Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding

Abstract

We compared the ability of two closely related truncated E2 glycoproteins (E2 660 ) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformationdependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2 660 formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2 660 chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2 660 , produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384-406) on H77 E2 660 , chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407-524 of Gla E2 660 , respectively, were recognized by conformationdependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407-524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2 660 aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N-G478S and/or D532N in Gla E2 660 had no effect on antigenicity or aggregation.

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

“…The enzyme-linked immunosorbent assay (ELISA) to detect MAb binding to E2 glycoprotein was performed essentially as described previously (40). Briefly, HEK293T cells were cotransfected with E1E2 coding sequence-containing plasmids, and the expressed glycoproteins present in clarified lysates of these cells were captured on to GNA (Galanthus nivalis) lectin-coated Immulon II enzyme immunoassay (EIA) plates (Thermolabsystems).…”

Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33

“…The branched peptides used in immunoassays corresponded to the AP33 epitope QLINTNG-SWHVN 22 encompassing amino acids 412-423 of the genotype 1a Glasgow strain, 36 the control peptide VNL-HDFRSDEIE, and a peptide HLANHQGKWRLH, which represented the most frequent peptide selected by MAb AP33 from a random peptide phage display library (below).…”

“…To detect MAb binding to E2 glycoprotein, an ELISA was performed essentially as previously described. 36 Briefly, E1E2 glycoproteins from the clarified lysates of transfected HEK 293FT cells were cap-tured onto GNA (Galanthus nivalis) lectin (Sigma)-coated microtiter plates then detected by MAb AP33 or 3/11, followed by an anti-species IgG-alkaline phosphatase conjugate (Sigma) and p-NPP substrate. Absorbance values were determined at 405 nm.…”

“…33 The epitope recognized by MAb AP33 has been mapped to a 12 amino acid region corresponding to residues 412 to 423 located immediately downstream of HVR1. 21,22,[34][35][36][37] This region is also recognized by rat MAb 3/11. 21 Like AP33, MAb 3/11 is capable of neutralizing HCVpp carrying strain H77 E1E2, although whether its neutralization capacity extends to genetically diverse E1E2 molecules is unknown.…”

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

“…The MAb mouse anti-cluster of differentiation 81 (CD81) clone JS-81 (BD Biosciences, Franklin Lakes, NJ), mouse isotype control IgG1 (R&D Systems, Minneapolis, MN), anti-HCV MAbs (HC33.4, HC84.26, AR3B, AR4A, and AR5A), and human anti-HIV antibody B6 have been described previously (15,16,44,45). A4 and H52 were provided by Jean Dubuisson (62,63).…”