Acta Pharmacologica Sinica 2008 November; 29 (11): 1327-1333; doi: 10.1111/j.1745-7254.2008.00876.x

Acta Pharmacologica Sinica 2008 November; 29 (11): 1327-1333; doi: 10.1111/j.1745-7254.2008.00876.x

Original Article

Amantadine as a regulator of internal ribosome entry site¹

Ying-ju CHEN², Shih-jhan ZENG², John TA HSU^3,4, Jim-tong HORNG⁵, Hong-ming YANG³, Shin-ru SHIH⁶, Yu-ting CHU², Tzong-yuan WU^2,7

²Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung-Li, Taiwan, China; ³Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan, China; ⁴Department of Chemical Engineering, National Tsing Hua Unversity, Hsinchu, Taiwan, China; ⁵Department of Biochemistry, Chang Gung University, Kweishan, Taoyuan, Taiwan, China; ⁶School of Medical Technology, Chang Gung University, Taoyuan, Taiwan & Clinical Virology Laboratory, Department of Clinical Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, China

Aim: Studies of eukaryotes have yielded 2 translation initiation mechanisms: a classical cap-dependent mechanism and a cap-independent mechanism proceeding through the internal ribosomal entry site (IRES). We hypothesized that it might be possible to identify compounds that may distinguish between cap-dependent translation and cap-independent IRES-mediated translation.

Methods: To facilitate compound screening, we developed bicistronic reporter constructs containing a β-galactosidase gene (β-gal) and a secreted human placental alkaline phosphatase (SEAP) reporter gene. Following transcription, the β-gal gene is translated by a cap-dependent mechanism, while SEAP expression is controlled by the IRES derived from either enterovirus 71 (EV-71) or encephalomyocarditis virus (EMCV). This assay could potentially identify compounds that inhibit SEAP expression (cap-independent) without affecting β-gal activity (cap-dependent).

Results: Using a bicistronic plasmid-based transient transfection assay in the COS-1 cells, we identified amantadine, a compound that inhibited the IRES of EV71- and EMCV-mediated cap-independent translation but did not interfere with cap-dependent translation when the dose of amantadine was lower than 0.25 mg/mL.

Conclusion: These results imply that amantadine may distinguish between cap-dependent translation and cap-independent IRES-mediated translation and can be used to regulate gene expression at a translational level.

Keywords: amantadine; Enterovirus 71; internal ribosome entry site; riboswich

¹This work was supported by Grants NSC-94-2317-B-033-001 and NSC-95-2317-B-033-001 to TY WU.

⁷Correspondence to Prof Tzong-yuan WU.
Phn/Fax 886-3-265-3599.
E-mail tywu@cycu.edu.tw
Received 2008-05-06 Accepted 2008-08-13

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