Detection and characterization of peptide NS3_1406–1415 in MHC class I ligands isolated from UNS3-4A/A2-27.35 cells.

<p>Naturally processed MHC class I ligands of UNS3-4A/A2-27.35 cells were separated by high performance liquid chromatography (HPLC) and analyzed by mass spectrometry. (A) A chromatogram of the total ion current (TIC) is shown. (B) The mass chromatogram of ionized peptides with m/z = 499.3 reveals a peak at retention time t = 49.5 min that correlates with (C) a signal of the synthetic peptide NS3_1406–1415 (KLVALGINAV) which elutes after 51.3 min. In all graphs, HPLC retention times are shown on the abscissa and relative signal intensity on the ordinate. Small insets in panels B and C show mass spectra of peptides eluted at the indicated time points. (D) MSMS spectrum of the synthetic peptide NS3_1406–1415 (KLVALGINAV). (E) MSMS spectrum of the natural peptide isolated from UNS3-4A/A2-27.35 cells, revealing amino acid sequence KLVALGINAV identical to the synthetic peptide shown in panel A. Identified amino acid sequences of peptide fragments are indicated on the top of the peaks. Due to the protonated N-terminal lysine residue, the b series of peptide fragments is dominating the MSMS spectra.</p

Cell lines UNS3-4A/A2-27.35 and UHCV/A2-27.

<p>(A) Indirect immunofluorescence microscopy of UNS3-4A/A2-27.35 and UHCV/A2-27 cells cultured for 48 h in the presence (+ tet) or absence (− tet) of tetracycline. Monoclonal antibody 1B6 against HCV NS3 was used as primary antibody. (B) Immunoblot analysis of UNS3-4A/A2-27.35 and UHCV/A2-27 cells were cultured for 48 h in the presence (+ tet) or absence (− tet) of tetracycline. Monoclonal antibodies 1B6 against HCV NS3 or 11H against NS5A were used as primary antibodies. (C) Left panel: HLA-A2 surface expression of UNS3-4A/A2-27.35 (blue histogram) compared to the founder cell line UNS3-4A-24 (red histogram). Right panel: HLA-A2 surface expression of UHCV/A2-27 (blue histogram) compared to the founder cell line UHCVcon-57.3 (red histogram).</p

Identification of naturally processed HLA-A2 ligands.

<p>Following large-scale expansion, cell pellets were lysed and MHC class I molecules with bound ligands were purified by immunoprecipitation. Subsequently, ligands were eluted and separated by high performance liquid chromatography (HPLC). Fractions of interest were sequenced by mass spectrometry.</p