Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7.
The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.
De Rocquigny
H
H
Département de Pharmacochimie Moléculaire et Structurale, INSERM U266-CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, Paris, France.
Caneparo
A
A
Dong
C Z
CZ
Delaunay
T
T
Roques
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D002213:Capsid / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D036022:Capsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D015683:Gene Products, gag / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D016336:Gene Products, vpr / Q000276:immunology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000276:immunology; D011233:Precipitin Tests; D011498:Protein Precursors / Q000276:immunology; D020349:Surface Plasmon Resonance / Q000379:methods; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D015683:Gene Products, gag; D016336:Gene Products, vpr; C071884:NCP7 protein, Human immunodeficiency virus 1; D010456:Peptides, Cyclic; D011498:Protein Precursors; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C059767:p55 gag precursor protein, Human immunodeficiency virus 1