RESUMO
Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG-I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG-I (ΔRIG-I), MAVS, TRIF, IKKε, and TBK1-induced activation of IFN-ß promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose-dependent manner inhibited IKKε and especially TBK1-induced IRF3 phosphorylation. GST pull-down experiments with GST-NS2 and in vitro-translated and cell-expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3-5D) activated readily IFN-ß promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses.
Assuntos
Hepacivirus/patogenicidade , Interações Hospedeiro-Patógeno , Quinase I-kappa B/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Centrifugação , Citocinas/antagonistas & inibidores , Hepacivirus/imunologia , Humanos , Quinase I-kappa B/imunologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
BACKGROUND: The viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in Sf9 insect cells by using the baculovirus expression system. RESULTS: The recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3) recognised NS5B. CONCLUSION: The data shows significant variation in the specificity in humoral immunity in chronic HCV patients.
Assuntos
Anticorpos Antivirais/sangue , Hepatite C Crônica/imunologia , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Antígenos Virais/imunologia , Western Blotting , Hepacivirus/imunologia , HumanosRESUMO
BACKGROUND: Hepatitis C virus (HCV) encodes several proteins that interfere with the host cell antiviral response. Previously, the serine protease NS3/4A was shown to inhibit IFN-beta gene expression by blocking dsRNA-activated retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3)-mediated signaling pathways. RESULTS: In the present work, we systematically studied the effect of all HCV proteins on IFN gene expression. NS2 and NS3/4A inhibited IFN gene activation. NS3/4A inhibited the Sendai virus-induced expression of multiple IFN (IFN-alpha, IFN-beta and IFN-lambda1/IL-29) and chemokine (CCL5, CXCL8 and CXCL10) gene promoters. NS2 and NS3/4A, but not its proteolytically inactive form NS3/4A-S139A, were found to inhibit promoter activity induced by RIG-I or its adaptor protein Cardif (or IPS-1/MAVS/VISA). Both endogenous and transfected Cardif were proteolytically cleaved by NS3/4A but not by NS2 indicating different mechanisms of inhibition of host cell cytokine production by these HCV encoded proteases. Cardif also strongly colocalized with NS3/4A at the mitochondrial membrane, implicating the mitochondrial membrane as the site for proteolytic cleavage. In many experimental systems, IFN priming dramatically enhances RNA virus-induced IFN gene expression; pretreatment of HEK293 cells with IFN-alpha strongly enhanced RIG-I expression, but failed to protect Cardif from NS3/4A-mediated cleavage and failed to restore Sendai virus-induced IFN-beta gene expression. CONCLUSION: HCV NS2 and NS3/4A proteins were identified as potent inhibitors of cytokine gene expression suggesting an important role for HCV proteases in counteracting host cell antiviral response.
Assuntos
Regulação da Expressão Gênica , Hepacivirus/química , Hepacivirus/imunologia , Interferon beta/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Citocinas/genética , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Membranas Mitocondriais/química , Regiões Promotoras Genéticas , RNA Helicases/genética , RNA Helicases/imunologia , RNA Helicases/metabolismo , Receptores Imunológicos , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologia , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
Development was monitored throughout the entire life span of silver birch (Betula pendula Roth.) leaves. The focus was on senescence-related changes in photosynthesis and gene expression. The youngest fully developed leaves were compared with older senescing leaves in two silver birch lines: the wild-type line R and a late-senescing line R3.1. Line R3.1 was found among transgenic lines produced with a plasmid containing sense-RbcS and nptII under the control of the 35S CaMV promoter. Compared with the wild type, line R3.1 showed no general change in the mRNA levels of RbcS or Rubisco protein; therefore, it can be considered a line whose phenotype is due to insertional mutagenesis. Leaf senescence started earlier in line R than in line R3.1. Senescence was characterized by declining photosynthesis as indicated by decreases in chlorophyll fluorescence, the amount and activity of Rubisco, and the level of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS1) mRNA. Some well-known senescence-associated genes (SAGs) encoding cysteine proteinases (Cyp1, Cyp2) and a pathogenesis-related gene (Pr1) were associated with leaf senescence. The expression pattern of Cyp1 indicated that it could serve as a molecular marker of leaf senescence in silver birch. Several genes related to energy metabolism, antioxidants and phenylpropanoid biosynthesis showed enhanced expression during leaf senescence. A distinct pattern in transcript abundance during leaf development was revealed for some of the identified SAGs.
