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

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.

Figure 1:. Schematic of the experimental procedure.

PCR is used to generate a DNA template with the desired elements. mRNA encoding a luciferase reporter gene is synthesized in vitro using a T7 RNA polymerase-based system. A Firefly luciferase (Fluc) mRNA is co-transfected with a Renilla luciferase (Rluc) mRNA into uninfected or VACV-infected cells. Luciferase activities are measured using a luminometer with dual luciferase capability.

Figure 2:. Primer design and PCR-based DNA amplification.

(A) The forward primer is synthesized to include an 8nt random sequence, T7 promoter followed by a desired 5′-UTR and part of the 5′ end of the luciferase reporter gene, while the reverse primer includes a T-tract to generate poly(A) tail and the 3′ end of the luciferase reporter gene. By overhang extension PCR using a plasmid template containing a luciferase gene, a DNA template is generated. (B) DNA band of the desired size from PCR reaction was detected using 1% agarose TAE gel electrophoresis.

Figure 3:. mRNA synthesis and transfection.

(A) Schematic of in vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5′-UTR of interest and the T7 promoter is used as the template. The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5′ to 3′ direction. Once mRNA is 25–30 nt long, m⁷G cap is added using an anti-reverse cap analog, ARCA. (B) RNA bands from in vitro transcription ware detected using 1.5% agarose TBE gel electrophoresis. (C) Schematic demonstrating the transfection of reporter mRNA into cells. Medium containing either the reporter mRNA or cationic lipid transfection reagent in separate tubes is allowed to equilibrate at room temperature for 5 min. The solutions are then mixed followed by incubation at room temperature for 15 min after which the RNA/transfection reagent mixture is added into cells in culture plates.

Figure 4:. Increased translational efficiency of mRNA containing a 5′-poly(A) leader.

(A) Fluc mRNA containing a poly(A) leader in the 5′-UTR and Rluc mRNA with the Kozak consensus sequence in the 5′-UTR are co-transfected into cells. (B) Fluc mRNA with 5′-poly(A) leader was transfected in uninfected and VACV-infected cells along with Rluc mRNA. 5 hpi, luciferase activity was measured using a luminometer. Rluc normalized Fluc activity is represented in uninfected and VACV-infected cells. Error bars indicate the standard deviations (SD) of at least three repeats. Student’s t-test was used to determine P-values; ***P-value < 0.001.