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 E2⌬HVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2⌬HVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2⌬HVR1-G had a reduced plaquing efficiency (ϳ50%) 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 E2⌬HVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2⌬HVR1-G pseudotypes were not sensitive to heparin (6 to 50 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.Hepatitis C virus (HCV) is a major causative agent of parentally transmitted hepatitis (9) and is associated with liver cirrhosis and hepatocellular carcinoma (1). Approximately 25% of infected individuals appear to clear viremia without therapeutic intervention (2, 31), and the mechanism leading to this natural resolution of HCV infection is not completely understood. However, the majority of HCV-infected individuals do not resolve infection, leading to the development of chronic hepatitis.The HCV genome is a linear, positive sense, single-stranded RNA molecule of ϳ9,500 nucleotides. It encodes a polyprotein precursor of ϳ3,000 amino acids (10). This polyprotein is cleaved by both host and viral proteases to generate several distinct polypeptides (24,29,36). The structural proteins, core, E1 and E2, of HCV physically interact and may have a role in virus assembly (4,11,36). The glycosylated polypeptides (E1 and E2) are most likely anchored onto the envelope lipid bilayer of the virus and facilitate virus entry by interaction with the cell surface. In vitro expression studies have suggested that the glycoproteins of HCV associate to form two types of complexes: (i) a heterodimer stabilized by no...

Coexpression of Hepatitis C Virus E1 and E2 Chimeric Envelope Glycoproteins Displays Separable Ligand Sensitivity and Increases Pseudotype Infectious Titer

“…Overexpression of the unmodified E1 and E2 may result in a loss of ER retention, and a portion of these recombinant viral glycoproteins were translocated onto the cell surface. However, the reported differences in susceptibilities of the cell types to MuLV or lentivirus pseudotype (5,52) and VSV pseudotype infectivity and the role of individual virus glycoproteins in pseudotype infectivity (7,32,36,37) remain unclear at this time. The cell surface expression levels of virus envelope glycoproteins significantly contribute to the pseudotype titer (11).…”

“…The pseudotype generated from the expression of both E1-G and E2-G was sensitive to the use of heparin as an inhibitor of plaque formation in a manner similar to that of the pseudotype generated with E2-G alone. The HVR1 of E2 may bind with heparan sulfate to provide additional means to facilitate virus attachment to host cells (4,7). Further examination with a negatively charged sulfated sialyl lipid, NMSO3, has suggested inhibition of VSV/HCV pseudotype virus infectivity which was significantly less than that of the parental VSVts045.…”

“…The G glycoprotein of the backbone VSVts045 used in pseudotype preparation does not appear to compromise HCV-specific infectivity, as we consistently observed a distinct functional role for HCV envelope glycoproteins located on pseudotype particles and their role in infectivity. Each of the pseudotype viruses have exhibited differential sensitivity to a number of ligands in comparison to the parental VSV (7,37). Recently, MuLV and lentivirus pseudotypes have been generated from 293T cells by expression of an unmodified HCV envelope genomic region (5,20,27).…”

“…Baby hamster kidney cells (BHK-21), human breast cancer cells (MCF-7), and human hepatoma cells (Huh-7, Hep3B, and HepG2) were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and antibiotics (100 U of penicillin and 100 g of streptomycin per ml). The plasmids expressing chimeric E1-G and E2-G from HCV genotype 1a (GenBank accession number M62321 [13]) under the control of a cytomegalovirus (CMV) or MuLV promoter have been described previously (7,32,38). BHK stable transfectants were generated first by the introduction of E1-G under the control of a CMV promoter.…”

“…However, the differences in susceptibilities of the cell types to MuLV or lentivirus pseudotype (5,52) and VSV pseudotype infectivity (32,7,9,37) and the reason for the lack of infectivity of lentivirus pseudotypes bearing chimeric HCV glycoproteins (27) remain unclear at this time. In the present study, we have investigated the role of the ectodomains from both E1 and E2 chimeric glycoproteins in mediating virus infectivity.…”