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J. Biol. Chem., Vol. 279, Issue 5, 3389-3397, January 30, 2004

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Identification of a Novel Internal Ribosome Entry Site in Giardiavirus That Extends to Both Sides of the Initiation Codon^*

Srinivas Garlapati and Ching C. Wang

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280

In Giardia lamblia, enhanced translation of luciferase mRNA, flanked between the 5'-untranslated region (UTR) and 3 '-end of giardiavirus transcript, requires the presence of the initial 264-nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264-nt downstream region alone is insufficient to function as an internal ribosome entry site (IRES) without including a portion of the 5 '-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nts 114–367) of the 5 '-UTR in combination with the downstream 264 nts. Specific mutations that disrupted the predicted secondary structural elements in either the 5 '-UTR or the 264-nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 517 nts that extends to both sides of the initiation site.

Received for publication, July 14, 2003 , and in revised form, November 7, 2003.

^* This work was supported by Grant AI-30475 from the National Institutes of Health. 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Figs. I and II.

To whom correspondence should be addressed. Tel.: 415-476-1321; Fax: 415-476-3382; E-mail: ccwang{at}cgl.ucsf.edu.

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