RESUMO
We report the discovery and characterization of a novel adenovirus, Zoothera dauma adenovirus (ZdAdV), from a wild bird species, Zoothera dauma (Scaly thrush). This new atadenovirus was discovered by metagenomic sequencing without virus cultivation. Analyses of the full genome sequence revealed that this new virus is a distinct member of the genus Atadenovirus and represents a novel species. ZdAdV has a genome of 34,760 bp with 28 predicted genes and 39% GC content. ZdAdV is the first atadenovirus to contain ORF19, a gene previously found only in aviadenoviruses. Phylogenetic analysis of ORF19 suggests that it was acquired by ZdAdV through horizontal gene transfer from an aviadenovirus. By analyzing all orthologous genes of aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus, we also found potential horizontal gene transfer for the E4 gene in Pigeon aviadenovirus B. Our study widens our knowledge concerning the genetic diversity and evolutionary history of atadenoviruses and their potential for cross-species transmission.
Assuntos
Infecções por Adenoviridae , Atadenovirus , Aviadenovirus , Animais , Atadenovirus/genética , Genoma Viral , Filogenia , Transferência Genética Horizontal , Adenoviridae/genética , Aviadenovirus/genética , Aves , Infecções por Adenoviridae/genéticaRESUMO
Oncolytic adenoviruses (OAds), most of which are based on species C human adenovirus serotype 5 (Ad5) (OAd5), have recently received much attention as potential anticancer agents. High seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based gene therapy. However, the impacts of anti-Ad5 neutralizing antibodies on OAd5-mediated transgene expression in the tumor and antitumor effects remain to be fully elucidated. In this study, we examined the impact of anti-Ad5 neutralizing antibodies on the OAd5-mediated antitumor effects and OAd5-mediated transgene expression. The luciferase expression of OAd-tAIB-Luc, which contains the cytomegalovirus promoter-driven luciferase gene, was inhibited in human cultured cells in the presence of human serum. Although the inhibitory effects of human serum possessing the low anti-Ad5 neutralizing antibody titers were overcome by long-term infection, the in vitro tumor cell lysis activities of OAd-tAIB-Luc were entirely attenuated by human serum containing the high titers of anti-Ad5 neutralizing antibodies. OAd-tAIB-Luc-mediated luciferase expression in the subcutaneous tumors 3 days after administration and tumor growth suppression levels following intratumoral administration were significantly lower in mice possessing the high titers of anti-Ad5 neutralizing antibodies, compared to those in control mice. These results suggested that pre-existing anti-Ad5 antibodies attenuated both transgene expression and potential antitumor effects of OAd5 following intratumoral administration.
Assuntos
Infecções por Adenoviridae , Adenovírus Humanos , Neoplasias , Humanos , Camundongos , Animais , Adenoviridae/genética , Vetores Genéticos/genética , Estudos Soroepidemiológicos , Infecções por Adenoviridae/genética , Transgenes , Adenovírus Humanos/genética , Anticorpos Antivirais , Luciferases/genética , Neoplasias/terapia , Neoplasias/genética , Anticorpos Neutralizantes/genéticaRESUMO
The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/genética , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Infecções por Adenoviridae/genética , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Poro Nuclear/metabolismo , Ligação Proteica , Proteômica , Ribonucleoproteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação , Vírion/metabolismo , Replicação Viral/fisiologiaRESUMO
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
Virus-host cell interactions include several skirmishes between the virus and its host, and the DNA damage response (DDR) network is one of their important battlegrounds. Although some aspects of the DDR are exploited by adenovirus (Ad) to improve virus replication, especially at the early phase of infection, a large body of evidence demonstrates that Ad devotes many of its proteins, including E1B-55K, E4orf3, E4orf4, E4orf6, and core protein VII, and utilizes varied mechanisms to inhibit the DDR. These findings indicate that the DDR would strongly restrict Ad replication if allowed to function efficiently. Various Ad serotypes inactivate DNA damage sensors, including the Mre11-Rad50-Nbs1 (MRN) complex, DNA-dependent protein kinase (DNA-PK), and Poly (ADP-ribose) polymerase 1 (PARP-1). As a result, these viruses inhibit signaling via DDR transducers, such as the ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, to downstream effectors. The different Ad serotypes utilize both shared and distinct mechanisms to inhibit various branches of the DDR. The aim of this review is to understand the interactions between Ad proteins and the DDR and to appreciate how these interactions contribute to viral replication.
