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.

1型人类免疫缺陷病毒（HIV-1）Gag通过其核衣壳（NC）结构域特异性识别病毒包装信号（Psi），导致将两个拷贝的基因组RNA（gRNA）衣壳化到病毒颗粒中。在病毒成熟过程中从Gag裂解而来的NCp7是一种核酸伴侣蛋白，可包覆并保护gRNA。在这项研究中，开发了一种基于RT-qPCR的方法，用于定量比较存在细菌或293T总RNA时Gag和NCp7的Psi选择性。Gag和NCp7对Psi的茎环（SL）3的结合亲和力也使用表面等离振子共振进行了比较。我们发现Gag从大肠杆菌BL21（DE3）和体外中选择的Psi-RNA比NCp7更多结合反应，并且Gag以比NCp7高的亲和力与SL3-RNA结合。此外，Gag含2个Zn ^2+，而NCp7含1个。NCp7的N末端锌指基序失去了大部分的Zn ²⁺结合活性。删除NCp7的N端氨基酸1-11导致Psi选择性，SL3-亲和力和Zn ²⁺含量增加。这些结果表明Gag的Zn ²⁺配位对于Psi结合和选择至关重要。在Gag切割过程中或之后从第一个锌指基序中去除Zn ²⁺以生成成熟的NCp7可能是调节Gag NC域和成熟NCp7功能的开关。我们的研究将有助于阐明Zn ²⁺的重要作用。 病毒在病毒的生命周期中发挥着重要作用，可能有助于进一步研究HIV-1 Gag和NCp7的功能。