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1.
Proc Natl Acad Sci U S A ; 120(44): e2310770120, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37883435

RESUMO

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.


Assuntos
Adenovírus Humanos , Proteínas Oncogênicas Virais , Animais , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Transformação Celular Neoplásica/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas Oncogênicas Virais/metabolismo , DNA , Mamíferos/genética
2.
Cancer Gene Ther ; 29(11): 1628-1635, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35596069

RESUMO

There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3' splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.


Assuntos
Infecções por Adenoviridae , Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Camundongos , Animais , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , Neoplasias/genética , Neoplasias/terapia , Replicação Viral/genética
3.
Viruses ; 14(3)2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35336871

RESUMO

The human adenovirus type C5 (HAdV-C5) E1B-55K protein is a multifunctional regulator of HAdV-C5 replication, participating in many processes required for maximal virus production. Its multifunctional properties are primarily regulated by post-translational modifications (PTMs). The most influential E1B-55K PTMs are phosphorylation at highly conserved serine and threonine residues at the C-terminus, and SUMO conjugation to lysines 104 (K104) and 101 (K101) situated in the N-terminal region of the protein, which have been shown to regulate each other. Reversible SUMO conjugation provides a molecular switch that controls key functions of the viral protein, including intracellular trafficking and viral immune evasion. Interestingly, SUMOylation at SUMO conjugation site (SCS) K104 is negatively regulated by another multifunctional HAdV-C5 protein, E4orf6, which is known to form a complex with E1B-55K. To further evaluate the role of E4orf6 in the regulation of SUMO conjugation to E1B-55K, we analyzed different virus mutants expressing E1B-55K proteins with amino acid exchanges in both SCS (K101 and K104) in the presence or absence of E4orf6. We could exclude phosphorylation as factor for E4orf6-mediated reduction of E1B-55K SUMOylation. In fact, we demonstrate that a direct interaction between E1B-55K and E4orf6 is required to reduce E1B-55K SUMOylation. Additionally, we show that an E4orf6-mediated decrease of SUMO conjugation to K101 and K104 result in impaired co-localization of E1B-55K and SUMO in viral replication compartments. These findings indicate that E4orf6 inhibits E1B-55K SUMOylation, which could favor assembly of E4orf6-dependent E3 ubiquitin ligase complexes that are known to degrade a variety of host restriction factors by proteasomal degradation and, thereby, promote viral replication.


Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , Humanos , Sumoilação , Replicação Viral
4.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071532

RESUMO

The adenovirus type 5 (HAdV-C5) E1 transcription unit encodes regulatory proteins that are essential for viral replication and transformation. Among these, E1A and E1B-55K act as key multifunctional HAdV-C5 proteins involved in various steps of the viral replication cycle and in virus-induced cell transformation. In this context, HAdV-C5-mediated dysregulations of cellular factors such as the tumor suppressors p53 and pRB have been intensively investigated. However, cellular components of downstream events that could affect infection and viral transformation are widely unknown. We recently observed that cellular FAM111B is highly regulated in an E1A-dependent fashion. Intriguingly, previous reports suggest that FAM111B might play roles in tumorigenesis, but its exact functions are not known to date. Here, we set out to investigate the role of FAM111B in HAdV-C5 infections. We found that (i) FAM111B levels are upregulated early and downregulated late during infection, that (ii) FAM111B expression is differentially regulated, that (iii) FAM111B expression levels depend on the presence of E1B-55K and E4orf6 and that (iv) a FAM111B knockdown increases HAdV-C5 replication. Our data indicate that FAM111B acts as an anti-adenoviral host factor that is involved in host cell defense mechanisms in productive HAdV-C5 infection. Moreover, these findings suggest that FAM111B might play an important role in the host antiviral immune response that is counteracted by HAdV-C5 E1B-55K and E4orf6 oncoproteins.


Assuntos
Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Células A549 , Adenovírus Humanos/classificação , Transformação Celular Viral , Humanos , Regulação para Cima , Replicação Viral
5.
PLoS One ; 14(4): e0214882, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30943256

RESUMO

The E1B 55kDa produced by human adenovirus type 5 is a multifunctional protein that participates in the regulation of several steps during the viral replication cycle. Previous studies suggest this protein plays an important role in postranscriptional regulation of viral and cellular gene expression, as it is required for the selective accumulation of maximal levels of viral late mRNA in the cytoplasm of the infected cell; however the molecular mechanisms that are altered or regulated by this protein have not been elucidated. A ribonucleoprotein motif that could implicate the direct interaction of the protein with RNA was initially predicted and tested in vitro, but the interaction with RNA could not be detected in infected cells, suggesting the interaction may be weak or transient. Here it was determined that the E1B 55kDa interacts with RNA in the context of the viral infection in non-transformed human cells, and its contribution to the adenovirus replication cycle was evaluated. Using recombinant adenoviruses with amino acid substitutions or a deletion in the ribonucleoprotein motif the interaction of E1B 55kDa with RNA was found to correlate with timely and efficient viral DNA replication and viral late mRNA accumulation and splicing.


