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J. Biol. Chem., Vol. 278, Issue 23, 20565-20573, June 6, 2003

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Synergism of the 3'-Untranslated Region and an Internal Ribosome Entry Site Differentially Enhances the Translation of a Plant Virus Coat Protein^*

Dora Chin-Yen Koh  , Sek-Man Wong  ¶ and Ding Xiang Liu || ** 

From the Department of Biological Sciences, The National University of Singapore, 14 Science Dr. 4, Singapore 117543, the ^||Institute of Molecular Agrobiology, 1 Research Link, Singapore 117604, and the ^**School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Block 5, Level 3, Singapore 637616, Republic of Singapore

The use of internal ribosome entry sites (IRESs) is one of the unorthodox mechanisms exploited by viruses to initiate the translation of internal genes. Herein, we report a plant virus exploiting an IRES and its 3'-untranslated region (UTR) to express its internal genes, notably the 3'-proximal viral coat protein gene. Hibiscus chlorotic ringspot virus (HCRSV), a positive-strand non-polyadenylated RNA virus, was demonstrated to harbor a unique 100-nucleotide (nt) IRES, located 124 nt upstream of the coat protein gene, that could function in wheat germ extract, rabbit reticulocyte lysate, and mammalian cells. In comparison with other known IRESs of picornaviruses and eukaryotic mRNAs, this 100-nt IRES is distinctively short and simple. The IRES activity was tested in homologous and heterologous bicistronic constructs, and the expression of the 3'-proximal gene was enhanced when the 3'-UTR was present. When the IRES element was bisected, each half still possessed IRES activity and could initiate internal translation on its own. Site-directed mutagenesis and deletion analyses revealed that the primary sequence within the 5' half was crucial for IRES activity, whereas the primary sequence of the second half and a GNRA motif were non-essential. To our knowledge, this is the first report describing a mechanism whereby an IRES, located in the 3' portion of the virus genome, co-operates with the 3'-UTR to enhance gene expression differentially.

Received for publication, October 5, 2002 , and in revised form, March 25, 2003.

^* This research was supported by the National Science and Technology Board, Singapore and the National University of Singapore (NUS) through Research Grant R-154-000-111-112. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the Singapore Millennium Foundation Scholarship and the President's Graduate Fellowship (NUS).

^¶ To whom correspondence may be addressed. Tel.: 65-68-74-2976; Fax: 65-77-95-671; E-mail: dbswsm{at}nus.edu.sg. To whom correspondence may be addressed. Tel.: 65-67-90-3738; Fax: 65-68-96-8032; E-mail: dxliu{at}ntu.edu.sg.

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