The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

Abstract

The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (0NCp7), 1 (NCp7) or 2 (2NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, 2NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas 0NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity

“…The nucleocapsid (NC; p7) protein, consisting of 55 amino acids [ 82 ], packages two copies of the viral RNA into rapidly assembling virions [ 83 ]. A prominent feature of NC is the presence of two highly conserved Cys-X2-Cys-X4-His-X4-Cys (CCHC) signatures that resemble zinc-finger motifs [ 83 ].…”

“…The nucleocapsid (NC; p7) protein, consisting of 55 amino acids [ 82 ], packages two copies of the viral RNA into rapidly assembling virions [ 83 ]. A prominent feature of NC is the presence of two highly conserved Cys-X2-Cys-X4-His-X4-Cys (CCHC) signatures that resemble zinc-finger motifs [ 83 ]. The zinc fingers, each comprising an aromatic residue (F16 in the N-terminal zinc finger and W37 in the C-terminal zinc finger) [ 84 ], coordinate a zinc ion [ 85 ] and are divided by a 406 RAPRKKG 412 basic domain linker [ 4 ].…”

“…Genome packaging in HIV-1 is dependent on interactions between an un-cleaved precursor PR55 NC domain and a 110 nucleotide Psi (ψ; RNA packaging signal) segment [ 94 ] in the 5′ RNA leader sequence [ 83 ]. The ψ segment comprises four stem loops (SL1–SL4) that are connected by short linkers [ 95 ].…”

Zn2+-dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses

“…Metal ions, represented by zinc, also play vital roles in virus packaging, inhibiting the replication of RNA viruses, antiviral immunity, and host-virus interactions [24][25][26]. The structural protein Gag precursor of human immunodeficiency virus type 1 (HIV-1) participates in the assembly of budding virion, the nucleocapsid (NC) domain of Gag binds to the 3'-leader of the genomic RNA to initiate the packaging of HIV virions [27]. The NC domain or its cleaved version contains two CCHC zinc-finger motifs, mutations of these zinc-finger motifs cause severe defects in viral RNA packaging and infectivity [27,28].…”

“…The structural protein Gag precursor of human immunodeficiency virus type 1 (HIV-1) participates in the assembly of budding virion, the nucleocapsid (NC) domain of Gag binds to the 3’-leader of the genomic RNA to initiate the packaging of HIV virions [ 27 ]. The NC domain or its cleaved version contains two CCHC zinc-finger motifs, mutations of these zinc-finger motifs cause severe defects in viral RNA packaging and infectivity [ 27 , 28 ]. Zn 2+ mediates the conformational changes of hepatitis B virus (HBV) CP to increase the production of virus-like particles (VLPs) [ 29 ].…”