Assuntos
Proteínas E1B de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , RNA Viral/metabolismo , Replicação Viral/fisiologia , Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/genética , Linhagem Celular , Humanos , RNA Viral/genética , Replicação Viral/genética
6.
Cancer Biol Ther ; 19(12): 1174-1184, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067431

RESUMO

Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.


Assuntos
Adenoviridae , Vetores Genéticos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos , Replicação Viral , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Fosforilação , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Virology ; 521: 118-128, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29906705

RESUMO

Adenovirus E1B 55-kilodalton (E1B-55K) mediated DAXX degradation represents a potential mechanism by which E1B-55K sensitizes cancer cells to chemotherapy. Here we report the effects of E1B-55K-mediated DAXX degradation in chemoresistant ovarian cancer cells on response to chemotherapy. Cells with E1B-55K expression were more sensitive to cisplatin than cells without E1B-55K expression. In vivo C13* xenograft studies showed that the combination of cisplatin and E1B-55K was markedly more effective to slow tumor growth and to confer prolonged survival of tumor-bearing mice than either cisplatin or E1B-55K alone. Our studies show that DAXX plays an important role in cisplatin resistance in ovarian cancer, and strategies that promote DAXX degradation such as E1B-55K expression in combination with cisplatin can overcome drug resistance and improve responses to standard chemotherapy. These results also indicate that E1B-55K might be a novel agent for enhancing treatment responses for cisplatin-resistant ovarian cancer.


Assuntos
Proteínas E1B de Adenovirus/genética , Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Adenovírus Humanos/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas Correpressoras , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Nus , Chaperonas Moleculares , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Inclusão em Parafina
8.
J Virol ; 92(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29695423

RESUMO

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.


Assuntos
Proteínas E1B de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas E1B de Adenovirus/genética , Infecções por Adenovirus Humanos/metabolismo , Proteínas Correpressoras , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão Intranuclear , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteína SUMO-1/genética , Sumoilação , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Replicação Viral
9.
Cancer Invest ; 36(1): 19-27, 2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29388837

RESUMO

Conditionally replicative adenoviruses (CRAds) replicate poorly in murine cancer cells; however, E1b-deleted CRAds may replicate effectively in HPV16-E6/E7-positive murine cancer cells (TC-1). The HPV16 E7 open reading frame encodes functions analogous to these deleted adenovirus E1 proteins. In this study, an E1b-deleted CRAd (Adhz60) was evaluated for its ability to replicate and induce oncolysis in TC-1 cells. Adhz60-mediated oncolysis was similar in TC-1 and HeLa cells. Productive viral replication was evident based on expression of E1A and hexon, production of infectious virus progeny, and Adhz60-induced apoptosis. The results suggest that TC-1 murine cancer cells allow Adhz60 replication and oncolysis.


Assuntos
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Apoptose/genética , Papillomavirus Humano 16/genética , Replicação Viral/genética , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Repressoras/genética
10.
J Gen Virol ; 98(6): 1377-1388, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28631589

RESUMO

Adenovirus has evolved strategies to usurp host-cell factors and machinery to facilitate its life cycle, including cell entry, replication, assembly and egress. Adenovirus continues, therefore, to be an important model system for investigating fundamental cellular processes. The role of adenovirus E1B-55k in targeting host-cell proteins that possess antiviral activity for proteasomal degradation is now well established. To expand our understanding of E1B-55k in regulating the levels of host-cell proteins, we performed comparative proteome analysis of wild-type, and E1B-55k-deletion, adenovirus-infected cancer cells. As such we performed quantitative MS/MS analysis to monitor protein expression changes affected by viral E1B-55k. We identified 5937 proteins, and of these, 69 and 58 proteins were down-regulated during wild-type and E1B-55k (dl1520) adenovirus infection, respectively. This analysis revealed that there are many, previously unidentified, cellular proteins subjected to degradation by adenovirus utilizing pathways independent of E1B-55k expression. Moreover, we found that ALCAM, EPHA2 and PTPRF, three cellular proteins that function in the regulation of cell-cell contacts, appeared to be degraded by E1B-55k/E4orf3 and/or E1B-55k/E4orf6 complexes. These molecules, like integrin α3 (a known substrate of E1B-55k/E4orf6), are critical regulators of cell signalling, cell adhesion and cell surface modulation, and their degradation during infection is, potentially, pertinent to adenovirus propagation. The data presented in this study illustrate the broad nature of protein down-regulation mediated by adenovirus.


