RESUMO
The development of smart immune evasion mechanisms is crucial for the establishment of acute and chronic viral hepatitis. Hepatitis is a major health problem worldwide arising from different causes, such as pathogens, metabolic disorders, and xenotoxins, with the five hepatitis viruses A, B, C, D, and E (HAV, HBV, HCV, HDV, and HEV) representing the majority of the cases. Most of the hepatitis viruses are considered enveloped. Recently, it was reported that the non-enveloped HAV and HEV are, in reality, quasi-enveloped viruses exploiting exosomal-like biogenesis mechanisms for budding. Regardless, all hepatitis viruses use exosomes to egress, regulate, and eventually escape from the host immune system, revealing another key function of exosomes apart from their recognised role in intercellular communication. This review will discuss how the hepatitis viruses exploit exosome biogenesis and transport capacity to establish successful infection and spread. Then, we will outline the contribution of exosomes in viral persistence and liver disease progression.
Assuntos
Vírus de Hepatite , Hepatite Viral Humana , Comunicação Celular , Hepatite Crônica , Humanos , ImunidadeRESUMO
HERV-K HML-2 (HK2) has been proliferating in the germ line of humans at least as recently as 250,000 years ago, with some integrations that remain polymorphic in the modern human population. One of the solitary HK2 LTR polymorphic integrations lies between exons 17 and 18 of RASGRF2, a gene that affects dopaminergic activity and is thus related to addiction. Here we show that this antisense HK2 integration (namely RASGRF2-int) is found more frequently in persons who inject drugs compared with the general population. In a Greek HIV-1-positive population (n = 202), we found RASGRF2-int 2.5 times (14 versus 6%) more frequently in patients infected through i.v. drug use compared with other transmission route controls (P = 0.03). Independently, in a United Kingdom-based hepatitis C virus-positive population (n = 184), we found RASGRF2-int 3.6 times (34 versus 9.5%) more frequently in patients infected during chronic drug abuse compared with controls (P < 0.001). We then tested whether RASGRF2-int could be mechanistically responsible for this association by modulating transcription of RASGRF2 We show that the CRISPR/Cas9-mediated insertion of HK2 in HEK293 cells in the exact RASGRF2 intronic position found in the population resulted in significant transcriptional and phenotypic changes. We also explored mechanistic features of other intronic HK2 integrations and show that HK2 LTRs can be responsible for generation of cis-natural antisense transcripts, which could interfere with the transcription of nearby genes. Our findings suggest that RASGRF2-int is a strong candidate for dopaminergic manipulation, and emphasize the importance of accurate mapping of neglected HERV polymorphisms in human genomic studies.
Assuntos
Células-Tronco de Carcinoma Embrionário/metabolismo , Retrovirus Endógenos/genética , Abuso de Substâncias por Via Intravenosa/genética , Transcrição Gênica , Integração Viral/genética , Fatores ras de Troca de Nucleotídeo Guanina/genética , Estudos de Casos e Controles , Criança , Estudos de Coortes , Células-Tronco de Carcinoma Embrionário/patologia , Feminino , Genoma Humano , Humanos , Masculino , Células Tumorais CultivadasRESUMO
Host lipid metabolism reprogramming is essential for hepatitis C virus (HCV) infection and progression to severe liver disease. Direct-acting antivirals (DAAs) achieve a sustained virological response (SVR) in most patients, but virus eradication does not always protect against hepatocellular carcinoma (HCC). Angiopoietin-like protein-3 (ANGPTL-3) and angiopoietin-like protein-4 (ANGPTL-4) regulate the clearance of plasma lipids by inhibiting cellular lipase activity and possess emerging roles in tumourigenesis. We used ELISA and RT-qPCR to investigate ANGPTL-3 and ANGPTL-4 expression in HCV patients with characterised fibrosis throughout the natural history of hepatitis C and in long-term HCV infection in vitro, before and after DAA treatment. ANGPTL-3 was decreased in patients with advanced fibrosis compared to other disease stages, while ANGPTL-4 was progressively increased from acute infection to cirrhosis and HCC, peaking at the advanced fibrosis stage. Only ANGPTL-3 mRNA was down-regulated during early infection in vitro, although both ANGPTLs were increased later. DAA treatment did not alter ANGPTL-3 levels in advanced fibrosis/cirrhosis and in HCV infection in vitro, in contrast to ANGPTL-4. The association between ANGPTLs and fibrosis in HCV infection was underlined by an inverse correlation between the levels of ANGPTLs and serum transforming growth factor- ß (TGF-ß). Collectively, we demonstrate the pivotal role of advanced fibrosis in defining the expression fate of ANGPTLs in HCV infection and after treatment and propose a role for ANGPTL-3 as a contributor to post-treatment deregulation of lipid metabolism that could predispose certain individuals to HCC development.
