RESUMO
Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Although vaccines against IHNV and IPNV have been commercialized in many countries, the prevalence of IHNV and IPNV is still widespread in modern aquaculture. In the present study, two IHNV recombinant viruses displaying IPNV VP2 protein (rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE) were generated using the RNA polymerase â ¡ system to explore the immunogenicity of IHNV and IPNV. The recombinant IHNV viruses were stable, which was confirmed by sequencing, indirect immunofluorescence assay, western blotting, transmission electron microscopy and viral growth curve assay. IHNV and IPNV challenge showed that the recombinant viruses had high protection rates against IHNV and IPNV with approximately 65% relative percent survival rates. Rainbow trout (mean weight 20â¯g) vaccinated with these two recombinant viruses showed a high level of antibodies against IHNV and IPNV infection. Taken together, our findings demonstrate that rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE might be promising vaccine candidates against IHNV and IPNV.
Assuntos
Doenças dos Peixes/imunologia , Oncorhynchus mykiss/imunologia , Proteínas Estruturais Virais/farmacologia , Vacinas Virais/farmacologia , Animais , Infecções por Birnaviridae/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Distribuição Aleatória , Infecções por Rhabdoviridae/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infections. The effect of antibiotic treatment is not satisfactory, and there is currently no vaccine to prevent C. trachomatis infection. Our results showed that Chlamydia virus CPG1 capsid protein Vp1 treatment significantly inhibited C. trachomatis growth in cell culture, and the inclusion numbers of different C. trachomatis serotypes were decreased. In addition, we conducted a preliminary investigation of the possible mechanisms behind the Vp1 inhibition effects and the C. trachomatis molecules targeted by Vp1. Using far-western blot and GST pull-down assay, we found that purified Vp1 can bind to the C. trachomatis outer membrane protein PmpI. PmpI polyclonal antibody treatment markedly reduced the inhibitory effect of Vp1 on C. trachomatis infectivity. On the basis of these experimental results, we infer that PmpI participates in the inhibitory effect of Vp1 and may be a potential receptor of Vp1 in the outer membrane of C. trachomatis. Our research provides clues regarding the molecular mechanisms underlying the interactions between chlamydia virus and chlamydia.
Assuntos
Anticorpos Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Chlamydia trachomatis/efeitos dos fármacos , Proteínas Estruturais Virais/farmacologia , Chlamydia trachomatis/patogenicidade , Feminino , Células HeLa , HumanosRESUMO
In the past decade, studies have revealed that the phosphatase and tensin homolog (PTEN) protein, a tumor suppressor, comprises a potential biological marker and therapeutic target for esophageal squamous cell carcinoma (ESCC). As such, the delivery of the PTEN gene represents a powerful strategy for ESCC therapy. The tegument protein VP22 of herpes simplex virus type 1 (HSV-1) has been reported to act as a transporter of heterologous proteins across the host cell membrane, thereby enhancing the biological functions of these proteins. In the present study, the intercellular delivery and antitumor activity of the fusion protein PTEN-VP22 were examined in the esophageal squamous cell carcinoma cell line Eca109 both in vitro and in vivo. VP22-mediated PTEN intercellular delivery was confirmed in the Eca109 cells by western blot analysis and by quantitation of immunofluorescence. VP22 alone did not exert antiproliferative effects or induce cell cycle arrest, induction of apoptosis, blockage of the Akt and focal adhesion kinase (FAK) pathways, tumor growth inhibition, or antiangiogenic effects in Eca109 cells. However, compared with PTEN alone, PTEN-VP22 exerted significantly higher antiproliferative effects and induced cell cycle arrest at G1 stage, apoptosis and antiangiogenic effects in Eca109 cells. Together, our findings demonstrate that VP22 alone does not exert antitumor activity directly; however, this protein mediates the intercellular delivery of PTEN and thereby increases its intracellular concentration to achieve a therapeutic steady state, leading to an overall increase in the antitumor activity of PTEN. This study provides further experimental data to confirm the potential of VP22-based intercellular delivery strategies for enhancing the efficacy of gene therapy for cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Portadores de Fármacos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , PTEN Fosfo-Hidrolase/farmacologia , Proteínas Estruturais Virais/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Quinase 1 de Adesão Focal/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.