Assuntos
Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Dano ao DNA , Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Humanos , Transdução de Sinais , Replicação ViralRESUMO
CD8 T cells contribute to effective clearance of mouse adenovirus type 1 (MAV-1) and to virus-induced pulmonary inflammation. We characterized effects of a CD8 T cell effector, TNF, on MAV-1 pathogenesis. TNF inhibited MAV-1 replication in vitro. TNF deficiency or immunoneutralization had no effect on lung viral loads or viral gene expression in mice infected intranasally with MAV-1. Absence of TNF delayed virus-induced weight loss and reduced histological evidence of pulmonary inflammation, although concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF) were not significantly affected. BALF concentrations of IL-10 were greater in TNF-deficient mice compared to controls. Our data indicate that TNF is not essential for control of viral replication in vivo, but virus-induced TNF contributes to some aspects of immunopathology and disease. Redundant CD8 T cell effectors and other aspects of immune function are sufficient for antiviral and pro-inflammatory responses to acute MAV-1 respiratory infection.
Assuntos
Infecções por Adenoviridae/virologia , Mastadenovirus/fisiologia , Pneumonia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Replicação Viral , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Mastadenovirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/genética , Pneumonia/virologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.
Assuntos
Infecções por Adenoviridae/veterinária , Doenças dos Bovinos/virologia , Nucléolo Celular/virologia , Mastadenovirus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Motivos de Aminoácidos , Animais , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Genoma Viral , Carioferinas/genética , Carioferinas/metabolismo , Mastadenovirus/química , Mastadenovirus/genética , Ligação Proteica , Transporte Proteico , Proteínas Virais/genéticaRESUMO
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
The adenovirus (Ad) genome is believed to be packaged into the virion by forming a chromatin-like structure. The replicated viral genome is likely to be condensed through binding with viral core proteins before encapsidation. Replicated viral genomes accumulate in the central region of the nucleus, which we termed virus-induced postreplication (ViPR) body. However, the molecular mechanism by which the nuclear structure is reorganized and its functional significance in virus production are currently not understood. In this study, we found that viral packaging protein IVa2, but not capsid proteins, accumulated in the ViPR body. In addition, nucleolar chromatin regulatory proteins, nucleophosmin 1 (NPM1), upstream binding factor, and nucleolin accumulated in the ViPR body in late-stage Ad infection. NPM1 depletion increased the nuclease-resistant viral genome and delayed the ViPR body formation. These results suggested that structural changes in the infected cell nucleus depend on the formation of viral chromatin by host chromatin regulatory proteins. Because NPM1 depletion decreases production of the infectious virion, we propose that host factor-mediated viral chromatin remodeling and concomitant ViPR body formation are prerequisites for efficient encapsidation of Ad chromatin.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/genética , Replicação do DNA , DNA Viral/genética , Proteínas Nucleares/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Células A549 , Infecções por Adenoviridae/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Viral/metabolismo , Genoma Viral , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Virais/genética , Montagem de VírusRESUMO