Assuntos
Proteína 4 Semelhante a Angiopoietina/biossíntese , Proteínas Semelhantes a Angiopoietina/biossíntese , Antivirais/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/metabolismo , Hepatite C Crônica , Cirrose Hepática , Proteína 3 Semelhante a Angiopoietina , Linhagem Celular Tumoral , Feminino , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/metabolismo , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , MasculinoRESUMO
Hepatitis B virus (HBV)-specific CD8+ T cells play an important role in the clearance of HBV infection. Programmed cell death-1 (PD-1), an immunosuppressive molecule that regulates T-cell activation and peripheral immune tolerance, is increasingly shown to influence the outcome of HBV infection. rs10204525, a single-nucleotide polymorphism in the 3'-untranslated region (3'-UTR) of PD-1, has been associated with susceptibility and disease progression of chronic HBV infection in far-eastern patients. The aim of our study was to assess the impact of rs10204525 variation on HBV infection in Moroccan patients. A total of 236 patients with chronic HBV infection and 134 individuals with spontaneous HBV resolution were genotyped using a Taqman assay. In addition, PD-1 mRNA expression in peripheral blood nuclear cells was determined by quantitative reverse-transcription polymerase chain reaction. We found that the AA genotype is protective (odds ratio, 0.43; 95% confidence interval, 0.19 to 0.97; P = 0.038) against HBV infection. Interestingly, PD-1 messenger RNA (mRNA) expression analysis has revealed that chronic HBV carriers with GG and GA displayed higher levels of PD-1 mRNA compared with corresponding genotypes in resolved subjects (P = 0.031 and 0.014, respectively). Our data suggest that Mediterranean HBV-infected patients carrying PD-1 GG and GA genotypes at rs10204525 have high PD-1 mRNA expression and may be more prone to installation of chronicity.
Assuntos
Regiões 3' não Traduzidas , Predisposição Genética para Doença , Vírus da Hepatite B/imunologia , Hepatite B/genética , Hepatite B/imunologia , Polimorfismo de Nucleotídeo Único , Receptor de Morte Celular Programada 1/genética , Adulto , Idoso , Feminino , Perfilação da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , MarrocosRESUMO
Autotaxin (ATX) is a secreted lysophospholipase D that catalyzes the production of lysophosphatidic acid (LPA), a pleiotropic growth-factor-like lysophospholipid. Increased ATX expression has been detected in various chronic inflammatory disorders and different types of cancer; however, little is known about its role and mode of action in liver fibrosis and cancer. Here, increased ATX expression was detected in chronic liver disease (CLD) patients of different etiologies, associated with shorter overall survival. In mice, different hepatotoxic stimuli linked with the development of different forms of CLDs were shown to stimulate hepatocyte ATX expression, leading to increased LPA levels, activation of hepatic stellate cells (HSCs), and amplification of profibrotic signals. Hepatocyte-specific, conditional genetic deletion and/or transgenic overexpression of ATX established a liver profibrotic role for ATX/LPA, whereas pharmacological ATX inhibition studies suggested ATX as a possible therapeutic target in CLDs. In addition, hepatocyte ATX ablation and the consequent deregulation of lipid homeostasis was also shown to attenuate hepatocellular carcinoma (HCC) development, thus implicating ATX/LPA in the causative link of cirrhosis and HCC. CONCLUSION: ATX is a novel player in the pathogenesis of liver fibrosis and cancer and a promising therapeutic target. (Hepatology 2017;65:1369-1383).
