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J Microbiol Biotechnol. 2019 Jan 28;29(1):127-140. doi: 10.4014/jmb.1811.11019.

Development of an RNA Expression Platform Controlled by Viral Internal Ribosome Entry Sites.

Author information

1
Department of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.
2
Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Yongin 17104, Republic of Korea.
3
Cancer Imaging Center; Seoul National University Hospital, Seoul 08826, Republic of Korea.
4
Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, Seoul 08826, Republic of Korea.

Abstract

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

KEYWORDS:

Internal ribosome entry sites; RNA expression platform; coxsackievirus B3; cricket paralysis virus; encephalomyocarditis virus; poly(A)

PMID:
30518020
DOI:
10.4014/jmb.1811.11019
[Indexed for MEDLINE]
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