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1.
Mem. Inst. Oswaldo Cruz ; 110(4): 534-542, 09/06/2015. graf
Artigo em Inglês | LILACS | ID: lil-748872

RESUMO

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.


Assuntos
Animais , Bovinos , Humanos , /fisiologia , Células Endoteliais/virologia , Hepacivirus/imunologia , Receptores de LDL/fisiologia , Proteínas do Envelope Viral/fisiologia , /imunologia , Linhagem Celular , Escherichia coli , Células Endoteliais/imunologia , Citometria de Fluxo , Proteínas de Membrana , Pichia , Proteínas Recombinantes , Receptores de LDL/imunologia
2.
Mem. Inst. Oswaldo Cruz ; 109(6): 748-756, 09/09/2014. graf
Artigo em Inglês | LILACS | ID: lil-723994

RESUMO

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.


Assuntos
Humanos , Hepacivirus/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas do Envelope Viral/metabolismo , Apoptose/genética , Arginase/metabolismo , Sobrevivência Celular , Escherichia coli/metabolismo , Fibrose , Expressão Gênica/genética , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Hepacivirus/imunologia , Antígenos da Hepatite C/metabolismo , Inflamação/metabolismo , /metabolismo , Pichia/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular/metabolismo
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