Assuntos
Benzoxazóis/farmacologia , Carcinoma Hepatocelular/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Diester Fosfórico Hidrolases/genética , Piperazinas/farmacologia , Animais , Biópsia por Agulha , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Diester Fosfórico Hidrolases/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: Chronic viral hepatitis is a prevalent disease with major health implications. Its underlying pathophysiological mechanisms are not fully understood. IL-1ß and the NLRP3 inflammasome involvement has been suggested in recent years, from in vitro data and data from peripheral blood samples. Therefore, we investigated IL-1ß and the NLRP3 inflammasome in liver tissues in an effort to clarify their role in the pathophysiology of chronic viral hepatitis. METHODS: We studied liver biopsies from patients with a new diagnosis of either chronic hepatitis B (CHB) and chronic hepatitis C (CHC) or patients with chronic hepatitis B in remission (CHB-rem). The biopsies were separated in two parts. The first part was sent to histology to determine the grade of inflammation and fibrosis. From the second part, RNA was extracted and converted to cDNA used in semi-quantitative Real-Time PCR to measure the levels of IL1B, CASP1, NLRP3, ASC and IL1RA. The cell lines used in the in vitro experiments were Huh7.5, LX2 and THP-1 in variety of combinations of monocultures, co-cultures and triple cultures with one of the cell lines infected with the JFH-1 HCV clone. From the cell cultures RNA was extracted and converted to cDNA. For cell lines, we focused in the expression of IL1B and NLRP3. RESULTS: The expression of IL1B, CASP1 and NLRP3 were found significantly different between our groups (pâ¯=â¯0.001, pâ¯=â¯0.001 and pâ¯=â¯0.038, respectively). CHB patients displayed significantly higher IL1B and CASP1 mRNA levels compared to both CHB-rem and CHC patients. IL1B expression significantly correlates with liver biochemical data in CHB patients (AST: pâ¯=â¯0.006, râ¯=â¯0.457; ALT pâ¯=â¯0.002, râ¯=â¯0.497). Finally, mRNA levels of IL1B in CHB patients significantly correlate with the degree of inflammation (pâ¯=â¯0.016) but not the stage of fibrosis (pâ¯=â¯0.362). Interestingly, the relative expression of IL1B in triple culture experiments in vitro was below of 1.5-fold, suggesting no activation of IL1B. Moreover, no activation of NLRP3 was demonstrated in all investigated in vitro conditions. CONCLUSION: IL-1ß might play an important role in the pathogenesis of chronic hepatic inflammation from HBV, but not from HCV.
Assuntos
Inflamassomos/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Fígado/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Adulto , Idoso , Caspase 1/metabolismo , Linhagem Celular , Feminino , Fibrose/metabolismo , Fibrose/virologia , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Humanos , Inflamação/virologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1-JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Janus Quinase 2/metabolismo , Mecanotransdução Celular , Osteoblastos/citologia , Fator de Transcrição STAT3/metabolismo , Canais de Cátion TRPP/metabolismo , Regulação para Cima/genética , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA/metabolismo , Humanos , Modelos Biológicos , Osteoblastos/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/químicaRESUMO
Hepatitis C virus (HCV) infection is associated with hepatic iron overload and elevated serum iron that correlate to poor antiviral responses. Hepcidin (HAMP), a 25-aa cysteine-rich liver-specific peptide, controls iron homeostasis. Its expression is up-regulated in inflammation and iron excess. HCV-mediated hepcidin regulation remains controversial. Chronic HCV patients possess relatively low hepcidin levels; however, elevated HAMP mRNA has been reported in HCV core transgenic mice and HCV replicon-expressing cells. We investigated the effect of HCV core protein on HAMP gene expression and delineated the complex interplay of molecular mechanisms involved. HCV core protein up-regulated HAMP promoter activity, mRNA, and secreted protein levels. Enhanced promoter activity was abolished by co-transfections of core with HAMP promoter constructs containing mutated/deleted BMP and STAT binding sites. Dominant negative constructs, pharmacological inhibitors, and silencing experiments against STAT3 and SMAD4 confirmed the participation of both pathways in HAMP gene regulation by core protein. STAT3 and SMAD4 expression levels were found increased in the presence of HCV core, which orchestrated SMAD4 translocation into the nucleus and STAT3 phosphorylation. To further understand the mechanisms governing the core effect, the role of the JAK/STAT-activating kinase CK2 was investigated. A CK2-dominant negative construct, a CK2-specific inhibitor, and RNAi interference abrogated the core-induced increase on HAMP promoter activity, mRNA, and protein levels, while CK2 acted in synergy with core to significantly enhance HAMP gene expression. Therefore, HCV core up-regulates HAMP gene transcription via a complex signaling network that requires both SMAD/BMP and STAT3 pathways and CK2 involvement.
