Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn2+ content
Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn2+ content
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HIV-1 Gag 和 NCp7 对包装信号的选择性、对茎环 3 的亲和力以及 Zn2 含量的比较。
DOI：
10.1016/j.biochi.2020.09.024
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发表时间：
2020-12-01
期刊：
影响因子：
3.9
通讯作者：
Wang, Ying
中科院分区：
文献类型：
--
作者：
Guo, Chao;Yao, Xiaohong;Wang, Ying
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)，导致两个拷贝的基因组RNA(GRNA)被包裹到病毒颗粒中。NCp7是病毒成熟过程中从Gag裂解而来的，是一种核酸伴侣，包裹和保护gRNA。在本研究中，建立了一种基于RT-qPCR的方法来定量比较GAG和NCp7在细菌或293T总RNA存在下的Psi选择性。用表面等离子体共振法比较了Gag和NCp7与Psi的茎环(SL)3的结合亲和力。我们发现，在大肠杆菌BL21(DE3)和体外结合反应中，GAG比NCp7选择了更多的Psi-RNA，并且GAG与SL3-RNA的亲和力高于NCp7。此外，GAG含有两个锌离子，而NCp7含有一个锌离子。NCp7的N-末端锌指基序失去了大部分的锌结合活性。NCp7 N端1-11位氨基酸的缺失导致Psi选择性、SL3亲和力和锌含量的增加。这些结果表明，GAG的锌配位对Psi的结合和选择是至关重要的。在Gag裂解过程中或之后，从第一个锌指基序中去除锌离子以产生成熟的NCp7，可能是调节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锌离子在病毒生命周期中的重要作用，并可能有助于进一步研究HIV-1 Gag和NCp7的功能。(C)2020年Elsevier B.V.和法国生物与分子生物学公司(SFBBM)。版权所有。
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. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.