Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.,Journal of Proteome Research

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Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-02-21 , DOI: 10.1021/acs.jproteome.9b00771
Shayli Varasteh Moradi ₁ , Dejan Gagoski ₂ , Sergey Mureev ₁ , Patricia Walden ₁ , Kerrie-Ann McMahon ₃ , Robert G Parton _{3,

4} , Wayne A Johnston ₁ , Kirill Alexandrov ₁

Affiliation

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 multi-host 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 Leishmania cell-free system and AlphaLISA interaction assay, pairwise protein interactions of all ZIKV proteins were systematically tested. We identified 33 intra-viral 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.

中文翻译：

无细胞表达的寨卡病毒蛋白之间的映射相互作用。

节肢动物传播的寨卡病毒的迅速传播构成了严重的公共卫生威胁，需要控制和治疗病毒感染的有效方法。反过来，这就需要更好地了解病毒装配的机制及其与宿主细胞的相互作用。为了促进此类工作，我们开发了一种新的多宿主表达载体pmCellFree，该载体可在任何体外翻译系统以及哺乳动物细胞中快速且多重地生产ZIKV蛋白。结合使用利什曼原虫无细胞系统中的体外表达和AlphaLISA相互作用测定，系统地测试了所有ZIKV蛋白的成对蛋白相互作用。我们确定了33种病毒内二进制蛋白相互作用，其中13种是新颖的相互作用。通过在哺乳动物HEK293T细胞系中表达所选蛋白对并评估其在细胞裂解液中的相互作用，进一步验证了这些发现。这些相互作用测定的结果与用体外表达的蛋白质获得的结果相同。观察到的新型蛋白质：蛋白质相互作用使用下拉测定法进一步验证。未公开的新型蛋白质相互作用可能指向ZIKV组装过程以前未曾意识到的复杂性，并且可能在感染过程中起重要作用。这些相互作用可能代表了抗病毒药物开发的新目标。使用下拉测定法进一步验证蛋白质相互作用。未公开的新型蛋白质相互作用可能指向ZIKV组装过程以前未曾意识到的复杂性，并且可能在感染过程中起重要作用。这些相互作用可能代表了抗病毒药物开发的新目标。使用下拉测定法进一步验证蛋白质相互作用。未公开的新型蛋白质相互作用可能指向ZIKV组装过程以前未曾意识到的复杂性，并且可能在感染过程中起重要作用。这些相互作用可能代表了抗病毒药物开发的新目标。

更新日期：2020-02-21

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