CSIRO-QUT Synthetic Biology Alliance, Centre for Tropical Crops and Bio-commodities, Queensland University of Technology, Brisbane QLD 4001, Australia.
Queensland Brain Institute, The University of Queensland, St Lucia, Brisbane QLD 4072, Australia.
J Proteome Res. 2020 Apr 3;19(4):1522-1532. doi: 10.1021/acs.jproteome.9b00771. Epub 2020 Mar 9.
The rapid spread of arthropod-borne Zika virus poses a serious public health threat that calls for effective ways of controlling and treating viral infection. This in turn necessitates better understanding of the mechanisms of virus assembly and its interaction with the host cells. In order to facilitate such efforts, we developed a new multihost expression vector pmCellFree that allows rapid and multiplexed production of ZIKV proteins in any in vitro translation system as well as in mammalian cells. Using a combination of in vitro expression in cell-free system and AlphaLISA interaction assay, pairwise protein interactions of all ZIKV proteins were systematically tested. We identified thirty-three intraviral binary protein interactions, of which 13 interactions are novel. These findings were further validated by expressing selected protein pairs in mammalian HEK293T cell line and assessing their interactions in the cellular lysate. The results of these interaction assays were identical to those obtained with in vitro expressed proteins. The observed novel protein-protein interactions were further validated using a pulldown assay. The unrevealed novel protein interactions may point to the previously unappreciated complexity of the ZIKV assembly process and may play an important role in the infection process. These interactions may represent new targets for antiviral drug development.
虫媒寨卡病毒的迅速传播对公共健康构成了严重威胁，需要采取有效的控制和治疗病毒感染的方法。这反过来又需要更好地了解病毒组装的机制及其与宿主细胞的相互作用。为了促进这些努力，我们开发了一种新的多宿主表达载体 pmCellFree，该载体允许在任何体外翻译系统以及哺乳动物细胞中快速且多路复用地生产 ZIKV 蛋白。我们使用无细胞系统中的体外表达和 AlphaLISA 相互作用测定的组合，系统地测试了所有 ZIKV 蛋白的两两蛋白相互作用。我们鉴定了三十三个病毒内的二聚体蛋白相互作用，其中 13 个相互作用是新的。通过在哺乳动物 HEK293T 细胞系中表达选定的蛋白对，并在细胞裂解物中评估它们的相互作用，进一步验证了这些发现。这些相互作用测定的结果与体外表达蛋白的结果相同。使用下拉测定进一步验证了观察到的新的蛋白-蛋白相互作用。未揭示的新蛋白相互作用可能表明 ZIKV 组装过程的先前未被认识到的复杂性，并可能在感染过程中发挥重要作用。这些相互作用可能代表抗病毒药物开发的新靶标。