Assuntos
Receptores ErbB/metabolismo , Ranavirus/patogenicidade , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Estruturais Virais/farmacologia , Animais , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Lisossomos/metabolismo , Camundongos , Proteólise/efeitos dos fármacos , Ranavirus/genética , Ranavirus/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de Proteína , Transcrição Gênica , Proteínas Estruturais Virais/metabolismo , Vírion , Replicação ViralRESUMO
UNLABELLED: Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-ß) in DF-1 cells pretreated with IFN-α/ß. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-ß expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-ß expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE: MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-ß did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-ß expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.
Assuntos
Proteínas Aviárias/genética , RNA Helicases DEAD-box/genética , Imunidade Inata/efeitos dos fármacos , Vírus da Doença Infecciosa da Bursa/imunologia , RNA Viral/antagonistas & inibidores , Proteínas Estruturais Virais/genética , Animais , Proteínas Aviárias/imunologia , Proteínas Aviárias/farmacologia , Ligação Competitiva , Linhagem Celular , Galinhas , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/farmacologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Vírus da Doença Infecciosa da Bursa/genética , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/imunologia , Ligação Proteica , RNA de Cadeia Dupla/antagonistas & inibidores , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA Viral/genética , RNA Viral/imunologia , Transdução de Sinais , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/farmacologiaRESUMO
Immune evasion by rabies virus depends on targeting of the signal transducers and activator of transcription 1 (STAT1) and STAT2 proteins by the viral interferon antagonist P protein, but targeting of other STAT proteins has not been investigated. Here, we find that P protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human-pathogenic viruses.
Assuntos
Receptor gp130 de Citocina/metabolismo , Evasão da Resposta Imune/genética , Interferons/antagonistas & inibidores , Fosfoproteínas/farmacologia , Vírus da Raiva/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Estruturais Virais/farmacologia , Animais , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Luciferases , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Chaperonas Moleculares , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Vírus da Raiva/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB. RESULTS: Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC50) of below 12 µM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 µl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further. CONCLUSIONS: The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.
Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Glicoproteínas/farmacologia , Mardivirus/crescimento & desenvolvimento , Doença de Marek/prevenção & controle , Peptídeos/farmacologia , Proteínas Estruturais Virais/farmacologia , Animais , Produtos Biológicos/genética , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/patologia , Membrana Corioalantoide/virologia , Fibroblastos/virologia , Glicoproteínas/genética , Concentração Inibidora 50 , Mardivirus/efeitos dos fármacos , Mardivirus/genética , Doença de Marek/virologia , Testes de Sensibilidade Microbiana/métodos , Vírus da Doença de Newcastle/genética , Peptídeos/genética , Ensaio de Placa Viral , Proteínas Estruturais Virais/genéticaRESUMO
Vaccines are often inefficient in old people and old mice. Few studies have focused on testing vaccines in old populations. Here we used DNA tumor antigen vaccines against melanoma and showed that old mice were not protected. Vaccines incorporating fusions of the tumor antigen with microbial adjuvant proteins OmpA (E. Coli) or Vp22 (Herpes simplex virus-1) dramatically improved protection of old mice. The mechanisms by which these adjuvant proteins act are distinct. TLR2 was not required for either OmpA or Vp22. Antigen processing and presentation were not boosted by these fusion constructs. However, fusion constructs with Vp22 gave a strong CD4 response to B16 melanoma and the OmpA response is MHC-II dependent. Both adjuvant fusion constructs stimulated CD4 and CD8 responses otherwise diminished in old mice.
Assuntos
Antígenos de Neoplasias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Anticâncer/imunologia , Melanoma/prevenção & controle , Proteínas Recombinantes de Fusão/farmacologia , Vacinas de DNA/imunologia , Proteínas Estruturais Virais/farmacologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Neoplasias/genética , Proteínas da Membrana Bacteriana Externa/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Interferon gama/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Vacinas de DNA/genética , Proteínas Estruturais Virais/genéticaRESUMO
The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-beta production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-beta promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-alpha and IL-1beta, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-alpha, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VDeltaC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VDeltaC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VDeltaC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-beta even though rPIV5VDeltaC-infected cells produced IFN-beta. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-kappaB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5.
