RESUMO
Receptor internalization and degradation (RID), is a transmembrane protein coded within the E3 region expression cassette of adenoviruses. RID downregulates the cell surface expression of epidermal growth factor receptor (EGFR), tumor necrosis factor receptor (TNFR), and apoptosis antigen 1 (FAS), causing a reduction of the effects of their respective ligands. In addition, RID inhibits apoptosis by decreasing the secretion of TNF-related apoptosis-inducing ligand (TRAIL) by normal tissue cells. In this article, we report that RID inhibited chemokine expression in human breast cancer cell line MDA-MB-231 but showed no effect in cell line MCF7. These dissimilar results may be due to the different molecular and functional properties of both cell lines. Therefore, it is necessary to replicate this study in other breast cancer cell models.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Neoplasias da Mama/metabolismo , Proteínas de Membrana/fisiologia , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
Human adenoviruses (HAdVs) cause widespread acute and persistent infections. Infections are usually mild and controlled by humoral and cell-based immunity. Reactivation of persistently infected immune cells can lead to a life-threatening disease in immunocompromised individuals, especially children and transplant recipients. To date, no effective therapy or vaccine against HAdV disease is available to the public. HAdV-C2 and C5 are the best-studied of more than 100 HAdV types. They persist in infected cells and release their progeny by host cell lysis to neighbouring cells and fluids, a process facilitated by the adenovirus death protein (ADP). ADP consists of about 100 amino acids and harbours a single membrane-spanning domain. It undergoes post-translational processing in endoplasmic reticulum and Golgi compartments, before localizing to the inner nuclear membrane. Here, we discuss the current knowledge on how ADP induces membrane rupture. Membrane rupture is essential for both progression of disease and efficacy of therapeutic viruses in clinical applications, in particular oncolytic therapy.
Assuntos
Adenoviridae/patogenicidade , Proteínas E3 de Adenovirus/fisiologia , Infecções por Adenovirus Humanos/patologia , Interações Hospedeiro-Patógeno/fisiologia , Adenoviridae/metabolismo , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Animais , Morte Celular , Retículo Endoplasmático/metabolismo , Humanos , Proteínas Mad2/metabolismo , Vírus Oncolíticos/genéticaRESUMO
BACKGROUND: Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture. RESULTS: No differences were observed in the kinetics of infected cell death, and virus progeny release or in the plaque size and dissemination phenotypes between cells infected with HAdV-3 E3-9K mutants or the parental virus. The ectopic expression of E3-10.9K orthologs with a hydrophobic domain did not compromise cell viability. CONCLUSIONS: Our data show that despite the remarkable similarities with HAdV-C E3-11.6K, HAdV-B1 ORF E3-10.9K does not encode a product with a "death-like" biological activity.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Proteínas E3 de Adenovirus/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Humanos , Mutação , Fases de Leitura AbertaRESUMO
BACKGROUND: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery. RESULTS: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors. CONCLUSIONS: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.
Assuntos
Adenovírus Humanos/fisiologia , Vetores Genéticos , Transcrição Gênica , Replicação Viral , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/fisiologia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/genética , Deleção de Genes , Terapia Genética/métodos , Genoma Viral , Humanos , Viabilidade Microbiana , Montagem de VírusRESUMO
Host-pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-alpha that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-alpha mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-alpha rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-alpha induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-alpha regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-alpha also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Colesterol/metabolismo , Proteínas de Membrana/fisiologia , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas E3 de Adenovirus/metabolismo , Animais , Autofagia/fisiologia , Células CHO , Linhagem Celular , Colesterol/fisiologia , Cricetinae , Cricetulus , Endocitose/fisiologia , Homeostase/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Doença de Niemann-Pick Tipo C/enzimologia , Doença de Niemann-Pick Tipo C/virologia , Ácido Palmítico/metabolismo , Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositol 3-Quinases/fisiologiaRESUMO
Virotherapy employing conditionally replicative adenovirus (CRAd) represents a novel targeted strategy for the hepatocellular carcinoma (HCC) treatment. In this study, we explored the potential influence of E3 region, which encodes several TRAIL-inhibiting proteins (E3-6.7K, E3-10.4K/14.5K and E3-14.7K), on CRAd mediated cytotoxicity to HCC cells. Two E1B-55 kDa-deleted CRAds containing E3 region (Ad.DeltaE1B) or no E3 region (Ad.DeltaE1B.DeltaE3) were fabricated. Ad.DeltaE1B.DeltaE3 exhibited higher cytocidal potency than Ad.DeltaE1B in all tested HCC cells (Hep3B, BEL-7404, BEL-7402, HuH7, PLC/PRF/5 and HepG2), suggesting that Ad.DeltaE1B.DeltaE3 mediated cytotoxicity was partly attributed to the absence of E3 region encoding TRAIL-inhibiting proteins. In representative Hep3B cells, Ad.DeltaE1B.DeltaE3 led to more drop of mitochondrial membrane potential (MMP) and much lower ATP level than Ad.DeltaE1B. Moreover, Ad.DeltaE1B.DeltaE3 induced early apoptotic cells and the late apoptotic/necrotic cells for three and four times more than those infected by Ad.DeltaE1B. The cytotoxicity to all TRAIL endogenously expressing HCC cells and MMP drop of Hep3B cells induced by Ad.DeltaE1B.DeltaE3 but not Ad.DeltaE1B could be significantly inhibited by z-vad-fmk, a pan caspase inhibitor, suggesting that the endogenous TRAIL-mediated apoptotic pathway may be implicated in the cytocidal potency of Ad.DeltaE1B.DeltaE3 on HCC cells although other unknown mechanisms may be also involved. Our findings provided the first evidence that CRAd without E3 region might be a smart choice for the virotherapy of HCC.
