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

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 ((0)NCp7), 1 (NCp7) or 2 ((2)NCp7) 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, (2)NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas (0)NCp7 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.