In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

• Published in: Journal of visualized experiments no. 147
• Main Authors: Dhungel, Pragyesh, Cantu, Fernando, Hernandez, Candy, Yang, Zhilong
• Format: Journal Article
• Language: English
• Published: United States 01.05.2019
• Subjects: DNA, Viral - genetics; Genes, Reporter; HeLa Cells; Humans; Luciferases - genetics; Poly A - metabolism; Poxviridae - physiology; Protein Biosynthesis; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Viral - genetics; Transcription, Genetic; Virus Replication
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/59626

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.