Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn²⁺ content

The human immunodeficiency virus type 1 (HIV-1) Gag recognizes viral packaging signal (Psi) specifically via its nucleocapsid (NC) domain, resulting in the encapsidation of two copies of genomic RNA (gRNA) into the viral particle. The NCp7, which is cleaved from Gag during viral maturation, is a nucleic acid chaperone, coating and protecting the gRNA. In this study, an RT-qPCR-based approach was developed to quantitatively compare the Psi-selectivity of Gag and NCp7 in the presence of bacterial or 293T total RNAs. The binding affinity of Gag and NCp7 to the stem-loop (SL) 3 of Psi was also compared using surface plasmon resonance. We found that Gag selected more Psi-RNA than NCp7 from both E. coli BL21 (DE3) and in vitro binding reactions, and Gag bound to SL3-RNA with a higher affinity than NCp7. Moreover, Gag contained two Zn²⁺ whereas NCp7 contained one. The N-terminal zinc-finger motif of NCp7 lost most of its Zn²⁺-binding activity. Deletion of N-terminal amino acids 1-11 of NCp7 resulted in increased Psi-selectivity, SL3-affinity and Zn²⁺ content. These results indicated that Zn²⁺ coordination of Gag is critical for Psi-binding and selection. Removal of Zn²⁺ from the first zinc-finger motif during or after Gag cleavage to generate mature NCp7 might serve as a switch to regulate the functions of Gag NC domain and mature NCp7. Our study will be helpful to elucidate the important roles that Zn²⁺ plays in the viral life cycle, and may benefit further investigations of the function of HIV-1 Gag and NCp7.