ASSESSMENT OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I INTERACTION WITH EPSTEIN-BARR-VIRUS AND HUMAN-IMMUNODEFICIENCY-VIRUS PEPTIDES BY ELEVATION OF MEMBRANE H-2 AND HLA IN PEPTIDE LOADING-DEFICIENT CELLS

Earlier findings indicate that peptides can affect the expression of major histocompatibility complex (MHC) class I molecules on the surface of cells with defective peptide loading mechanism.We have used peptide induced increase of class I antigen expression to assess peptide interaction with MHC class I molecules. A panel of 41 overlapping synthetic peptides derived from the human immunodeficiency virus-1 (HIV-1) gag protein and 33 nonoverlapping peptides from Epstein-Barr virus (EBV) proteins EBNA-1, 2, 3, 4, 5, 6, LMP, BZLF2, BILF2, BSLF2, BALF4 and BcLF1 was assessed for the ability to enhance the expression of HLA-A2. 1, H-2D(b), K(b) and D(d) on the murine RMA-S and human 721.174/T2 (.174/T2) lines by indirect immunofluorescence. Considering doubling of the fluorescence intensity in the peptide-treated samples as positivity, 6 of 39 HIV and 1 of 32 EBV peptides were found to bind to A2.1, 6 of 39 HIV gag and 7 of 16 EBV peptides to Db, 8 of 39 HIV gag and 5 of 16 EBV peptides to K(b) and 2 of 39 HIV gag and 1 of 17 EBV peptides to D(d). The sensitivity of the method is comparable to the in vitro class I assembly assay with conformation-dependent monoclonal antibody and is more discriminating than the solid-phase assay. Due to its simplicity this method can also serve for testing large peptide panels for binding capacity to various class I molecules. Moreover, the method provides information about the relevance of in vitro tests for class I assembly in living cells.