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Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Zhenlan Yao·Melody Li
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Abstract

Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.

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Chikungunya virusImmunoprecipitationViral ProteinsHumansEpitopesAnimalsMass SpectrometryProteomicsProtein Interaction MappingHost-Pathogen InteractionsProtein BindingAP-MSEpitope taggingMacrophageMulti-fragment assemblyTagged virusJournal ArticleResearch Support, N.I.H., Extramural

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Zhenlan Yao, Melody Li. 2027. Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.. https://doi.org/10.1007/978-1-0716-5496-5_13

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