Assuntos
Regulação da Expressão Gênica , Interleucina-6/metabolismo , Vírus da Parainfluenza 5/patogenicidade , Proteínas Estruturais Virais/farmacologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Genes Reporter , Células HeLa , Humanos , Interleucina-6/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
Activity-dependent neuroprotective protein (ADNP) is essential for brain formation. Here, we investigated the potential neuroprotective effects of recombinant ADNP under stress conditions. The human ADNP cDNA was sub-cloned into a vector that contains VP22, a Herpes virus protein that may allow penetration of fused proteins through cellular membranes. When incubated with pheochromocytoma (PC12) cells, a neuronal model, VP22-ADNP was associated with the cells after a 25-min incubation period. Pre-incubation with VP22-ADNP enriched protein fractions protected against beta amyloid peptide toxicity and oxidative stress (H2O2) in PC12 cells. VP22 by itself was devoid of protective activity. Furthermore, the pro-apoptotic protein p53 increased by 3.5-fold from control levels in the presence of H2O2, while treatment with VP22-ADNP prior to H2O2 exposure significantly reduced the p53 protein levels. ADNP expression was previously shown to oscillate as a function of the estrus cycle in the mouse arcuate nucleus, these oscillations are now correlated with increased cellular protection.
Assuntos
Estresse Oxidativo/fisiologia , Amiloide/toxicidade , Animais , Clonagem Molecular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/farmacologiaRESUMO
Human papillomaviruses (HPVs) are a causative agent of cervical cancer and are implicated in several other types of malignant disease including cancer of the vulva, oral cancer, and skin cancer. In HPV-transformed cells, expression of the viral E6 and E7 oncogenes increases cell proliferation and inhibits apoptosis. Expression of the viral E2 protein in HPV-transformed cells represses transcription of E6 and E7 and induces apoptosis and/or growth arrest. We have shown previously that herpes simplex virus type 1 (HSV-1) VP22-HPV E2 fusion proteins can traffic between cells and induce apoptosis. Here we show that replication-defective adenoviruses can be used to deliver VP22-E2 fusion proteins to target cells. We show that the use of adenoviral vectors to deliver VP22-E2 proteins leads to high levels of apoptosis. Interestingly, VP22-E2 proteins produced in adenovirus-infected cells are able to enter uninfected cells and induce apoptosis. Trafficking between cells and the induction of apoptosis in bystander cells are detectable in a three-dimensional tumor model. These results suggest that adenoviral vectors expressing VP22-E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases.
Assuntos
Proteínas de Ligação a DNA/farmacologia , Terapia Genética/métodos , Vetores Genéticos/farmacologia , Proteínas Oncogênicas Virais/farmacologia , Neoplasias do Colo do Útero/terapia , Proteínas Estruturais Virais/farmacologia , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Efeito Espectador , Meios de Cultivo Condicionados/farmacologia , Proteínas de Ligação a DNA/genética , Difusão , Feminino , Vetores Genéticos/genética , Células HeLa/virologia , Papillomavirus Humano 16/patogenicidade , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias do Colo do Útero/genética , Proteínas Estruturais Virais/genética , Replicação Viral/genéticaRESUMO
BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.
Assuntos
Ganciclovir/farmacologia , Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Pró-Fármacos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Timidina Quinase/genética , Proteínas Estruturais Virais/farmacologia , Adenoviridae/genética , Animais , Células Cultivadas , Estudos de Viabilidade , Feminino , Genes Transgênicos Suicidas , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Lentivirus/genética , Camundongos , Camundongos Nus , Pró-Fármacos/farmacologia , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Transgenes , Células Tumorais Cultivadas , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
In some species DNA vaccines elicit potent humoral and cellular immune responses. However, their performance in humans and non-human primates is less impressive. There are suggestions in the literature that an increase in the intercellular distribution of protein expressed from a DNA vaccine may enhance immunogenicity. We incorporated the Herpes Simplex Virus type 1 (HSV) VP22 gene, which encodes a protein that has been described as promoting intercellular spread, into a DNA vector in which it was fused to enhanced green fluorescent protein (EGFP). Following transfection of the plasmid DNA into mammalian cells, distribution of the fusion protein VP22-EGFP was not increased compared to EGFP alone. Furthermore, we found no evidence to suggest that VP22 was capable of mediating intercellular spread. However, when these constructs were used as DNA vaccines to immunise mice, antibody levels specific to EGFP were significantly enhanced when EGFP was fused to VP22. These data suggest that amplification of the immune response may occur via mechanisms other than VP22-mediated intercellular spread of antigen.
