Sheng N, Pettit S C, Tritch R J, Ozturk D H, Rayner M M, Swanstrom R, Erickson-Viitanen S
Molecular Biology Department, The DuPont Merck Pharmaceutical Co., Wilmington, Delaware 19880-0336, USA.
J Virol. 1997 Aug;71(8):5723-32. doi: 10.1128/JVI.71.8.5723-5732.1997.
During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.
在人类免疫缺陷病毒1型（HIV-1）病毒体组装过程中，病毒蛋白酶对Gag前体的切割导致一种名为p15NC的核衣壳-p1-p6中间产物短暂出现。利用体外转录-翻译系统产生或在大肠杆菌中表达后纯化的p15NC前体蛋白，我们已证明RNA是HIV p15NC有效切割所必需的。凝胶迁移率变动和硝酸纤维素滤膜结合实验表明，纯化的p15NC蛋白以估计为1.5 nM的解离常数（Kd）特异性结合其相应的mRNA。锌或乙二胺四乙酸（EDTA）的存在与否不影响结合。此外，两个半胱氨酸-组氨酸阵列中任何一个内的半胱氨酸残基诱变对RNA结合或病毒蛋白酶的RNA依赖性切割均无影响。相比之下，在两个锌指之间区域存在的碱性氨基酸三联体中一个或多个残基发生含p15NC突变的情况下，观察到RNA结合减少以及体外切割敏感性降低。此外，我们发现特定序列和二级结构的21至24个碱基的DNA和RNA寡核苷酸可替代p15 RNA增强p15NC切割。携带NC碱性残基三联体突变（P3BE）的病毒颗粒显示p15NC切割延迟且核心形成存在缺陷，尽管最终出现了完全加工的病毒体蛋白。这些结果确定了导致蛋白酶介导的切割增强的p15NC-RNA相互作用的决定因素，并证明了碱性残基三联体在病毒核心形成中的重要性。