Assuntos
Caseína Quinase II/metabolismo , Regulação Enzimológica da Expressão Gênica , Hepacivirus/metabolismo , Hepcidinas/metabolismo , Proteínas do Core Viral/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , Células Hep G2 , Homeostase , Humanos , Ferro/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Regulação para CimaRESUMO
BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.
Assuntos
Angiopoietinas/genética , Hepacivirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , DNA/metabolismo , Regulação para Baixo , Feminino , Humanos , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Receptores Nucleares Órfãos/fisiologia , Regiões Promotoras Genéticas , Receptores X de Retinoides/fisiologiaRESUMO
Mechanical forces trigger biological responses in bone cells that ultimately control osteoblastogenesis and bone program. Although several mechanosensors have been postulated, the precise mechanotransduction pathway remains obscure as the initial mechanosensing event has not yet been identified. Studies in kidney cells have shown that polycystin-1 (PC1), via its extracellular N-terminal part, may function as an "antenna-like" protein providing a linkage between environmental cues and their conversion into biochemical responses that regulate various cellular processes via the calcineurin/NFAT pathway. Here we explored the involvement of PC1 in mechanical load (stretching)-induced signaling cascades that control osteoblastogenesis/bone formation. FACS and TransAM Transcription Factor ELISA analyses employing extracts from primary human osteoblast-like, PC1 expressing cells subjected to mechanical stretching (0-6 h) revealed that unphosphorylated/DNA-binding competent NFATc1 increased at 0.5-1 h and returned to normal at 6 h. In accordance with the activation mechanism of NFATc1, stretching of cultured cells pre-treated with cyclosporin A (CsA, a specific inhibitor of the calcineurin/NFAT pathway) abrogated the observed decrease in the abundance of the cytoplasmic pNFATc1 (phosphorylated/inactive) species. Furthermore, stretching of osteoblastic cells pre-treated with an antibody against the mechanosensing N-terminal part of PC1 also abrogated the observed decrease in the cytoplasmic levels of the inactive pNFATc1 species. Importantly, under similar conditions (pre-incubation of stretched cells with the inhibitory anti-PC1 antibody), the expression of the key osteoblastic, NFATc1-target gene runx2 decreased compared to untreated cells. Therefore, PC1 acts as chief mechanosensing molecule that modulates osteoblastic gene transcription and hence bone-cell differentiation through the calcineurin/NFAT signaling cascade.
Assuntos
Calcineurina/metabolismo , Mecanotransdução Celular/fisiologia , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/fisiologia , Canais de Cátion TRPP/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Humanos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Estimulação Física , RNA Mensageiro/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Hepatitis C virus non-enveloped particles circulate in the serum of HCV-infected patients and are believed to be involved in viral persistence. It was previously demonstrated that recombinant HCVne particles can efficiently enter T cells. In this study we investigated the effect of this entry on the phenotype and function of PBMCs, focused on the CD4+ and CD8+ T-cells. We have generated recombinant HCVne in the absence of other viral proteins. PBMCs from healthy donors were sampled after incubation either with HCVne or the control at different time points. Levels of expression of CD107a, CD25, CTLA-4, and T regulatory cells were estimated and cytokine expression and secretion were also monitored. Peripheral T cells expressed elevated CD127. The intracellular expression of the inhibitory marker CTLA-4 (CD152) increased significantly on peripheral T cells at late hours post-treatment, compared to the respective non-treated group. Despite the fact that there was an initial immune response due to HCVne uptake, T cells were driven to a partial exhausted phenotype. A significant induction of CD4+CD25+(hi)CD127-regulatory T cells at late hours was observed. Consistently, Foxp3+CD4+ T cells were also increased. In parallel, a significant transcriptional activation and increased secretion of IL-2, IL-10, and IFN-γ, was recorded. Moreover, mRNA transcription of TGF-ß was considerably elevated. HCVne particles have the potential to shape the immune response by modifying specific phenotypic and functional markers mainly on CD4+ T cells and driving them to partial exhaustion as well as to Treg expansion.