Assuntos
Adjuvantes Imunológicos , Vacinas de DNA/imunologia , Proteínas Estruturais Virais/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Western Blotting , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/imunologia , Metanol , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/imunologia , Fixação de Tecidos , Transfecção , Proteínas Virais de Fusão/farmacologiaRESUMO
Herpesvirus tegument protein VP22 has been shown to have biotherapeutic potential in tumor gene therapy. Some studies indicate that VP22 may enhance the transfer efficiency of therapeutic proteins by delivering them to more cells while trafficking. Our previous study showed that bovine herpesvirus VP22 (BVP22) enhanced equine herpesvirus thymidine kinase-ganciclovir (Etk-GCV) suicide gene therapy by an unknown intracellular effect. In this study, the interaction between BVP22 and host tumor cells was studied in neuroblastoma NXS2 cells. Cell cycle analysis was performed to determine whether BVP22 possesses biotherapeutic potential by altering the cell cycle, making cells more sensitive to therapeutic genes. As a result, the cell cycle was not affected by the transfection of BVP22 into NXS2 cells. However, cytotoxicity induced by BVP22 was observed in NXS2 cells on the second and third days after transient transfection. Further, analyses of caspase-3 activity and apoptosis suggested that BVP22 induces apoptosis in host tumor cells by upregulating the expression ratio of Bax to Bcl-2.
Assuntos
Apoptose , Ciclo Celular , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Estruturais Virais/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Bovinos , Humanos , Camundongos , Neuroblastoma/patologia , Fosfoproteínas/farmacologia , Deleção de Sequência , Transfecção , Células Tumorais Cultivadas , Regulação para Cima , Proteínas Estruturais Virais/genética , Proteína X Associada a bcl-2RESUMO
A specific interaction of ASFV p54 protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural protein p54 in apoptosis, transient expression experiments of p54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, p54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of p54. This is the first reported ASFV protein which induces apoptosis.
Assuntos
Vírus da Febre Suína Africana , Apoptose/efeitos dos fármacos , Proteínas Virais/farmacologia , Proteínas Estruturais Virais/farmacologia , Vírus da Febre Suína Africana/genética , Animais , Sequência de Bases , Caspases/metabolismo , Chlorocebus aethiops , Primers do DNA , Ativação Enzimática , Genes Virais , Mitocôndrias/virologia , Reação em Cadeia da Polimerase , Deleção de Sequência , Transfecção , Células Vero , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
BACKGROUND: Both viral and non-viral gene transfer vectors transduce vascular endothelial cells (EC) with low efficiency compared with other cell types such as hepatocytes. Generation of EC-selective vectors would enhance the clinical utility of gene therapy for diverse vascular-targeted applications. METHODS: 12mer peptides derived by in vitro phage display with EC binding specificity [MTPFPTSNEANL (MTP) and MSLTTPPAVARP (MSL)] were inserted at position T542 in the exposed HI loop of the adenovirus (Ad) serotype 5 fiber using overlapping oligonucleotides; in combination with a double point mutation (KO1) to ablate virus : cell binding via the coxsackie-adenovirus receptor (CAR). The resulting modified viruses were tested in vitro and in vivo for their ability to direct endothelial-specific gene transfer. RESULTS: Peptide insertion was not deleterious to fiber trimerisation or virion maturation. In vitro gene transfer studies using a panel of cell types demonstrated that both peptide-targeted Ad vectors mediated efficient CAR-independent gene transfer to vascular EC compared with non-modified Ads. Neither peptide supported gene delivery to non-EC. Upon systemic injection into mice and subsequent evaluation of transgene expression we failed to observe a reduction in hepatic Ad accumulation but observed a significant elevation in beta-galactosidase in blood vessels with the MSLTTPPAVARP-targeted Ad vector. CONCLUSIONS: We have genetically engineered two novel Ads that transduce human EC selectively in vitro, one of which leads to altered Ad biodistribution in vivo. The successful generation of genetically engineered tropism for EC has broad implications for cardiovascular gene therapy. Further modifications to the Ad capsid will be required to improve in vivo biodistribution profiles.
