ArticleJournal of visualized experiments : JoVE2019
In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells.
Article in Journal of visualized experiments : JoVE, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- A Site-Specific Self-Association of a Protein Hub Drives Its Phase Separation.ACS chemical biology · 2026Article
- A structural element within the 5'UTR of β-catenin mRNA modulates its translation under hypoxia.Nucleic acids research · 2025Article
- Current Analytical Strategies for mRNA-Based Therapeutics.Molecules (Basel, Switzerland) · 2025Review
- Development of a reporter feline herpesvirus-1 for antiviral screening assays.Veterinary research · 2024Article
- La-related protein 4 is enriched in vaccinia virus factories and is required for efficient viral replication in primary human fibroblasts.Microbiology spectrum · 2023Article
- La-related protein 4 is enriched in vaccinia virus factories and is required for efficient viral replication in primary human fibroblasts.bioRxiv : the preprint server for biology · 2023Article
- Article
- Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs.PLoS pathogens · 2020Article
- Vaccinia Virus as a Master of Host Shutoff Induction: Targeting Processes of the Central Dogma and Beyond.Pathogens (Basel, Switzerland) · 2020Review
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Authors and funding
4 authors.
Funding
Abstract
Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. To dissect the role of 5'-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5'-UTR and 3'-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.
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