Adenoviruses (AdVs) cause respiratory, ocular, and gastrointestinal tract infection and inflammation in immunocompetent people and life-threatening disease upon immunosuppression. AdV vectors are widely used in gene therapy and vaccination. Incoming particles attach to nuclear pore complexes (NPCs) of post-mitotic cells, then rupture and deliver viral DNA (vDNA) to the nucleus or misdeliver to the cytosol. Our genome-wide RNAi screen in AdV-infected cells identified the RING-type E3 ubiquitin ligase Mind bomb 1 (Mib1) as a proviral host factor for AdV infection. Mib1 is implicated in Notch-Delta signaling, ciliary biogenesis, and RNA innate immunity. Mib1 depletion arrested incoming AdVs at NPCs. Induced expression of full-length but not ligase-defective Mib1 in knockout cells triggered vDNA uncoating from NPC-tethered virions, nuclear import, misdelivery of vDNA, and vDNA expression. Mib1 is an essential host factor for AdV uncoating in human cells, and it provides a new concept for licensing virion DNA delivery through the NPC.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/genética , Genoma Viral , Poro Nuclear/virologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Adenoviridae/imunologia , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/imunologia , DNA Viral/genética , Células HEK293 , Células HeLa , Humanos , Poro Nuclear/genética , Ligação Proteica , Interferência de RNA , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , VírionRESUMO
Last advances in the treatment of pediatric tumors has led to an increase of survival rates of children affected by primitive neuroectodermal tumors, however, still a significant amount of the patients do not overcome the disease. In addition, the survivors might suffer from severe side effects caused by the current standard treatments. Oncolytic virotherapy has emerged in the last years as a promising alternative for the treatment of solid tumors. In this work, we study the anti-tumor effect mediated by the oncolytic adenovirus VCN-01 in CNS-PNET models. VCN-01 is able to infect and replicate in PNET cell cultures, leading to a cytotoxicity and immunogenic cell death. In vivo, VCN-01 increased significantly the median survival of mice and led to long-term survivors in two orthotopic models of PNETs. In summary, these results underscore the therapeutic effect of VCN-01 for rare pediatric cancers such as PNETs, and warrants further exploration on the use of this virus to treat them.
Assuntos
Infecções por Adenoviridae/genética , Adenoviridae/genética , Tumores Neuroectodérmicos Primitivos/genética , Vírus Oncolíticos/genética , Infecções por Adenoviridae/terapia , Infecções por Adenoviridae/virologia , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Modelos Animais de Doenças , Humanos , Estimativa de Kaplan-Meier , Camundongos , Tumores Neuroectodérmicos Primitivos/terapia , Tumores Neuroectodérmicos Primitivos/virologia , Terapia Viral Oncolítica , Pediatria , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Viruses are believed to be ubiquitous; however, the diversity of viruses is largely unknown because of the bias of previous research toward pathogenic viruses. Deep sequencing is a promising and unbiased approach to detect viruses from animal-derived materials. Although cranes are known to be infected by several viruses such as influenza A viruses, previous studies targeted limited species of viruses, and thus viruses that infect cranes have not been extensively studied. In this study, we collected crane fecal samples in the Izumi plain in Japan, which is an overwintering site for cranes, and performed metagenomic shotgun sequencing analyses. We detected aviadenovirus-like sequences in the fecal samples and tentatively named the discovered virus crane-associated adenovirus 1 (CrAdV-1). We determined that our sequence accounted for approximately three-fourths of the estimated CrAdV-1 genome size (33,245 bp). The GC content of CrAdV-1 genome is 34.1%, which is considerably lower than that of other aviadenoviruses. Phylogenetic analyses revealed that CrAdV-1 clusters with members of the genus Aviadenovirus, but is distantly related to the previously identified aviadenoviruses. The protein sequence divergence between the DNA polymerase of CrAdV-1 and those of other aviadenoviruses is 45.2-46.8%. Based on these results and the species demarcation for the family Adenoviridae, we propose that CrAdV-1 be classified as a new species in the genus Aviadenovirus. Results of this study contribute to a deeper understanding of the diversity and evolution of viruses and provide additional information on viruses that infect cranes, which might lead to protection of the endangered species of cranes.
