Conformational changes between human immunodeficiency virus type 1 nucleocapsid protein NCp7 and its precursor NCp15 as detected by anti-NCp7 monoclonal antibodies.
The nucleocapsid protein NCp15 of human immunodeficiency virus type 1 (HIV-1) is a small basic protein with two zinc fingers. It is required for virion morphogenesis and synthesis of proviral DNA. As the first step in our study of the structural domains involved in the various functions of this protein, 18 monoclonal antibodies (MAbs) were isolated. The epitopes of NCp7 recognized by the MAbs were mapped using synthetic peptides representing overlapping sequences and truncated forms of NCp7. These anti-NCp7 MAbs were investigated by ELISA and real-time biospecific interaction analysis (BIAcore). Five classes of anti-NCp7 MAbs were characterized. Three classes (14 MAbs) were directed against continuous epitopes, one in the N-terminal part, another next to the second zinc finger and the third in the C-terminal part of the protein. Two other classes comprised four MAbs reacting only with the entire NCp7 and not with any of the small overlapping peptides used, suggesting that these MAbs were directed against conformational epitopes. The anti-NCp7 MAbs directed against linear epitopes were able to react efficiently with both NCp7 and NCp15, the NCp7 precursor, whereas the anti-NCp7 MAbs directed against conformational epitopes did not react with NCp15. Interestingly, most of the anti-NCp7 MAbs directed against conformational epitopes were capable of inhibiting the tight interaction between NCp7 and the HIV-1 replication primer tRNA(Lys,3). In contrast, most of the MAbs directed against linear epitopes did not inhibit this interaction.
Tanchou
V
V
Signalisation, Inflammation et Transformation Cellulaire, U332 INSERM, Paris, France.
Delaunay
T
T
Bodéus
M
M
Roques
B
B
Darlix
J L
JL
Benarous
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002213:Capsid / Q000737:chemistry* / Q000276:immunology; D036022:Capsid Proteins; D018604:Epitope Mapping; D005260:Female; D015683:Gene Products, gag / Q000737:chemistry* / Q000276:immunology; D015483:HIV Antibodies; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry; D012357:RNA, Transfer, Lys / Q000032:analysis; D051381:Rats; D014764:Viral Proteins; D016335:Zinc Fingers / Q000276:immunology; D054301:gag Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D015683:Gene Products, gag; D015483:HIV Antibodies; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D010455:Peptides; D011498:Protein Precursors; D012357:RNA, Transfer, Lys; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1