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.
Highlights
Hepatitis C virus (HCV) is a small, enveloped RNA virus
We aimed to study the binding interactions between recombinant envelope 2 (E2) proteins and low-density lipoprotein receptor (LDLr) and CD81, two well known binding partners of E2, in two different endothelial cell lines: human umbilical vein endothelial cells (HUVEC), which have a high concentration of LDLr but no CD81 receptors, and ECV304 cells, which are a derivative of the human urinary bladder carcinoma T24 cell line and possess a high concentration of LDLr and a low concentration of CD81
Sequence amplification and correspondence to E2 recombinant proteins - A sequence was amplified that corresponded to an E2 protein without a transmembrane domain (GenBank accession AF009606); when including restriction sites, this resulted in an 852-bp-long sequence
Summary
Hepatitis C virus (HCV) is a small, enveloped RNA virus. A single open reading frame encodes a precursor polyprotein that is cleaved via the signalling mechanisms of host and viral proteases, generating 10 different structural [core (C), envelope 1 (E1) and envelope 2 (E2)] and nonstructural 2-5 proteins, in addition to the protein P7 (Bartenschlager & Lohmann 2000, Penin et al 2004). Vascular changes in the cirrhotic livers of patients with chronic hepatitis C have attracted increasing interest because little is known about the relationship of HCV with endothelial cells As this pathology is associated with major complications and prognostic implications, the necessity of acquiring a more detailed characterisation of the interactions between HCV and cells during infection is required. We aimed to study the binding interactions between recombinant E2 proteins and LDLr and CD81, two well known binding partners of E2, in two different endothelial cell lines: HUVEC, which have a high concentration of LDLr but no CD81 receptors, and ECV304 cells, which are a derivative of the human urinary bladder carcinoma T24 cell line and possess a high concentration of LDLr and a low concentration of CD81. To investigate how human LDL and differences in protein structure (glycosylated or not) affected binding efficiency, we examined recombinant proteins that were either associated or not associated with lipoproteins
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