Vol 51(2017) N 2 p. 283-292; DOI 10.1134/S002689331702011X
F. Ghassemi1, O. Madadgar1, F. Roohvand2, M. Rasekhian3, M.H. Etemadzadeh2, G.R.N. Boroujeni1, A.G. Langroudi1, K. Azadmanesh2*
Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines1Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2Department of Virology, Pasteur Institute of Iran, Tehran, Iran
3Department of Pharmacognosy and Pharmaceutical Biotechnology, School of Pharmacy, Kermanshah University of Medical Science, Kermanshah, Iran
Received - 2015-11-14; Accepted - 2016-01-03
Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were -544.00 and -469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.
BVDV-IRES, EMCV-IRES, T7 RNA polymerase, translational efficiency