Assuntos
Capsídeo/metabolismo , Regulação da Expressão Gênica , Leucócitos Mononucleares/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/virologia , Antígeno CTLA-4/metabolismo , Citocinas/metabolismo , Hepacivirus/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Leucócitos Mononucleares/virologia , Fenótipo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.
Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/fisiologia , Lisina/metabolismo , Hepatite C/genética , Histona Desmetilases/metabolismo , Epigênese Genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.
Assuntos
Infecções por Coronavirus , Coronavirus Bovino , Coronavirus , Humanos , Bovinos , Animais , Fármacos Fotossensibilizantes/farmacologia , Cloreto de Tolônio/farmacologia , Azul de Metileno , Pandemias , Antivirais/farmacologia , MamíferosRESUMO
Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.
Assuntos
Proteínas do Capsídeo/farmacologia , Hepacivirus , Proteínas de Domínio MADS/metabolismo , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fatores de Regulação Miogênica/metabolismo , Vírion/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Células Hep G2 , Hepacivirus/fisiologia , Humanos , Proteínas de Domínio MADS/fisiologia , MAP Quinase Quinase 5/fisiologia , Fatores de Transcrição MEF2 , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Modelos Biológicos , Fatores de Regulação Miogênica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Spodoptera , Proteínas do Core Viral/farmacologia , Proteínas do Envelope Viral/farmacologia , Proteínas não Estruturais Virais/farmacologiaRESUMO
Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response.
Assuntos
Capsídeo/metabolismo , Hepacivirus/fisiologia , Interleucina-2/genética , Linfócitos T/virologia , Ativação Transcricional , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
The emerging SARS-CoV and SARS-CoV-2 belong to the family of "common cold" RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue-L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific-yet to be discovered-functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Interações entre Hospedeiro e Microrganismos , Humanos , Peptídeos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2RESUMO
Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients. The HCV core particle serves as a protective capsid shell for the viral genome and recombinant in vitro assembled HCV core particles induce strong specific immunity. We investigated the post-binding mechanism of recombinant core particle uptake and its intracellular fate. In hepatic cells, these particles are internalized, most likely in a clathrin-dependent pathway, reaching early to late endosomes and finally lysosomes. The endocytic acidic milieu is implicated in trafficking process. Using specific phosphoantibodies, signaling pathway inhibitors and chemical agents, ERK(1/2) was found to be activated in a sustained way after endocytosis, followed by downstream immediate early genes (c-fos and egr-1) modulation. We propose that the intriguing properties of cellular internalization of HCV non-enveloped particles can induce specific ERK(1/2)-MAPKs events that could be important in HCV life cycle and pathogenesis of HCV infection.
Assuntos
Endocitose , Hepacivirus/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Vírion/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular , Clatrina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Endossomos/virologia , Ativação Enzimática , Genes Precoces , Genes fos , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/virologia , Microtúbulos/fisiologia , Ativação TranscricionalRESUMO
Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease.
Assuntos
Enterócitos/patologia , Hepatite C/patologia , Hepatócitos/patologia , Homeostase , Imunidade Inata , Ferro/metabolismo , Macrófagos/patologia , Técnicas de Cocultura , Citocinas/metabolismo , Enterócitos/imunologia , Enterócitos/metabolismo , Enterócitos/virologia , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologiaRESUMO
Hepcidin, a 25-amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron-regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down-regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A-expressing stable cell line, which also exhibited down-regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well-characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A-driven reduction of hepcidin levels, we ruled out any NS5A-induced alterations in the expression of the well-known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF-1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Hepcidinas/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/isolamento & purificação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Inata/genética , Ferro/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Fator MTF-1 de TranscriçãoRESUMO
Hepatitis C virus (HCV) is the major agent causing chronic liver disease. The core gene is the most conserved sequence in the HCV genome and proved immunoreactive when expressed in bacteria and antigenic in humans. In order to test the ability of plants to express the core gene for the production of core antigen, transgenic tobacco plants carrying the core gene were generated. The core protein was stably synthesized in T(0) and T(1) generations and was found to be immunoreactive, not only with anti-core polyclonal and monoclonal antibodies, but also was able to recognize the HCV virus in infected human serum. The prospects of producing a plant based vaccine and/or a food vaccine for this important virus are discussed.