H-1-NMR STRUCTURE AND BIOLOGICAL STUDIES OF THE HIS(23)-]CYS MUTANT NUCLEOCAPSID PROTEIN OF HIV-1 INDICATE THAT THE CONFORMATION OF THE FIRST ZINC-FINGER IS CRITICAL FOR VIRUS INFECTIVITY

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which has key functions in the virus life cycle, possesses two zinc fingers of the CX(2)CX(4)HX(4)C type characterized by three successive loops containing a tetrahedrally coordinated zinc atom, The replacement of any cysteine by a serine in either finger has been shown to result in the production of noninfectious viruses, probably by impairing the biological functions of NCp7. In order to more precisely elucidate the structural role of the zinc finger motif, His(23) was replaced by Cys in the proximal finger of the peptide (13-64)NCp7 which retains NCp7 activities in vitro. The peptide Cys(23)(13-64)NCp7 was synthesized by solid phase and studied by 2D H-1 NMR and molecular modeling. The His to Cys modification causes important structural modifications of the N-terminal zinc finger which impair the spatial proximity of the two zinc fingers as shown by the disappearance of several interresidue NOEs. The side chains of Val(13), Lys(14), Phe(16), Thr(24), Ala(25), Trp(37), Gln(45), and Met(46), which are thought to be involved in nucleic acid recognition, are no longer found clustered in the Cys(23)(13-64)NCp7 mutant as they are in the wild-type NCp7 structure. In vitro, Cys(23)(13-64)NCp7 is unable to tightly interact with the viral RNA or replication primer tRNA(Lys,3). The Cys(23)(NCp7) mutation was introduced into an infectious HIV-1 molecular clone, and virions produced upon DNA transfection into cells were analyzed for their viral protein and RNA compositions as well as for their infectivity. Results show that, while the Cys(23)(NCp7) mutation does not impair virion production, viruses contain a low amount of degraded viral RNA and are not infectious. These findings suggest that a bona fide conformation of the HIV-1 NCp7 is critical for the packaging of viral RNA, its stability in virions, and virus infectivity.