Assuntos
Betula/crescimento & desenvolvimento , Betula/genética , Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Proteínas de Plantas/genética , Clorofila/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Fotossíntese/fisiologia , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Fatores de TempoRESUMO
Activation of host innate antiviral responses are mediated by retinoic-acid inducible gene I (RIG-I)-like receptors, RIG-I and melanoma differentiation-associated gene 5, and TLRs 3, 7, 8 and 9, recognising different types of viral nucleic acids. The major components of the RIG-I- and TLR pathways have putatively been identified, but previously unrecognised kinases may contribute to virus infection-induced activation of the IFN response. Here, we screened a human kinase cDNA library, termed the kinome, using an IFN-λ1 promoter-driven luciferase reporter assay in HEK293 cells during Sendai virus infection. Of the 568 kinases analysed, nearly 50 enhanced IFN-λ1 gene expression at least twofold in response to Sendai virus infection. The best activators were FYN (FYN oncogene related to SRC, FGR, YES), serine/threonine kinase 24, activin A receptor type 1 and SRPK1 (SFRS protein kinase 1). These kinases enhanced RIG-I-dependent IFN-λ1 promoter activation via IFN-stimulated response and NF-κB elements, as confirmed using mutant IFN-λ1 promoter constructs. FYN and SRPK1 enhanced IFN-λ1 and CXCL10 protein production via the RIG-I pathway, and stimulated RIG-I and MyD88-dependent phosphorylation of IRF3 and IRF7 transcription factors, respectively. We conclude that several previously unrecognised kinases, particularly FYN and SRPK1, positively regulate IFN-λ1 and similarly regulated cytokine and chemokine genes during viral infection.
Assuntos
RNA Helicases DEAD-box/metabolismo , Interleucinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Infecções por Respirovirus/metabolismo , Vírus Sendai/imunologia , Receptores de Ativinas Tipo I/metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Biblioteca Gênica , Células HEK293 , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon , Interferons , Interleucinas/genética , Mutagênese Sítio-Dirigida , Receptores Imunológicos , Infecções por Respirovirus/imunologia , Transdução de Sinais/genética , Receptores Toll-Like/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Narcolepsy cataplexy syndrome, characterised by excessive daytime sleepiness and cataplexy, is strongly associated with a genetic marker, human leukocyte antigen (HLA) DQB1*06:02. A sudden increase in the incidence of childhood narcolepsy was observed after vaccination with AS03-adjuvanted Pandemrix influenza vaccine in Finland at the beginning of 2010. Here, we analysed whether the coinciding influenza A H1N1pdm pandemic contributed, together with the Pandemrix vaccination, to the increased incidence of childhood narcolepsy in 2010. The analysis was based on the presence or absence of antibody response against non-structural protein 1 (NS1) from H1N1pdm09 virus, which was not a component of Pandemrix vaccine. METHODS: Non-structural (NS) 1 proteins from recombinant influenza A/Udorn/72 (H3N2) and influenza A/Finland/554/09 (H1N1pdm09) viruses were purified and used in Western blot analysis to determine specific antibody responses in human sera. The sera were obtained from 45 patients who fell ill with narcolepsy after vaccination with AS03-adjuvanted Pandemrix at the end of 2009, and from controls. FINDINGS: Based on quantitative Western blot analysis, only two of the 45 (4.4%) Pandemrix-vaccinated narcoleptic patients showed specific antibody response against the NS1 protein from the H1N1pdm09 virus, indicating past infection with the H1N1pdm09 virus. Instead, paired serum samples from patients, who suffered from a laboratory confirmed H1N1pdm09 infection, showed high levels or diagnostic rises (96%) in H1N1pdm virus NS1-specific antibodies and very high cross-reactivity to H3N2 subtype influenza A virus NS1 protein. CONCLUSION: Based on our findings, it is unlikely that H1N1pdm09 virus infection contributed to a sudden increase in the incidence of childhood narcolepsy observed in Finland in 2010 after AS03-adjuvanted Pandemrix vaccination.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/uso terapêutico , Influenza Humana/complicações , Influenza Humana/imunologia , Narcolepsia/etiologia , Adolescente , Formação de Anticorpos , Criança , Pré-Escolar , Finlândia/epidemiologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Pandemias , Testes Sorológicos , Proteínas não Estruturais Virais/imunologiaRESUMO
Transgenic silver birch lines carrying extra copies of endogenous small subunit of Rubisco (RbcS)-gene under 35S CaMV promoter were used to study the carbon use efficiency of silver birch (Betula pendula Roth). A five week greenhouse experiment was carried out with four transgenic lines, R3.2, R7.2, E5 and E25, and their corresponding wild types (wt). The first fully developed leaves were used for analyses. Three of the produced lines, R3.2, E5 and E25, differed from the wt lines. Line R3.2 showed an altered growth rhythm; its chlorophyll content, Rubisco amount and activity as well as photosynthetic characteristics were reduced at the beginning of the experiment, which resulted in decreased biomass and growth. In lines E25 and E5, the biomass accumulation was shifted to roots, and in line E25, the total biomass was also reduced. In line E25, the differences were particularly marked in the dry mass, indicating a difference in water use, seen as increased transpiration. Introduction of sense RbcS decreased the Rubisco amount in birch leaves to 80% of wt at times during the tree development, but the lower amount of Rubisco was usually not seen in photosynthesis. The accumulation and distribution of biomass within the plants was altered.
