TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China.
TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, 23 Hongda Street, TEDA, Tianjin, 300457, China; Tianjin Key Laboratory of Microbial Functional Genomics, 23 Hongda Street, TEDA, Tianjin, 300457, China.
Biochimie. 2020 Dec;179:135-145. doi: 10.1016/j.biochi.2020.09.024. Epub 2020 Sep 26.
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 Zn whereas NCp7 contained one. The N-terminal zinc-finger motif of NCp7 lost most of its Zn-binding activity. Deletion of N-terminal amino acids 1-11 of NCp7 resulted in increased Psi-selectivity, SL3-affinity and Zn content. These results indicated that Zn coordination of Gag is critical for Psi-binding and selection. Removal of Zn 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 Zn plays in the viral life cycle, and may benefit further investigations of the function of HIV-1 Gag and NCp7.
人类免疫缺陷病毒 1 型（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），导致两个基因组 RNA（gRNA）拷贝被包裹到病毒颗粒中。在病毒成熟过程中从 Gag 中切割出来的 NCp7 是一种核酸伴侣，覆盖并保护 gRNA。在这项研究中，开发了一种基于 RT-qPCR 的方法来定量比较 Gag 和 NCp7 在存在细菌或 293T 总 RNA 时对 Psi 的选择性。还使用表面等离子体共振比较了 Gag 和 NCp7 与 Psi 的茎环（SL）3 的结合亲和力。我们发现，与 NCp7 相比，Gag 从大肠杆菌 BL21（DE3）和体外结合反应中选择了更多的 Psi-RNA，并且 Gag 与 SL3-RNA 的结合亲和力高于 NCp7。此外，Gag 含有两个 Zn，而 NCp7 含有一个。NCp7 的 N 端锌指模体失去了大部分 Zn 结合活性。NCp7 的 N 端氨基酸 1-11 的缺失导致 Psi 选择性、SL3 亲和力和 Zn 含量增加。这些结果表明，Gag 的 Zn 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模体中去除 Zn，可能会成为调节 Gag NC 结构域和成熟 NCp7 功能的开关。我们的研究将有助于阐明 Zn 在病毒生命周期中所起的重要作用，并可能有助于进一步研究 HIV-1 Gag 和 NCp7 的功能。
