A HIDDEN REGION IN THE 3RD VARIABLE DOMAIN OF HIV-1 IIIB GP120 IDENTIFIED BY A MONOCLONAL-ANTIBODY

The third variable domain (V3 domain) of HIV-1 gp120 is involved in vi rus neutralization by antibody, in determination of cell tropism, and in syncytium-inducing/non-syncytium-inducing capacity. Antibodies are highly specific tools to delineate the role of different V3 amino acid sequences in these processes, and to dissect events occurring during synthesis of gp120/160, gp120-CD4 interaction, cellular infection, and syncytium formation. We describe here an IgG1 murine monoclonal antib ody (MAb), coded IIIB-V3-01, that was raised with a synthetic peptide (FVTIGKIGNMRQAHC) derived from the carboxy-terminal flank of the HIV-1 IIIB V3 domain. The binding site of this antibody was mapped to the s equence IGKIGNMRQ, using Pepscan analysis. In ELISA, this antibody bin ds to E. coli-derived gp120 from HIV-1 IIIB, which is denatured and no t glycosylated. The antibody showed no neutralizing activity against H IV-1 IIIB, MN, SF2, or RF in a virus neutralization assay and in a syn cytium formation inhibition assay. In addition, this antibody did not react with gp120 expressed on the surface of IIIB-infected MOLT-3 cell s in FACS analysis. To assess whether the epitope defined by MAb IIIB- V3-01 is hidden on native gp120, reactivity of the antibody with SDS-D TT-denatured or DTT-denatured glycosylated gp120 (CHO cell produced) w as tested. Both these treatments exposed the epitope for binding. From these data we conclude that the epitope defined by MAb IIIB-V3-01 is hidden on glycosylated recombinant gp120, and is not accessible on gp1 20 expressed on the membrane of HIV-1, IIIB-infected cells. The result s described contribute to the understanding of the three-dimensional s tructure of native gp120.