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Eukaryotic Cell, April 2005, p. 742-754, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.742-754.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Structural Elements in the 5'-Untranslated Region of Giardiavirus Transcript Essential for Internal Ribosome Entry Site-Mediated Translation Initiation{dagger}

Srinivas Garlapati and Ching C. Wang*

Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94107-2280

Received 3 February 2005/ Accepted 15 February 2005

Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5'-untranslated region (5'-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5'-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5'-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5'-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.

* Corresponding author. Mailing address: Dept. of Pharmaceutical Chemistry, University of California San Francisco, Mission Bay Genentech Hall, 600 16th St., San Francisco, CA 94107-2280. Phone: (415) 476-1321. Fax: (415) 476-3382. E-mail: ccwang{at}cgl.ucsf.edu.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, April 2005, p. 742-754, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.742-754.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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