolução Molecular , Interações Hospedeiro-Patógeno , Mastadenovirus , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Motivos de Aminoácidos , Animais , Mamíferos/virologia , Mastadenovirus/química , Mastadenovirus/genética , Mapeamento de Interação de ProteínasRESUMO
Interferons (IFNs) are one of the hallmarks of host antiviral immunity. IFNs exert their antiviral activities through the induction of IFN-stimulated genes (ISGs) and antiviral proteins; however, the mechanism by which ISGs inhibit adenovirus (Ad) replication is not clearly understood. IFNs repress Ad immediate early gene expression and, consequently, all subsequent aspects of the viral life cycle. In this study, we found that IFN-induced protein with tetratricopeptide repeats 3, IFIT3 (ISG60), restricts Ad replication. IFIT3 repressed Ad E1A immediate early gene expression but did not alter Ad genome entry into the nucleus. Expression of IFIT3 led to phosphorylation of TBK1, IRF3, and STAT1; increased expression of IFNß and ISGs; and required IFIT1 and IFIT2 partner proteins. During RNA virus infections, it is known that IFIT3 stimulates IFN production through mitochondrial antiviral signaling (MAVS)-mediated activation of TBK1 which synergizes activation of IRF3 and NF-κB. MAVS or TBK1 depletion in cells expressing IFIT3 blocked IFN signaling and reversed the Ad replication restriction. In addition, STING depletion phenocopied the effect suggesting that IFIT3 activates the STING pathway with cross talk to the MAVS pathway. This occurs independently of viral pathogen-associated molecular patterns (PAMPs). These results demonstrate that the expression of a single ISG, IFIT3, activates IFN signaling and establishes a cellular antiviral state independent of viral PAMPs. IMPORTANCE IFITs belong to a family of IFN-induced proteins that have broad antiviral functions, primarily studied with RNA viruses leaving a gap of knowledge on the effects of these proteins on DNA viruses. In this study we show that IFIT3, with its partner proteins IFIT1 and IFIT2, specifically restricts replication of human Ad, a DNA virus, by stimulating IFNß production via the STING and MAVS pathways. This effect enhanced the IFN response and is independent of viral PAMPs. These results reveal a novel mechanism of activation of IFN signaling to enhance cellular antiviral responses.
Assuntos
Proteínas E1A de Adenovirus/genética , Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/genética , Interferon beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas E1A de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon beta/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologiaRESUMO
Papillomaviruses, polyomaviruses and adenoviruses are collectively categorized as the small DNA tumour viruses. Notably, human adenoviruses were the first human viruses demonstrated to be able to cause cancer, albeit in non-human animal models. Despite their long history, no human adenovirus is a known causative agent of human cancers, unlike a subset of their more famous cousins, including human papillomaviruses and human Merkel cell polyomavirus. Nevertheless, seminal research using human adenoviruses has been highly informative in understanding the basics of cell cycle control, gene expression, apoptosis and cell differentiation. This review highlights the contributions of human adenovirus research in advancing our knowledge of the molecular basis of cancer.
Assuntos
Adenovírus Humanos , Neoplasias , Adenoviridae/genética , Proteínas E1A de Adenovirus , Proteínas E1B de Adenovirus , Adenovírus Humanos/genética , Animais , Neoplasias/terapiaRESUMO
Adenovirus infections tend to be mild, but they may pose a serious threat for young and immunocompromised individuals. The treatment is complicated because there are no approved safe and specific drugs for adenovirus infections. Here, we present evidence that 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 chaperone, decreases the rate of human adenovirus 5 (HAdV-5) replication in cell cultures by 95%. 17-AAG inhibited the transcription of early and late genes of HAdV-5, replication of viral DNA, and expression of viral proteins. 6 h after infection, Hsp90 inhibition results in a 6.3-fold reduction of the newly synthesized E1A protein level without a decrease in the E1A mRNA level. However, the Hsp90 inhibition does not increase the decay rate of the E1A protein that was constitutively expressed in the cell before exposure to the inhibitor. The co-immunoprecipitation proved that E1A protein interacted with Hsp90. Altogether, the presented results show, for the first time. that Hsp90 chaperones newly synthesized, but not mature, E1A protein. Because E1A serves as a transcriptional co-activator of adenovirus early genes, the anti-adenoviral activity of the Hsp90 inhibitor might be explained by the decreased E1A level.
Assuntos
Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Replicação Viral/fisiologia , Células A549 , Adenoviridae/efeitos dos fármacos , Adenoviridae/genética , Benzoquinonas/farmacologia , Replicação do DNA/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Virais/metabolismo , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain.