Assuntos
Infecções por Adenoviridae/patologia , Adenoviridae/crescimento & desenvolvimento , Proteínas E1B de Adenovirus/genética , Deleção de Genes , Interações Hospedeiro-Patógeno , Proteoma/análise , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Linhagem Celular Tumoral , Humanos , Proteômica , Espectrometria de Massas em Tandem , Fatores de Tempo
11.
Virology ; 504: 12-24, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28135605

RESUMO

Several of the functions of the human adenovirus type 5 E1B 55kDa protein are fulfilled via the virus-specific E3 ubiquitin ligase it forms with the viral E4 Orf6 protein and several cellular proteins. Important substrates of this enzyme have not been identified, and other functions, including repression of transcription of interferon-sensitive genes, do not require the ligase. We therefore used immunoaffinity purification and liquid chromatography-mass spectrometry of lysates of normal human cells infected in parallel with HAdV-C5 and E1B 55kDa protein-null mutant viruses to identify specifically E1B 55kDa-associated proteins. The resulting set of >90 E1B-associated proteins contained the great majority identified previously, and was enriched for those associated with the ubiquitin-proteasome system, RNA metabolism and the cell cycle. We also report very severe inhibition of viral genome replication when cells were exposed to both specific or non-specific siRNAs and interferon prior to infection.


Assuntos
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/genética , Replicação Viral/genética , Células A549 , Proteínas E1B de Adenovirus/genética , Sequência de Aminoácidos/genética , DNA Viral/biossíntese , Genoma Viral/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3
12.
J Virol ; 91(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27795433

RESUMO

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. IMPORTANCE: It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.


Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/genética , Transformação Celular Viral , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Células-Tronco Mesenquimais/virologia , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Adenovírus Humanos/crescimento & desenvolvimento , Adenovírus Humanos/metabolismo , Animais , Linhagem Celular Transformada , Proliferação de Células , Aberrações Cromossômicas , Células Epiteliais/patologia , Células Epiteliais/virologia , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cariótipo , Lentivirus/genética , Lentivirus/metabolismo , Células-Tronco Mesenquimais/patologia , Oncogenes , Cultura Primária de Células , Ratos , Transfecção
13.
Viruses ; 8(2)2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26848680

RESUMO

The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Replicação Viral , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/genética , Transformação Celular Viral , Citomegalovirus/genética , Humanos
14.
PLoS One ; 11(1): e0147173, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26799485

RESUMO

CAR is a transmembrane protein that is expressed in various epithelial and endothelial cells. CAR mediates adenoviral infection, as well as adenovirus-mediated oncolysis of AxdAdB-3, an E1A/E1B double-restricted oncolytic adenovirus, in prostate cancer cells. This study further assessed the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, in prostate cancer. Susceptibility of prostate cancer cells LNCaP, PC3, and DU145 to adenovirus infection was associated with CAR expression. All of the prostate cancer cell lines expressed integrin αvß3 and αvß5. AxdAdB-3 was more cytopathic in CAR-positive prostate cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused potent oncolysis in both CAR-positive and CAR-negative prostate cancer cells. In contrast, AxdAdB3-F/RGD was not cytopathic against normal prostate epithelial cells, RWPE-1. Intratumoral injection of AxdAdB3-F/RGD into CAR-negative prostate cancer cell xenografts in nude mice inhibited tumor growth. The current study demonstrates that E1A/E1B double-restricted oncolytic adenovirus with an RGD-fiber modification enhances infection efficiency and anti-tumor activity in CAR-deficient prostate cancer cells, while sparing normal cells. Future studies will evaluate the therapeutic potential of AxdAdB3-F/RGD in prostate cancer.


