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To study the immunological and immunohistochemicai nature of HIV-1 Nef.Monoclonal anti-Nef antibodies were generated and used to identify antigenic epitopes in Nef, to study immunological cross-reactivity between Nef from different isolates and to reveal the subcelluiar localization of Nef.Monoclonal antibodies against recombinant HIV-1 Nef protein (BRU isolate) were generated in BALB/c mice. The epitope mapping was carried out with the use of overlapping 15–20mer lipopeptides linked to a lipid group at the amino-terminus. Immunoperoxidase method was used for histochemical studies.Ten stable antibody-producing clones, mainly of the immunoglobulin (Ig) G1 subtype, with strong Western blot and enzyme-linked immunosorbent assay reactivity toward the recombinant Nef protein, were obtained. The epitopes recognized were located on amino-acid sequences 21–41, 31–50, 51–71, 61–80, 151–170, 161–180, and 171–190. All 10 monoclonal antibodies also reacted with the native Nef of HIV-1BRU, and eight reacted with native HIV-1IIIB. Most antibodies also reacted with Nef from more divergent HIV-1 strains. In Western blotting, two forms of Nef (24 and 27 kDa) were observed with most isolates studied. Immunohistochemical staining of HIV-1 -infected H9 or MT-4 lymphoid cells demonstrated that Nef was expressed mainly in the Golgi complex and at the nuclear membrane, but occasionally also in the nucleus. The nuclear localization of Nef was especially frequent in the HIV-1-infected MT-4 cells.Our findings suggest that Nef is expressed in two isomorphic forms, and that it may also act as a nuclear protein and thus have a direct regulatory function at the RNA/DNA level.