Assuntos
Infecções por Adenoviridae/genética , Aviadenovirus/genética , Doenças das Aves/genética , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/isolamento & purificação , Doenças das Aves/virologia , Aves/genética , Aves/virologia , Fezes/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Japão , FilogeniaRESUMO
Cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD) and dampens antiviral response, which increases viral infections and leads to COPD acute exacerbation (AECOPD). Adenovirus, a nonenveloped DNA virus, is linked with AECOPD, whose DNAs trigger innate immune response via interacting with pattern recognition receptors (PRRs). Stimulator of interferon genes (STING), as a cytosolic DNA sensor, participates in adenovirus-induced interferon ß (IFNß)-dependent antiviral response. STING is involved in various pulmonary diseases, but role of STING in pathogenesis of AECOPD is not well documented. In the present study, we explored relationship between STING and AECOPD induced by recombinant adenovirus vectors (rAdVs) and CS in wild type (WT) and STING-/- mice; and also characterized the inhibition of STING- IFNß pathway in pulmonary epithelium exposed to cigarette smoke extract (CSE). We found that CS or CSE exposure alone dramatically inhibited STING expression, but not significantly effected IFNß production. Moreover, CS or CSE-exposed significantly suppressed activation of STING-IFNß pathway induced by rAdVs and suppressed clearance of rAdVs DNA. Inflammation, fibrosis and emphysema of lung tissues were exaggerated when treated with CS plus rAdVs, which further deteriorate in absences of STING. In A549â¯cells with knockdown of STING, we also observed enhancing apoptosis related to emphysema, especially CSE and adenovirus vectors in combination. Therefore, STING may play a protective role in preventing the progress of COPD.
Assuntos
Infecções por Adenoviridae/genética , Vetores Genéticos/genética , Interferon beta/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Células A549 , Adenoviridae/efeitos dos fármacos , Infecções por Adenoviridae/tratamento farmacológico , Animais , Linhagem Celular , Linhagem Celular Tumoral , Vetores Genéticos/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Inflamação/tratamento farmacológico , Inflamação/genética , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/virologia , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/genética , Enfisema Pulmonar/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fumar/efeitos adversosRESUMO
Human adenovirus (HAdV) is recognized as a serious pathogen after allogeneic hematopoietic stem cell transplantation (HSCT), causing morbidity and mortality. Currently, there is no universal agreement regarding routine HAdV surveillance after HSCT. We assessed the impact of HAdV weekly monitoring by polymerase chain reaction (PCR) on HAdV viremia rates and the risk factors that influence survival. Three-hundred and fifty-six pediatric allogeneic HSCT were done between 2007 and 2015. Until July 2011, HAdV testing was performed based on clinical suspicion (cohort 1, n = 175) and from August 2011, weekly blood-HAdV monitoring was done (cohort 2, n = 181) until day +100. Twenty-three patients (4 [2.3%] from cohort 1 and 19 [10.5%] from cohort 2, p = .001) were found with HAdV viremia and seven of them died. Both cohorts had a similar incidence of HAdV-associated mortality (3/175; 1.7% in cohort 1 and 4/181; 2.2% in cohort 2). Respiratory failure was the cause of death in all patients. Clinical symptoms appeared prior to or within 5 days of HAdV detection in cohort 2. In summary, weekly monitoring was associated with higher detection of HAdV. The study could not assess survival benefit due to small numbers of HAdV-positive cases. In many instances, symptoms occurred with the development of positive HAdV blood PCR results and hence, symptomatology could have triggered the test. Future studies are needed to provide data that help establishing a uniform approach for regular monitoring of HAdV post-transplant.
Assuntos
Infecções por Adenoviridae , Adenovírus Humanos , DNA Viral , Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Infecções por Adenoviridae/sangue , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/mortalidade , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , DNA Viral/sangue , DNA Viral/genética , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Humanos , Lactente , Masculino , Fatores de Risco , Viremia/sangue , Viremia/genética , Viremia/mortalidadeRESUMO
The adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCE Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.