Assuntos
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Mutação , Oligopeptídeos/genética , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Humanos , Técnicas In Vitro , Masculino , Camundongos
15.
Oncogene ; 35(24): 3178-89, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-26477309

RESUMO

Human adenoviruses (HAdV) are used as a model system to investigate tumorigenic processes in mammalian cells where the viral oncoproteins E1A and E1B-55K are absolutely required for oncogenic transformation, because they simultaneously accelerate cell cycle progression and inhibit tumor suppressor proteins such as p53, although the underlying mechanism is still not understood in detail. In our present study, we provide evidence that E1B-55K binding to the PML-NB component Sp100A apparently has an essential role in regulating adenovirus-mediated transformation processes. Specifically, when this E1B-55K/Sp100A complex recruits p53, Sp100A-induced activation of p53 transcriptional activity is effectively abolished. Hence, Sp100A exhibits tumor-suppressive activity, not only by stabilizing p53 transactivation but also by depressing E1A/E1B-55K-mediated transformation. E1B-55K counteracts this suppressive activity, inducing Sp100A SUMOylation and sequestering the modified cellular factor into the insoluble matrix of the nucleus or into cytoplasmic inclusions. These observations provide novel insights into how E1B-55K modulates cellular determinants to maintain growth-promoting activity during oncogenic processes and lytic infection.


Assuntos
Proteínas E1B de Adenovirus/fisiologia , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Transformação Celular Viral/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1B de Adenovirus/genética , Transformação Celular Viral/genética , Humanos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/genética
16.
Virology ; 487: 249-59, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26561948

RESUMO

Oncolytic adenoviruses (OAds) are very promising for the treatment of lung cancer. However, OAd-based monotherapeutics have not been effective during clinical trials. Therefore, the effectiveness of virotherapy must be enhanced by combining OAds with other therapies. In this study, the therapeutic potential of OAd in combination with temozolomide (TMZ) was evaluated in lung cancer cells in vitro and in vivo. The combination of OAd and TMZ therapy synergistically enhanced cancer cell death; this enhanced cancer cell death may be explained via three related mechanisms: apoptosis, virus replication, and autophagy. Autophagy inhibition partially protected cancer cells from this combined therapy. This combination significantly suppressed the growth of subcutaneous H441 lung cancer xenograft tumors in athymic nude mice. In this study, we have provided an experimental rationale to test OAds in combination with TMZ in a lung cancer clinical trial.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Apoptose/efeitos dos fármacos , Dacarbazina/análogos & derivados , Neoplasias Pulmonares/terapia , Terapia Viral Oncolítica/métodos , Adenoviridae/fisiologia , Infecções por Adenoviridae/virologia , Proteínas E1B de Adenovirus/genética , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada/métodos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/uso terapêutico , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vírus Oncolíticos/fisiologia , Temozolomida , Proteínas Supressoras de Tumor/metabolismo , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
17.
Oncogene ; 35(1): 69-82, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-25772236

RESUMO

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.


Assuntos
Adenoviridae/genética , Transformação Celular Viral/genética , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Animais , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Isoformas de Proteínas , Ratos , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética
18.
Viruses ; 7(11): 5767-79, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26561828

RESUMO

Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.


Assuntos
Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/deficiência , Vírus Oncolíticos/fisiologia , Replicação Viral , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Deleção de Genes , Humanos , Vírus Oncolíticos/genética
19.
J Virol ; 89(20): 10260-72, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26223632

RESUMO

UNLABELLED: Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here, we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation-deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through noncovalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue ΔE1B-55K/ΔE4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3-associated E2A domains is strikingly different from that in wild-type Ad5-infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3-associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, PIAS2, or PIAS4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionarily conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication. IMPORTANCE: SUMO is a key posttranslational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection.


Assuntos
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Genoma Viral , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Osteoblastos/metabolismo , Osteoblastos/virologia , Proteínas Inibidoras de STAT Ativados/genética , Alinhamento de Sequência , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitinas/genética , Replicação Viral
20.
J Control Release ; 205: 134-43, 2015 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-25575865

RESUMO

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX.


Assuntos
Adenoviridae/patogenicidade , Neoplasias/terapia , Neovascularização Fisiológica , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/patogenicidade , Microambiente Tumoral , Adenoviridae/genética , Adenoviridae/imunologia , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Animais , Hipóxia Celular , Movimento Celular , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Células HEK293 , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Imunidade Inata , Células MCF-7 , Camundongos Nus , Mutação , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/virologia , Terapia Viral Oncolítica/efeitos adversos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Peptídeos/química , Polietilenoglicóis/química , Ratos Sprague-Dawley , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transdução Genética , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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