RESUMO
Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination.
Assuntos
Adenoviridae/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Recombinação Genética , Replicação Viral , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Linhagem Celular , Células Cultivadas , Dano ao DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Cinética , UbiquitinaçãoRESUMO
One problem encountered in the use of adenoviral vectors for gene therapy is their toxicity. Although many studies have analyzed this question in vivo, few researches have investigated adenovirus vector effects at the cellular level using a large-scale approach. In particular, no such data are available for helper-dependent adenovirus vectors (HD), which are promising adenovirus vectors for clinical applications since they are devoid of all viral genes and can host large transgene cassettes. The present study used gene chips to examine (Affymetrix HG-U95Av2 interrogating 12,626 unique human transcripts) the effect on liver cells of HD vectors versus that of DeltaE1/E3 adenovirus vector and wild type Adenovirus (Ad5). The effects of the DeltaE1/E3 adenovirus and of HD vectors were comparable, and significantly milder than that of Ad5. Interestingly the expression signatures of DeltaE1/E3 adenovirus and HD vectors were non-overlapping both at the single gene and the pathway level, suggesting specific and different interactions between the host cell and the two gene therapy vectors.
Assuntos
Adenoviridae/fisiologia , Regulação da Expressão Gênica , Vetores Genéticos , Transcrição Gênica , Adenoviridae/genética , Linhagem Celular , Deleção de Genes , Perfilação da Expressão Gênica , Genes Virais/genética , Vírus Auxiliares , Hepatócitos/virologia , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.
Assuntos
Adenocarcinoma , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias Pancreáticas , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adenoviridae/genética , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Regulação para Baixo , Genes Virais , Humanos , Irinotecano , Proteína Homóloga a MRE11/biossíntese , GencitabinaRESUMO
In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered lambda by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286-393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286-393) includes the principal receptor-binding motif ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda Pb (286-393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.
Assuntos
Bacteriófago lambda/genética , Proteínas do Capsídeo/genética , Capsídeo , Fibroblastos/metabolismo , Receptores Virais/metabolismo , Transdução Genética/métodos , Bacteriófago lambda/metabolismo , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Endossomos/metabolismo , Terapia Genética/métodos , Proteínas de Fluorescência Verde , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/genéticaRESUMO
Given the biological complexity of viral infections, the variability of the host response, and the safety concerns related to viral-mediated gene transfer, recent studies have made use of DNA mircoarrays to integrate multi-layered experimental approaches aimed at completely clarifying virus-host interactions. Particular attention has been given to those viruses that are implicated in clinical use and/or in life-threatening diseases. Examples of such use can be divided into three main categories, including: (i) the use of microarrays to study viral expression; (ii) the use of microarrays to analyze the host response to viral infection; and (iii) the use of microarrays to characterize the host response to viral vector-mediated transduction. Significant information on virus- and viral vector-host interactions can be obtained with the microarray approach, including the recognition of master pathways of virally-induced responses, the identification of new target genes for specific viruses, and indications on the molecular toxicity of specific gene transfer vectors currently used for gene therapy trials (in particular, adeno-associated viruses and adenovirus-derived vectors). We predict that the development of accessible repositories containing most of the DNA microarray data on viral infections will certainly help to elucidate the puzzling pictures of different viral infections. This will be crucially important for the correct handling of viral diseases and the intelligent amelioration of viral vectors for gene therapy.
Assuntos
Terapia Genética , Vetores Genéticos , Viroses/terapia , Vírus/genética , Animais , DNA Viral/análise , Técnicas de Transferência de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Viroses/virologiaRESUMO
Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922-947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922-947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.
Assuntos
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Antineoplásicos/uso terapêutico , Mitoxantrona/uso terapêutico , Taxoides/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Citometria de Fluxo , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitoxantrona/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxoides/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs.
Assuntos
Adenovírus Humanos/metabolismo , Regulação da Expressão Gênica , Príons/metabolismo , Replicação Viral/fisiologia , Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/genética , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Proteínas PrPSc/metabolismo , Príons/genética , Deleção de SequênciaRESUMO
In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G(1)/S and G(2)/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G(2)/M checkpoint since dlE4 Ad5-infected cells arrested in G(2) while ONYX-015-infected cells did enter mitosis.
Assuntos
Adenoviridae/patogenicidade , Interfase , Mitose , Proteína Supressora de Tumor p53/fisiologia , Replicação Viral , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Endotélio Vascular/citologia , Humanos , Proteínas Mad2 , Vírus Oncolíticos/patogenicidade , Vírus Oncolíticos/fisiologia , Poliploidia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/fisiologia , Vacinas ViraisRESUMO
Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of alpha3beta1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin alpha3beta1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-alpha3 and anti-beta1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with alpha3beta1, which involved multiple additional contact sites.