Assuntos
Betula/genética , Betula/metabolismo , Fotossíntese/genética , Ribulose-Bifosfato Carboxilase/genética , Betula/crescimento & desenvolvimento , Clorofila/metabolismo , Dosagem de Genes , Genes de Plantas , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
The hepatitis C virus (HCV) non-structural (NS) 3/4A protein complex inhibits the retinoic acid inducible gene I (RIG-I) pathway by proteolytically cleaving mitochondria-associated CARD-containing adaptor protein Cardif, and this leads to reduced production of beta interferon (IFN-beta). This study examined the expression of CCL5 (regulated upon activation, normal T-cell expressed and secreted, or RANTES), CXCL8 (interleukin 8) and CXCL10 (IFN-gamma-activated protein 10, or IP-10) chemokine genes in osteosarcoma cell lines that inducibly expressed NS3/4A, NS4B, core-E1-E2-p7 and the entire HCV polyprotein. Sendai virus (SeV)-induced production of IFN-beta, CCL5, CXCL8 and CXCL10 was downregulated by the NS3/4A protein complex and by the full-length HCV polyprotein. Expression of NS3/4A and the HCV polyprotein reduced the binding of interferon regulatory factors (IRFs) 1 and 3 and, to a lesser extent, nuclear factor (NF)-kappaB (p65/p50) to their respective binding elements on the CXCL10 promoter during SeV infection. Furthermore, binding of IRF1 and IRF3 to the interferon-stimulated response element-like element, and of c-Jun and phosphorylated c-Jun to the activator protein 1 element of the CXCL8 promoter, was reduced when NS3/4A and the HCV polyprotein were expressed. In cell lines expressing NS3/4A and the HCV polyprotein, the subcellular localization of mitochondria was changed, and this was kinetically associated with the partial degradation of endogenous Cardif. These results indicate that NS3/4A alone or as part of the HCV polyprotein disturbs the expression of IRF1- and IRF3-regulated genes, as well as affecting mitogen-activated protein kinase kinase- and NF-kappaB-regulated genes.
Assuntos
Quimiocinas/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/química , Hepatite C/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Quimiocinas/genética , Regulação para Baixo , Hepacivirus/genética , Hepatite C/metabolismo , Hepatite C/virologia , Mitocôndrias/fisiologia , Poliproteínas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas não Estruturais ViraisRESUMO
BACKGROUND: Type I interferon (IFN-alpha/beta) response is one of the major host defence mechanisms against viruses. Some recent reports suggest that IFNs may interfere with the efficacy of both non-viral and virus-vector-mediated therapeutic gene transfer. METHODS: The type I IFN response upon different gene transfer methods in human tumor and primary cell lines was studied by analysing IFN-beta mRNA expression, secretion of type I IFNs and accumulation of IFN-alpha/beta-induced MxA protein (myxovirus resistance protein A). RESULTS: Infection with avirulent Semliki Forest virus A7[74] induced MxA protein accumulation and increased the IFN-beta mRNA level, whereas none of the studied virus vectors (adenovirus, CRAd, lentivirus or AAV) induced IFN response. However, plasmid DNA induced the accumulation of MxA protein when transfected with several commercial transfection reagents. RNA transfection appeared to be an efficient inducer of type I IFN response: replicating alphaviral RNA, eukaryotic total RNA, or mRNA all induced both MxA protein accumulation and IFN-beta expression. siRNA transfection failed to induce MxA response. CONCLUSIONS: The non-viral gene transfer methods have gained more interest in recent years due to their better safety profiles when compared to their viral counterparts. However, the efficiency of non-viral gene transfer is well below those reached by viral vector systems. The type I interferon response induced by non-viral methods may in part contribute to this inefficiency, while most currently used viral gene transfer vectors fail to induce or are able to suppress type I IFN response.
Assuntos
Terapia Genética/métodos , Interferon Tipo I/metabolismo , Neoplasias/imunologia , Transfecção , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Genes Virais , Vetores Genéticos , Humanos , Proteínas de Resistência a Myxovirus , Neoplasias/terapia , RNA Mensageiro/metabolismo , Vírus da Floresta de Semliki/genéticaRESUMO
Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.