Assuntos
Dano ao DNA/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Virais/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/genética , Proteínas E4 de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Reparo do DNA/fisiologia , DNA Viral/genética , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Transdução de Sinais , Proteínas Virais/fisiologia , Replicação Viral/fisiologiaRESUMO
Familial hypercholesterolemia (FH) is a genetic hyperlipidemia characterized by elevated concentrations of plasma LDL cholesterol. Statins are not always effective for the treatment of FH patients; unresponsive patients have poor prognosis and rely on LDL apheresis. In the past, we developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDL receptor (LDLR) fused with a rabbit transferrin dimer (LDLR-TF). We evaluated the efficacy of the LDLR-TF chimeric protein in CHOLDLA7, a cell line lacking LDLR expression, restoring the ability to uptake LDL. Subsequently, we administered intravenously 1 × 10E13 vp/kg of this vector in LDLR-deficient mice and observed amelioration of lipid profile and reduction of aortic atherosclerosis. Finally, we studied LDL distribution after HD-Ad vector-mediated expression of LDLR-TF in LDLR-deficient mice and found LDL accumulation in liver, and in heart and intestine. These results support the possibility of lowering LDL-C levels and reducing aortic atherosclerosis using a secreted therapeutic transgene; the present strategy potentially can be modified and adapted to non-systemic gene transfer with expression of the secreted chimeric protein in muscle or other tissues. Intramuscular or local administration strategies could improve the safety profile of this strategy and facilitate applicability.
Assuntos
Terapia Genética/métodos , Receptores de LDL/genética , Transferrina/genética , Adenoviridae/genética , Infecções por Adenoviridae/genética , Animais , Aorta/patologia , Aterosclerose/genética , Linhagem Celular , LDL-Colesterol/sangue , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/fisiopatologia , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Lipídeos/sangue , Camundongos , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Transferrina/metabolismo , TransgenesRESUMO
The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection.IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.
Assuntos
Infecções por Adenoviridae/genética , Adenoviridae/patogenicidade , Proteínas Culina/genética , MicroRNAs/metabolismo , RNA Viral/genética , Células A549 , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Células HEK293 , Células HeLa , Humanos , Análise em Microsséries , Proteólise , Proteínas Proto-Oncogênicas c-fos/química , Interferência de RNA , RNA Viral/metabolismo , Replicação ViralRESUMO
Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.
Assuntos
Infecções por Adenoviridae/terapia , Adenoviridae/imunologia , Anticorpos/imunologia , Fibrossarcoma/terapia , Terapia Genética , Ribonucleoproteínas/fisiologia , Vacinação , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/imunologia , Animais , Fibrossarcoma/genética , Fibrossarcoma/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transgenes , Células Tumorais CultivadasRESUMO
Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibroblasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/metabolismo , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Aminoácidos/metabolismo , Linhagem Celular , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Cinética , Redes e Vias Metabólicas/genética , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Inclusion body hepatitis and hydropericardium-hepatitis syndrome caused by serotype 4 fowl adenovirus (FAdV-4) have emerged in China since 2013. FAdV is usually propagated in primary chicken embryonic liver cells or embryo yolk sac. The aim of this work was to develop an immortalized CEL cell line by stable expression of human mitochondrial ribosomal protein 18S-2, named CEL-hMRP18S-2â¯cells, for the propagation of FAdV-4. The maximum cell density of CEL-hMRP18S-2â¯cells could reach 2.65â¯×â¯106â¯cells/ml in four-days culture. According to the mRNA levels of cell-cycle related genes in CEL-hMRP18S-2â¯cells tested by qRT-PCR, we speculated that the transformation of hMRP18S-2 into CEL cells caused the functional inactivation of p53 and the significant down-regulation of p15INK4b might cause the hyperphosphorylated form of Rb, releasing E2F-1 factor and enhancing the E2F-dependent transcription for cell cycle progression. It was suspected that the up-regulated c-Myc mRNA level at the initial period of immortalization might prompt transformed cells through the G0-G1 checkpoint. The normal CPE was observed in CEL-hMRP18S-2â¯cells infected by FAdV-4 and microcarrier suspension culture performed for FAdV-4 propagation with 9.0 lgTCID50/ml suggested that CEL-hMRP18S-2â¯cells could be a useful continuous cell line for isolation of wild FAdV and production of FAdV-inactivated vaccine.