Assuntos
Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/fisiologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Terapia Viral Oncolítica/métodos , Trifosfato de Adenosina/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/virologia , Potencial da Membrana Mitocondrial/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.
Assuntos
Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Terapia Genética , Vetores Genéticos , Interferon-alfa/genética , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/biossíntese , Proteínas E3 de Adenovirus/fisiologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Interferon-alfa/biossíntese , Interferon-alfa/fisiologia , Camundongos , Mutação , Neoplasias/genética , Neoplasias/patologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Tetraciclina/farmacologiaRESUMO
The receptor internalization and degradation (RID) complex of adenovirus plays an important role in modulating the immune response by downregulating the surface levels of tumour necrosis factor receptor 1 (TNFR1), thereby inhibiting NF-kappaB activation. Total cellular content of TNFR1 is also reduced in the presence of RID, which can be inhibited by treatment with lysosomotropic agents. In this report, surface biotinylation experiments revealed that, although RID and TNFR1 were able to form a complex on the cell surface, the rate of TNFR1 endocytosis was not affected by RID. However, the degradation of internalized TNFR1 was enhanced significantly in the presence of RID. Therefore, these data suggest that RID downregulates TNFR1 levels by altering the fate of internalized TNFR1 that becomes associated with RID at the plasma membrane, probably by promoting its sorting into endosomal/lysosomal degradation compartments.
Assuntos
Adenoviridae/metabolismo , Complexos Multiproteicos/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Virais/fisiologia , Proteínas E3 de Adenovirus/fisiologia , Linhagem Celular , Endocitose , Humanos , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Chamariz do Fator de Necrose Tumoral , Regulação para CimaRESUMO
The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Genes Virais , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/fisiologia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Substituição de Aminoácidos , Linhagem Celular , DNA Viral/química , DNA Viral/genética , Humanos , Mutagênese , Mutação Puntual , Raios Ultravioleta , Ensaio de Placa Viral , Proteínas Virais/análiseRESUMO
Replication-competent adenovirus-mediated suicide gene therapy has proven to be safe in humans when delivered intraprostatically. Although signs of efficacy are emerging, it is likely that further improvements will be needed before this technology will have widespread applicability in the clinic. Toward this end, we have developed a second-generation, replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-ADP) containing an improved yeast cytosine deaminase (yCD)/mutant(SR39) herpes simplex virus thymidine kinase fusion (yCD/mutTK(SR39)) gene and the adenovirus death protein (ADP) gene. Relative to the first-generation Ad5-CD/TKrep adenovirus, Ad5-yCD/mutTK(SR39)rep-ADP demonstrated greater tumor cell kill in vitro and significantly greater tumor control in preclinical models of human cancer. Quantification of transgene volume following direct injection of fadenovirus into human tumor xenografts and the naïve canine prostate demonstrated that ADP enhanced adenoviral spread in vivo. Toxicology studies were performed to determine whether the improved yCD/mutTK(SR39) fusion and ADP genes increased toxicity. Intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-ADP did not result in significantly increased toxicity relative to the parental Ad5-CD/TKrep adenovirus, the latter of which has proven to be safe in two Phase I prostate cancer clinical trials. Together, these results provide the scientific basis for evaluating the safety and efficacy of the second-generation Ad5-yCD/mutTK(SR39)rep-ADP adenovirus in humans.