Assuntos
Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Ciclo Celular , Ativação Enzimática , Células HEK293 , Humanos , Domínios ProteicosRESUMO
Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.
Assuntos
Proteínas Correpressoras/metabolismo , Chaperonas Moleculares/metabolismo , Terapia Viral Oncolítica/métodos , Replicação Viral/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/fisiologia , Proteínas E2 de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/fisiologia , Linhagem Celular Tumoral , Proteínas Correpressoras/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
BACKGROUND/AIM: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for self-replication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd. MATERIALS AND METHODS: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds. RESULTS: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds. CONCLUSION: These results provide important clues for the development of safe and efficient OAds.
Assuntos
Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/genética , Deleção de Sequência , Survivina/genética , Telomerase/genética , Adenoviridae/genética , Apoptose , Linhagem Celular Tumoral , Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Regiões Promotoras Genéticas , Replicação ViralRESUMO
Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have a deletion of early region 1 (E1), which is complemented by the cell line. Most commonly, these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell's genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the growth defect observed with dl312 and resulted in higher virus growth.IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans Here, we show that even complete genetic complementation of a viral gene does not result in full protein complementation, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.
Assuntos
Proteínas E1A de Adenovirus/genética , Adenovírus Humanos/genética , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/crescimento & desenvolvimento , Regulação Viral da Expressão Gênica , Teste de Complementação Genética , Células HEK293 , Humanos , Mutação , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , RNA Mensageiro/genética , Compartimentos de Replicação Viral/metabolismo , Replicação ViralRESUMO
Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteína de Ligação a CREB/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/genética , Proteína de Ligação a CREB/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Ligação Proteica , Domínios ProteicosRESUMO
Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor )-controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor -AIM2 adenoviruses (Ad-CAIXpromotor -AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad-CAIXpromotor , Ad-CAIXpromotor -AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786-O cells or OSRC-2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC-2-xenograft mice. The tumour growth was remarkably inhibited in Ad-CAIXpromotor -AIM2-treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD-CMK resulted in the reduction of anti-tumour activity by Ad-CAIXpromotor -AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad-CAIXpromotor -AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour-specific Ad-CAIXpromotor -AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2-based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.
Assuntos
Adenovírus Humanos/fisiologia , Anidrase Carbônica IX/genética , Carcinoma de Células Renais/terapia , Proteínas de Ligação a DNA/fisiologia , Neoplasias Renais/terapia , Terapia Viral Oncolítica , Regiões Promotoras Genéticas/genética , Proteínas E1A de Adenovirus/biossíntese , Proteínas E1A de Adenovirus/genética , Adenovírus Humanos/genética , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Carcinoma de Células Renais/secundário , Caspase 1/análise , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/análise , Proteínas de Ligação a DNA/genética , Genes Sintéticos , Humanos , Inflamassomos/efeitos dos fármacos , Neoplasias Renais/patologia , Túbulos Renais/citologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaio Tumoral de Célula-Tronco , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 50 kDa N-terminal product of the cellular transcription factor E4F1 (p50E4F1) mediates E1A289R trans-activation of the adenovirus E4 gene, and suppresses E1A-mediated transformation by sensitizing cells to cell death. This report shows that while both E1A289R and E1A243R stimulate p50E4F1 DNA binding activity, E1A289R trans-activation, as measured using GAL-p50E4F1 fusion proteins, involves a p50E4F1 transcription regulatory (TR) region that must be promoter-bound and is dependent upon E1A CR3, CR1 and N-terminal domains. Trans-activation is promoter-specific, as GAL-p50E4F1 did not stimulate commonly used artificial promoters and was strongly repressive when competing against GAL-VP16. p50E4F1 and E1A289R stably associate in vivo using the p50E4F1 TR region and E1A CR3, although their association in vitro is indirect and paradoxically disrupted by MAP kinase phosphorylation of E1A289R, which stimulates E4 trans-activation in vivo. Multiple cellular proteins, including TBP, bind the p50E4F1 TR region in vitro. The mechanistic implications for p50E4F1 function are discussed.
