PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 42734796

Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Zhenlan Yao·Melody Li
View original source ↗Export metadata ↓

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.

Explore related subjects

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

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

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

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2027

Overview of Chikungunya Virus Epidemiology, Biology, and Pathogenesis.

Chikungunya virus (CHIKV), an arthropod-borne alphavirus within the Togaviridae family, is transmitted primarily by Aedes aegypti and Aedes albopictus. The virus causes an acute febrile illness characterized by severe, often bilateral polyarthralgia, with potential progression to chronic musculoskeletal pain and rare systemic complications involving cardiovascular and neurological systems. CHIKV exhibits a spherical, enveloped virion (~70 nm) with T = 4 icosahedral symmetry, incorporating E1/E2 glycoprotein heterodimers that mediate receptor binding and membrane fusion. Its positive-sense RNA genome (~11.8 kb) encodes nonstructural proteins for replication and structural proteins for virion assembly. Replication occurs in cytoplasmic spherules, involving synthesis of genomic and subgenomic RNAs, followed by glycoprotein maturation and budding at the plasma membrane. Epidemiologically, CHIKV has expanded beyond Africa and Asia, with major outbreaks driven by adaptive mutations enhancing transmission via A. albopictus. Since introduction to the Americas in 2013, the global incidence remains high, with >180,000 confirmed cases reported in 2025. Preventive strategies rely on vector control and vaccination; VLP-based vaccines (e.g., Vimkunya) show promise, while live-attenuated formulations face safety concerns. No licensed antivirals exist; current management is supportive, though investigational therapies targeting viral replication and immune modulation are under development.

Chikungunya virus↗
PubMed2027

Nanopore Sequencing for Chikungunya Virus: Principles and Application.

Nanopore sequencing is transforming viral genomics through real-time, portable, long-read analysis of RNA and DNA. Unlike traditional short-read platforms, it detects nucleotide sequences by measuring ionic current changes as nucleic acids pass through nanoscale pores, enabling direct single-molecule sequencing and base modification detection. Its simplicity, flexibility, and capacity for ultra-long reads make it ideal for resolving complex genomic regions, structural variants, and full viral genomes. These advantages have accelerated its use in pathogen surveillance and outbreak response, especially in resource-limited settings. For chikungunya virus (CHIKV), nanopore sequencing allows rapid, culture-independent recovery of complete genomes from clinical and vector samples, enabling real-time tracking of viral diversity, evolution, and spread. Experiences from Ebola, Zika, and COVID-19 have demonstrated the power of portable sequencing, now applied to CHIKV monitoring. Advances in tools such as Guppy, Dorado, Minimap2, and Medaka enhance read quality, consensus accuracy, and downstream analyses. Despite challenges in basecalling and error correction, robust quality control pipelines ensure reliable results. Ongoing improvements in chemistry, flow cell design, and machine learning will further enhance fidelity and throughput, establishing nanopore sequencing as a cornerstone of CHIKV genomic surveillance and epidemic preparedness.

Chikungunya virus↗
PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