The hypervariable region 1 of the E2 glycoprotein of hepatitis C virus binds to glycosaminoglycans, but this binding does not lead to infection in a pseudotype system

Abstract

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein is a 27-amino-acid sequence located at its N terminus. In this study, we investigated the functional role of HVR1 for interaction with the mammalian cell surface. The C-terminal truncated E2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein for generation of the chimeric E2-G gene construct. A deletion of the HVR1 sequence from E2 was created for the construction of E2DeltaHVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2DeltaHVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2DeltaHVR1-G had a reduced plaquing efficiency ( approximately 50%) in HepG2 cells compared to that for the E2-G virus. Cells prior treated with pronase (0.5 U/ml) displayed a complete inhibition of infectivity of the E2DeltaHVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2DeltaHVR1-G pseudotypes were not sensitive to heparin (6 to 50 micro g/ml) as an inhibitor of plaque formation compared to the E2-G pseudotype virus. Although the HVR1 sequence itself does not match with the known heparin-binding domain, a synthetic peptide representing 27 amino acids of the E2 HVR1 displayed a strong affinity for heparin in an enzyme-linked immunosorbent assay. This binding was competitively inhibited by a peptide from the V3 loop of a human immunodeficiency virus glycoprotein subunit (gp120) known to bind with cell surface heparin. Taken together, our results suggest that the HVR1 of E2 glycoprotein binds to the cell surface proteoglycans and may facilitate virus-host interaction for replication cycle of HCV.

FIG. 1.

Expression of HCV E2-G chimeric glycoprotein on BHK cell surface. Expression from E2ΔHVR1-G (A and C) and E2-G (B and D) in BHK cells (nonpermeabilized) was observed by indirect immunofluorescence with a conformation-independent MAb H52 (A and B) and a conformation-dependent MAb H53 (C and D). (E and F) Mock-transfected BHK cells were similarly used as negative controls.

FIG. 2.

Plaquing efficiency of the pseudotype virus in mammalian cells. The E2-G or E2ΔHVR1-G pseudotype virus was generated from stable transfectants of BHK cells expressing chimeric glycoprotein by infecting with VSVts045. Pseudotype virus titers were determined by plaque assay on BHK cells, and a known titer of the virus (∼60 PFU) was used for determining the plaquing efficiency in two other epithelial cell lines, HepG2 and MCF7. VSVts045 was also included in this assay to distinguish the plaquing efficiency of the pseudotype virus on specific cell types. Plaque assay was done at 32°C, and the results from three independent assays are presented with the standard deviations indicated.

FIG. 3.

Infectivity of pseudotype virus following pronase (A) and heparinase (B) treatment of HepG2 cells. The indicated doses of enzymes were used to treat HepG2 cells for 1 h at 37°C. The cells were washed extensively and incubated with a known titer (ca. 80 to 120 PFU) of E2-G, E2ΔHVR1-G, or VSVts045 for plaque assay. The results presented are the means from three independent experiments, together with the standard deviations.

FIG. 4.

Effect of heparin and suramin on the infectivity of the pseudotype virus. E2-G, E2ΔHVR1-G, or VSVts045 pseudotype virus of known titer were pretreated with various concentrations of heparin (A) or suramin (B) at 32°C for 60 min and examined for plaque formation. The results are presented as mean from three independent assays, together with standard deviations.

FIG. 5.

(A) Concentration-dependent binding of biotinylated heparin to HVR1. HVR1 peptide (10 μg/well in MES [pH 6.0]) was used to coat ELISA plates. The binding of biotinylated heparin was measured at the indicated concentration. A peptide from HIV GP 120 (aa 308 to 331) known to bind with heparin was used similarly as a positive control. Two other peptides from different regions of E2 (aa 554 to 569) and HIV GP120 (aa 474 to 497) were included as negative controls in this assay. (B) Competitive inhibition of HVR1-heparin binding in ELISA. Serially diluted unlabeled recombinant E2, GP 120 (aa 308 to 331), heparin, or HVR1 was added as a competitor to HVR1- coated wells of a microtiter plate, and biotin-conjugated heparin at a fixed concentration was added immediately into wells. The binding of biotinylated heparin was measured after incubation with a streptavidin-HRP conjugate. The results from similar analyses with negative control peptides, E2 (aa 554 to 569) and GP120 (aa 474 to 497), are also shown. (C) Competitive inhibition of HVR1-HepG2 cell binding in ELISA. Serially diluted unlabeled HVR1, HIV GP120 (aa 308 to 331), E2 (aa 554 to 569) peptide, and heparin as competitors wereadded to cells fixed on a microtiter plate. Biotin-conjugated HVR1 at a fixed concentration was immediately added into the wells. The binding of biotinylated HVR1 was measured after incubation with streptavidin-HRP conjugate. The results are presented as the means, together with the standard deviations, from three independent assays.

FIG. 5.

(A) Concentration-dependent binding of biotinylated heparin to HVR1. HVR1 peptide (10 μg/well in MES [pH 6.0]) was used to coat ELISA plates. The binding of biotinylated heparin was measured at the indicated concentration. A peptide from HIV GP 120 (aa 308 to 331) known to bind with heparin was used similarly as a positive control. Two other peptides from different regions of E2 (aa 554 to 569) and HIV GP120 (aa 474 to 497) were included as negative controls in this assay. (B) Competitive inhibition of HVR1-heparin binding in ELISA. Serially diluted unlabeled recombinant E2, GP 120 (aa 308 to 331), heparin, or HVR1 was added as a competitor to HVR1- coated wells of a microtiter plate, and biotin-conjugated heparin at a fixed concentration was added immediately into wells. The binding of biotinylated heparin was measured after incubation with a streptavidin-HRP conjugate. The results from similar analyses with negative control peptides, E2 (aa 554 to 569) and GP120 (aa 474 to 497), are also shown. (C) Competitive inhibition of HVR1-HepG2 cell binding in ELISA. Serially diluted unlabeled HVR1, HIV GP120 (aa 308 to 331), E2 (aa 554 to 569) peptide, and heparin as competitors wereadded to cells fixed on a microtiter plate. Biotin-conjugated HVR1 at a fixed concentration was immediately added into the wells. The binding of biotinylated HVR1 was measured after incubation with streptavidin-HRP conjugate. The results are presented as the means, together with the standard deviations, from three independent assays.