title: Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors.
abstract: 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.
authors.affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Urbaczek
authors.affiliation: Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Valdecir Farias
authors.initials: VF
authors.lastname: Ximenes
authors.affiliation: Unidade de Parasitologia Médica e Microbiologia,, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Afonso
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
authors.forename: Wesley Cardoso
authors.initials: WC
authors.lastname: Generoso
authors.affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Camila Tita
authors.initials: CT
authors.lastname: Nogueira
authors.affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Aline
authors.initials: A
authors.lastname: Tansini
authors.affiliation: Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brasil.
authors.forename: Luciana Teresa Dias
authors.initials: LT
authors.lastname: Cappelini
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
authors.forename: Wilson
authors.initials: W
authors.lastname: Malagó Júnior
authors.affiliation: Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
authors.forename: Flávio Henrique
authors.initials: FH
authors.lastname: da Silva
authors.affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Luiz Marcos
authors.initials: LM
authors.lastname: da Fonseca
authors.affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil.
authors.forename: Paulo Inácio
authors.initials: PI
authors.lastname: da Costa
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D042783:Endothelial Cells / Q000276:immunology / Q000821:virology*; D004926:Escherichia coli; D005434:Flow Cytometry; D016174:Hepacivirus / Q000276:immunology*; D006801:Humans; D008565:Membrane Proteins; D010843:Pichia; D011973:Receptors, LDL / Q000276:immunology / Q000502:physiology*; D011994:Recombinant Proteins; D060225:Tetraspanin 28 / Q000276:immunology / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000502:physiology*
chemical_list: C554798:CD81 protein, human; D008565:Membrane Proteins; D011973:Receptors, LDL; D011994:Recombinant Proteins; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins