Identification of hepatitis G virus particles in human serum by E2-specific monoclonal antibodies generated by DNA immunization

Abstract

In order to elucidate the structure and morphology of hepatitis G virus (HGV), a recently isolated flavivirus, we generated a panel of eight monoclonal antibodies (MAbs) against the putative second envelope protein (E2) following DNA immunization. The MAbs were shown to be specific for four different epitopes on recombinant E2. MAb Mc6 was the only antibody able to detect the linear epitope LTGGFYEPL. In addition, Mc6 was able to immunoprecipitate viral particles in human blood samples as detected by reverse transcription-PCR amplification of HGV RNA. This precipitation could be competed by addition of saturating amounts of the linear peptide or abolished by addition of Nonidet P-40. We conclude that, albeit lacking the N-terminal sequence of a functional core protein, HGV builds classical viral particles displaying E2 envelope protein on their outer surfaces.

FIG. 1

Reaction profile of MAb Mc6 with synthetic overlapping peptides. Eleven peptides, spanning residues 253 to 305 of HGV E2, are shown. The amino acid residue numbers indicate the position of the first residue from each 13-mer; numbering starts with the first residue of the putative mature E2 protein. Only peptides 277 and 281, which share amino acids 281 to 289 (LTGGFYEPL), were recognized by Mc6.

FIG. 2

Immunoprecipitation of viral particles. HGV RNA-positive sera were incubated with either MAbs or human sera. Following precipitation by protein G-agarose, RNA was isolated and detected by RT-PCR. Shown are representative examples of three independent experiments. (a) HGV RNA-positive serum BM7822 was incubated with eight murine E2-specific MAbs, anti-β-Gal, FLAG-specific M1 (final concentration, 5 μg/ml), anti-E2-positive serum SB9700575, and anti-E2-negative serum SB315. Only Mc6 and SB9700575 were able to immunoprecipitate HGV. (b) HGV RNA-positive sera SB373, SB388, and BM7822 were incubated with anti-E2-positive serum SB9700575, anti-E2-negative serum SB315, E2-specific Mc6, and FLAG-specific M1. SB9700575 and Mc6 were able to immunoprecipitate HGV from all sera. (c) BM7822 was incubated with Mc6 in the presence of peptides 229, 273, 277, and 281 (final concentration, 1 μg/ml). For comparison, immunoprecipitation was performed with Mc6 without addition of a peptide and with anti-β-Gal. Peptides 277 and 281 competed for binding sites on viral E2. (d) BM7822 was incubated with Mc6 in the presence of different concentrations of NP-40. No viral RNA could be detected in the precipitate (bound) and supernatant (unbound fraction) at 0.025 and 0.0025% NP-40. ECL, electrochemiluminescence.