Assuntos
Proteínas E3 de Adenovirus/genética , Adenovírus Humanos/genética , Genes Transgênicos Suicidas/fisiologia , Vetores Genéticos/toxicidade , Neoplasias Experimentais/terapia , Neoplasias Experimentais/virologia , Vírus Oncolíticos/genética , Replicação Viral/genética , Proteínas E3 de Adenovirus/administração & dosagem , Proteínas E3 de Adenovirus/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , Cães , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/patologia , Vírus Oncolíticos/fisiologiaRESUMO
Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDalpha/beta) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDbeta tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDbeta was mutated to alanine or phenylalanine. Both RIDbeta mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDbeta mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDbeta mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDbeta and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the mu2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving mu2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Regulação para Baixo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas E3 de Adenovirus/genética , Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Correpressoras , Endossomos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Chamariz do Fator de Necrose Tumoral , Replicação ViralRESUMO
The majority of proteins encoded in the early 3 (E3) region of human subgroup C adenoviruses function to modulate the host immune response. For example, gp19K, one of these E3 proteins, prevents the major histocompatibility complex type I (MHC-I) from presenting viral antigens on the surface of the infected cell. Other E3 proteins, such as the RID and 14.7K proteins, counteract the effector phase of the cellular immune response. In order to study further the effects of these proteins, we constructed an E1-/E3- adenovirus vector, Ad/E3, that contains all the E3 genes with the exception of the cytolytic adp gene, inserted into the deleted E1 region. The transcription of the E3 genes in this vector is driven by a CMV promoter in place of the native E3 promoter. Ad/E3 expressed close to wild-type adenovirus levels of all E3 proteins, and these proteins appear to function normally in cell culture. For example, in Ad/E3-infected cells, surface expression of MHC-I was down-regulated, as was cell surface display of death receptors Fas and TRAIL Receptor 1. A human cell line of lung origin (A549), which was rapidly rejected after transplantation into C57BL/6 mice, was protected for an extended time from the host immune response after infection with an Ad/E3, and went through a number of divisions in immunocompetent mice. These latter results indicate that the E3 proteins protect cells from destruction by the immune system.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Rejeição de Enxerto , Terapia de Imunossupressão , Transplantes , Proteínas E3 de Adenovirus/genética , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Viral , Feminino , Vetores Genéticos , Humanos , Imunocompetência , Camundongos , Camundongos Endogâmicos C57BL , Transplante HeterólogoRESUMO
Adenoviruses employ multiple genes to inhibit the host antiviral responses. There is increasing evidence that these immunoregulatory genes may function either during lytic or latent infection. Adenovirus early transcription region 3 (E3) encodes at least seven proteins, five of which block the acquired or innate immune response. Previous findings from this laboratory demonstrated that the E3 proteins 10.4K and 14.5K, which form a complex in the plasma membrane, inhibit tumor necrosis factor (TNF)-induced activation of NF-kappaB and the synthesis of chemokines. To determine the mechanism of inhibition of these pathways by the adenovirus E3 10.4K/14.5K proteins, we have examined the effects of this viral complex on the inhibition of AP-1 and NF-kappaB activation by TNF and found a reduction in assembly of the TNF receptor 1 (TNFR1) signaling complex at the plasma membrane accompanied by downregulation of surface levels of TNFR1.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/análise , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Células Cultivadas , Regulação para Baixo , Humanos , Soluções Hipertônicas/farmacologia , NF-kappa B/metabolismo , Subunidades Proteicas , Fator de Transcrição AP-1/metabolismoRESUMO
Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Apoptose , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores do Ligante Indutor de Apoptose Relacionado a TNFRESUMO
We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.
Assuntos
Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/fisiologia , Neoplasias/terapia , Neoplasias/virologia , Proteínas Proto-Oncogênicas/genética , Adenoviridae/genética , Proteínas E3 de Adenovirus/biossíntese , Proteínas E3 de Adenovirus/genética , Proteínas E4 de Adenovirus/biossíntese , Proteínas E4 de Adenovirus/genética , Animais , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Endotélio Vascular/metabolismo , Endotélio Vascular/virologia , Vetores Genéticos/genética , Humanos , Camundongos , Neoplasias/genética , Plasmídeos/genética , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Replicação Viral , Proteínas Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This manuscript will contain a brief review of adenovirus (Ad) biology including its structure and the spectrum of clinical diseases that it causes. Newer findings about the interactions of Ad early region 3 (E3) proteins with host proteins and signal transduction processes, which control the inflammatory response, will be described. Some of these processes affect the strategies for using Ads as vectors for gene therapy. There are many excellent reviews of some of these aspects 1. The history of the discoveries that led to the use of Ads as vectors as well as key experiments that resulted in improvements to various aspects of these systems have also been reviewed recently 2. The use of Ad E3 immunoregulatory genes to facilitate allogeneic transplantation and prevent autoimmune diabetes will be described.