Assuntos
Proteínas E1A de Adenovirus/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Fosforilação , Domínios Proteicos , Proteínas Repressoras/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genéticaRESUMO
Viruses alter a multitude of host-cell processes to create a more optimal environment for viral replication. This includes altering metabolism to provide adequate substrates and energy required for replication. Typically, viral infections induce a metabolic phenotype resembling the Warburg effect, with an upregulation of glycolysis and a concurrent decrease in cellular respiration. Human adenovirus (HAdV) has been observed to induce the Warburg effect, which can be partially attributed to the adenovirus protein early region 4, open reading frame 1 (E4orf1). E4orf1 regulates a multitude of host-cell processes to benefit viral replication and can influence cellular metabolism through the transcription factor avian myelocytomatosis viral oncogene homolog (MYC). However, E4orf1 does not explain the full extent of Warburg-like HAdV metabolic reprogramming, especially the accompanying decrease in cellular respiration. The HAdV protein early region 1A (E1A) also modulates the function of the infected cell to promote viral replication. E1A can interact with a wide variety of host-cell proteins, some of which have been shown to interact with metabolic enzymes independently of an interaction with E1A. To determine if the HAdV E1A proteins are responsible for reprogramming cell metabolism, we measured the extracellular acidification rate and oxygen consumption rate of A549 human lung epithelial cells with constitutive endogenous expression of either of the two major E1A isoforms. This was followed by the characterization of transcript levels for genes involved in glycolysis and cellular respiration, and related metabolic pathways. Cells expressing the 13S encoded E1A isoform had drastically increased baseline glycolysis and lower maximal cellular respiration than cells expressing the 12S encoded E1A isoform. Cells expressing the 13S encoded E1A isoform exhibited upregulated expression of glycolysis genes and downregulated expression of cellular respiration genes. However, tricarboxylic acid cycle genes were upregulated, resembling anaplerotic metabolism employed by certain cancers. Upregulation of glycolysis and tricarboxylic acid cycle genes was also apparent in IMR-90 human primary lung fibroblast cells infected with a HAdV-5 mutant virus that expressed the 13S, but not the 12S encoded E1A isoform. In conclusion, it appears that the two major isoforms of E1A differentially influence cellular glycolysis and oxidative phosphorylation and this is at least partially due to the altered regulation of mRNA expression for the genes in these pathways.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/metabolismo , Células Epiteliais/virologia , Pulmão/virologia , Células A549 , Proteínas E1A de Adenovirus/genética , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Células Epiteliais/metabolismo , Glicólise , Humanos , Pulmão/metabolismo , Fosforilação Oxidativa , Oxigênio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/patogenicidade , Proteínas E3 de Adenovirus/metabolismo , Endorribonucleases/metabolismo , Regulação Viral da Expressão Gênica/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/genética , Doença Crônica , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Hospedeiro Imunocomprometido , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Latência Viral , Liberação de Vírus/genética , Proteína 1 de Ligação a X-Box/genéticaRESUMO
OBJECTIVE: The study was undertaken to investigate the effects of polyethyleneimine (PEI)-mediated adenovirus 5 early region 1A (E1A) on radiosensitivity of human hepatic carcinoma cell in vitro and to disclosure the underlying mechanism. MATERIALS AND METHODS: Human hepatic carcinoma SMMC-7721 cell line was transfected with E1A gene using PEI vector. Untransfected cells (SMMC-7721 group), cells transfected with blank-vector (SMMC-7721-vect group), and cells transfected with E1A gene (SMMC-7721-E1A group) were treated with 6 MV X-ray irradiation at doses of 0, 1, 2, 4, 8 and Gy, respectively. Radiosensitivity was determined by MTT assay and quantified by calculating the cell survival rate. Cell-cycle distribution and apotosis rate were monitored by flow cytometry. RESULTS: The survival rate of SMMC-7721-E1A was significantly lower than that of SMMC-7721 cell. Apoptosis rate of SMMC-7721-E1A group was significantly higher than that of SMMC-7721group (P<0.01).The ratio of S stage in cell cycle of SMMC-7721-E1A was significantly lower than that in SMMC-7721 cell. The ratio of G2/M stage in cell cycle of SMMC-7721-E1A was significantly higher than that in SMMC-7721 cell (P<0.01). CONCLUSION: PEI could transfect E1A gene into hepatic carcinoma cells PEI-mediated E1A could effectively enhance radiosensitivity of hepatic carcinoma cells which may be related to its effects on apoptosis promoting leading to S phase suppression and G2/M phase arrest.
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Assuntos
Proteínas E1A de Adenovirus/genética , Carcinoma Hepatocelular/radioterapia , Neoplasias Hepáticas/radioterapia , Polietilenoimina/química , Tolerância a Radiação/genética , Proteínas E1A de Adenovirus/administração & dosagem , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Células Tumorais CultivadasRESUMO
Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.