Nucleic Acids Research, 2002, Vol. 30, No. 20 4398-4405
© 2002 Oxford University Press
IRES-driven translation is stimulated separately by the FMDV 3'-NCR and poly(A) sequences
1 Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas—Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain and 2 CISA-INIA, Valdeolmos, 28130 Madrid, Spain
*To whom correspondence should be addressed. Tel: +34 91 3975533; Fax: +34 91 3974799; Email: emartinez{at}cbm.uam.es
Present address:
Sonia López de Quinto, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg 69117, Germany
The 3' end region of foot-and-mouth disease virus (FMDV) consists of two distinct elements, a 90 nt untranslated region (3'-NCR) and a poly(A) tract. Removal of either the poly(A) tract or both the 3'-NCR and the poly(A) tract abrogated infectivity in susceptible cells in the context of a full-length cDNA clone. We have addressed the question of whether the impairment of RNA infectivity is related to defects at the translation level using a double approach. First, compared to the full-length viral RNA, removal of the 3' sequences reduced the efficiency of translation in vitro. Secondly, a stimulatory effect of the 3' end sequences on IRES-dependent translation was found in vivo using bicistronic constructs. RNAs carrying the FMDV 3' end sequences linked to the second cistron showed a significant stimulation of IRES-dependent translation, whereas cap-dependent translation was not affected. Remarkably, IRES-dependent stimulation exerted by the poly(A) tract or the 3'-NCR seems to be the result of two separate events, as the 3'-NCR alone enhanced IRES activity on its own. Under conditions of FMDV Lb protease-induced translation shut-off, the stimulation of IRES activity reached values above 6-fold in living cells. A northern blot analysis indicated that IRES stimulation was not the consequence of a change in the stability of the bicistronic RNA produced in transfected cells. Analysis of the RNA-binding proteins interacting with a mixture of 3' end and IRES probes showed an additive pattern. Altogether, our results strongly suggest that individual signals in the viral 3' end ensure stimulation of FMDV translation.
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