Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Retrovirology. 2012 Feb 20;9:17. doi: 10.1186/1742-4690-9-17.
Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.
A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 μM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway.
The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.
锚蛋白是许多重要蛋白质-蛋白质相互作用的细胞中介。与内抗体不同，锚蛋白由高度结构化的重复模块组成，其特征是不依赖二硫键折叠。设计用于靶向病毒成分的人工锚蛋白分子可能作为细胞内抗病毒剂，有助于细胞对 HIV-1 等病毒病原体的免疫。
构建了一个噬菌体展示的人工锚蛋白文库，并在 HIV-1 Gag 前体的融合基质和衣壳域（MA-CA）的多蛋白上进行筛选。分离出一个具有三个模块的锚蛋白 Ank(GAG)1D4（16.5 kDa）。Ank(GAG)1D4 和 MA-CA 形成了一个 1:1 摩尔比的蛋白质复合物，解离常数 K(d)~1 μM，Ank(GAG)1D4 的结合位点位于 CA 的 N 端结构域内，残基 1-110 之间。与对照细胞相比，稳定表达 N-豆蔻酰化和非 N-豆蔻酰化两种形式的 Ank(GAG)1D4 的 SupT1 细胞中的 HIV-1 产量显著降低。Ank(GAG)1D4 的表达还降低了 MLV 的产生，MLV 是一种系统发育上较远的逆转录病毒。发现 Ank(GAG)1D4 介导的抗 HIV-1 作用发生在整合后步骤，但不涉及 Gag 前体加工或细胞运输。我们的数据表明，Ank(GAG)1D4-CA 对 Gag 组装和出芽途径的负干扰导致 HIV-1 后代产量降低。
表达 Ank(GAG)1D4 的细胞对 HIV-1 的抗性表明，靶向 CA 的锚蛋白 Ank(GAG)1D4 可以作为设计基于锚蛋白重复模块的新型 HIV-1 组装细胞内抑制剂的蛋白质平台。