Assuntos
Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/fisiologia , Adjuvantes Imunológicos/fisiologia , Proteínas/fisiologia , Adenoviridae/classificação , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/terapia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/imunologia , Animais , Quimiocinas/fisiologia , Humanos , NF-kappa B/fisiologia , Proteínas/imunologia , Transdução de Sinais/fisiologiaRESUMO
In the evolutionary battle between viruses and their hosts, viruses have armed themselves with weapons to defeat the host's attacks on infected cells. Various proteins encoded in the adenovirus (Ad) E3 transcription unit protect cells from killing mediated by cytotoxic T cells and death-inducing cytokines such as tumor necrosis factor (TNF), Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL). The viral protein E3-gp19 K blocks MHC class-I-restricted antigen presentation, which diminishes killing by cytotoxic T cells. The receptor internalization and degradation (RID) complex (formerly E3-10.4 K/14.5 K) stimulates the clearance from the cell surface and subsequent degradation of the receptors for Fas ligand and TRAIL, thereby preventing the action of these important immune mediators. RID also downmodulates the epidermal growth factor receptor (EGFR), although what role, if any, this function has in immune regulation is uncertain. In addition, RID antagonizes TNF-mediated apoptosis and inflammation through a mechanism that does not primarily involve receptor downregulation. E3-6.7 K functions together with RID in downregulating some TRAIL receptors and may block apoptosis independently of other E3 proteins. Furthermore, E3-14.7 K functions as a general inhibitor of TNF-mediated apoptosis and blocks TRAIL-induced apoptosis. Finally, after expending great effort to maintain cell viability during the early part of the virus replication cycle, Ads lyse the cell to allow efficient virus release and dissemination. To perform this task subgroup C Ads synthesize a protein late in infection named ADP (formerly E3-11.6 K) that is required for efficient virus release. This review focuses on recent experiments aimed at discovering the mechanism of action of these critically important viral proteins.
Assuntos
Adenoviridae/patogenicidade , Proteínas E3 de Adenovirus/fisiologia , Adenoviridae/genética , Adenoviridae/fisiologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/fisiopatologia , Infecções por Adenoviridae/virologia , Proteínas E3 de Adenovirus/genética , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Vetores Genéticos , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Transcrição GênicaRESUMO
Adenoviruses contain genes that have evolved to control the host immune and inflammatory responses; however, it is not clear whether these genes function primarily to facilitate survival of the virus during acute infection or during its persistent phase. These issues have assumed greater importance as the use of adenoviruses as vectors for gene therapy has been expanded. This review will focus on the mechanism of immune evasion mediated by the proteins encoded within the early region 3 (E3) transcription region, which affect the functions of a number of cell surface receptors including Fas, intracellular cell signaling events involving NF-kappaB, and the secretion of pro-inflammatory molecules such as chemokines. The successful use of E3 genes in facilitating allogeneic transplantation and in preventing autoimmune diabetes in several transgenic mouse models will also be described.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/imunologia , Adenoviridae/imunologia , Infecções por Adenoviridae/virologia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/fisiologia , Animais , Doenças Autoimunes/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica , HumanosRESUMO
The adenovirus (Ad) early transcription unit E3 encodes immunosubversive functions. The E3 transmembrane proteins 10.4 and 14.5 form a complex that down-regulates the epidermal growth factor receptor and apoptosis receptors from the cell surface by diverting them to endosomes/lysosomes for degradation. The latter process protects infected cells from ligand-induced apoptosis. The mechanism by which 10.4-14.5 mediate re-routing remains elusive. We examined the role of putative YXX Phi and dileucine (LL) transport motifs within Ad2 10.4-14.5 for target protein modulation. By generating stable E3 transfectants expressing 10.4-14.5 proteins with alanine substitutions in these motifs, we show that 3 of the 5 motifs are essential for functional activity. Whereas tyrosine 74 in 14.5 appears to be important for efficient 10.4-14.5 interaction, the 122YXX Phi motif in 14.5 and the dileucine motif Leu 87-Leu88 in 10.4 constitute genuine transport motifs: disruption of either motif abolished binding to the cellular adaptor proteins AP-1 and AP-2, as shown by surface plasmon resonance spectroscopy, and caused missorting, dramatically altering cell surface appearance and the intracellular location of viral proteins. Fluorescence-activated cell sorter analysis and immunofluorescence data provide evidence that Tyr122 in 14.5 is essential for rapid endocytosis of the 10.4-14.5 complex, whereas the 10.4LL motif acts down-stream and protects 10.4-14.5 from extensive degradation by rerouting it into a recycling pathway. Infection of primary cells with adenoviruses carrying the relevant point mutations confirmed the crucial role of these transport motifs for down-regulation of Fas, TRAIL-R1, TRAIL-R2, and epidermal growth factor receptor. Thus, two distinct transport motifs present in two proteins synergize for efficient target removal and immune evasion.
Assuntos
Proteínas E3 de Adenovirus/fisiologia , Apoptose , Regulação para Baixo , Receptores ErbB/metabolismo , Sinais Direcionadores de Proteínas , Receptores de Superfície Celular/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Transporte Proteico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Transfecção , Receptor fas/metabolismoRESUMO
Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.