Affiliation:
1. Unité de Régulation de la Traduction Eucaryote et Virale, CNRS URA 1966, Institut Pasteur, Paris, France
2. Department of Biochemistry, University of Cambridge, Cambridge, England
Abstract
ABSTRACT
Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5′ noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5°C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Reference39 articles.
1. Agol, V. I. 1991. The 5′-untranslated region of picornaviral genomes. Adv. Virus Res. 40 : 103-180.
2. Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma
3. Andino, R., G. E. Rieckhof, and D. Baltimore. 1991. A functional ribonucleoprotein complex forms around the 5′end of poliovirus RNA. Cell 63 : 369-380.
4. Barton, D. J., B. J. O'Donnell, and J. B. Flanegan. 2001. 5′ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J. 20 : 1439-1448.
5. Beales, L. P., D. J. Rowlands, and A. Holzenburg. 2001. The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy. RNA 7 : 661-670.
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