Assuntos
Adenoviridae/genética , Células Endoteliais/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Anticorpos/farmacologia , Ligação Competitiva , Linhagem Celular , Clonagem Molecular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Células Endoteliais/virologia , Vetores Genéticos/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Oligopeptídeos/genética , Oligopeptídeos/isolamento & purificação , Biblioteca de Peptídeos , Receptores Virais/metabolismo , Transdução Genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Proteínas Estruturais Virais/farmacologiaRESUMO
Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA-protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double-stranded DNA genome of bacteriophage phi29. Necessary for this stretching is a protein covalently bound at both 5' termini of phi29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from approximately 50% overstretched to approximately 50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some phi29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single-molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.
Assuntos
Fagos Bacilares/metabolismo , DNA Viral/química , DNA Viral/ultraestrutura , Proteínas de Ligação a DNA/farmacologia , Proteínas Estruturais Virais/farmacologia , Fagos Bacilares/genética , DNA Concatenado/química , DNA Concatenado/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Microscopia de Fluorescência , Proteínas Estruturais Virais/metabolismoRESUMO
The capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells could improve gene therapy approaches, especially in cancer therapy. However, there are conflicting data about VP22-mediated intercellular trafficking in different studies. To assess the role of VP22 in gene therapy of hepatocellular carcinomas (HCCs) we constructed expression vectors for N- and C-terminal versions of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells in vitro by fluorescence microscopy, but reporter gene transactivation assays demonstrated intercellular trafficking of functional VP22-p53 in vitro. For in vivo experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the in vitro experiments intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments. Because spread of p53-VP22 in liver tumors was correlated with enhanced apoptosis of hepatoma cells VP22-mediated trafficking of potential therapeutic proteins may improve the results of gene therapy of HCCs.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Estruturais Virais/farmacologia , Adenoviridae/genética , Animais , Apoptose , Células COS , Carcinoma Hepatocelular/tratamento farmacológico , Genes Reporter , Genes p53 , Terapia Genética/métodos , Proteínas de Fluorescência Verde , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Plasmídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
It has been reported that interferon (IFN)-alpha/gamma signal transduction pathway is blocked in several cell lines persistently infected with mumps virus (MV) through decrease of STAT-1alpha. Expression of the MV structural V protein (MV-V) or C terminal CYS-RICH region of the V protein (MV-Vsp) inhibited the establishment of the antivirus state induced by IFN, but not by expression of the MV-P protein. Suppression of IFN-induced STAT-1alpha, STAT-2, and IRF-9 (p48) induction was also recognized in the cells transfected with expression vector of the MV-V (pTM-V) or MV-Vsp (pTM-Vsp) protein, even though it was in the absence of the other virus protein. It is supposed that the cysteine-rich domain of V protein (Vsp) is involved in the suppression of the IFN signal transduction pathway.
Assuntos
Interferon-alfa/antagonistas & inibidores , Interferon gama/antagonistas & inibidores , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/virologia , Proteínas de Ligação a DNA/metabolismo , Fator Gênico 3 Estimulado por Interferon , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon gama/metabolismo , Interferon gama/farmacologia , Vírus da Caxumba/fisiologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição STAT2 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transfecção , Células Vero , Ensaio de Placa Viral , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/farmacologiaRESUMO
To develop an animal model of hepatitis C virus (HCV) infection, transgenic mice carrying part of the HCV cDNA (C980) encoding HCV-core and envelope proteins under control of the mouse class I major histocompatibility complex gene (H-2K) regulatory region were produced. HCV-C980 RNA and HCV-core protein were present in livers from line H36 as determined by RNase protection assay and immunostaining, respectively. More than 40 animals from line H36 were examined histologically. Most of these H36 mice after 10 months of age developed spontaneous focal infiltration of lymphocytes, hepatocyte necrosis, degeneration, and altered foci with mitotic hepatocytes. These pathological lesions were absent in livers from the age-matched control littermates. Liver cells from these H36 mice were sensitive to damage induced by intravenous administration of an anti-Fas antibody. It is suggested that HCV-C980 proteins by themselves may be one causative agent of liver cell injury in subjects with HCV infection.