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Structural analysis of a RNA viral element and its translation initiation factor partner, both controlling cap-independent translation of viral RNAs

AutorMiras, Manuel ; Querol-Audí, Jordi ; Truniger, Verónica ; Silva-Espiña, Cristina ; Verdaguer, Núria ; Aranda, Miguel A.
Fecha de publicación11-jul-2015
Citación35th Annual Meeting American Society for Virology (2015)
ResumenIn animal viruses, internal ribosome entry sites at the 5¿-unstranslated regions (UTRs) of viral RNAs are the most frequently found translation control elements. In contrast to animal viruses, plant positive-strand RNA viruses, in the absence of a 5¿cap, have evolved 3¿ cap-independent translational enhancers (3¿-CITEs) in their 3¿-UTRs. It has been reported that these 3¿-CITEs require and directly bind eukaryotic translation initiation factors (eIFs) for their function. We have shown that capindependent translation of Melon necrotic spot virus (MNSV) RNAs is controlled by a 3¿-CITE in cis (Truniger et al., 2008). Remarkably, MNSV 3¿-CITEs are diverse, including at least M¿5TE, M264TE and CXTE (Truniger et al., 2008; Miras et al., 2014). Genetic evidence indicates that the eIF4E subunit of melon eIF4F is necessary for cap-independent translation of MNSV RNAs harboring M¿5TE (Nieto et al., 2006); in contrast, M264TE and CXTE are both eIF4E independent, conferring in cis translational competence to viral RNAs in the absence of this factor (Miras et al., 2014). In this study, we have performed structural and functional analysis of M¿5TE. Thus, we defined the minimal size of the 3¿-CITE in ¿in vivo¿ translation assays to a sequence of 45 nucleotides. Its secondary structure in solution was studied by Selective 2¿-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and compared with those of the other two types of 3¿-CITEs. On the other hand, we have expressed, crystalized and determined by X-ray crystallography the 3D structure of melon eIF4E and a truncated version of eIF4F complex (eIF4E-eIF4G1003-1092). Binding to eIF4G1003-1092 significantly alters eIF4E conformation. UV-crosslinking assays indicated the formation revealed eIF4F-binding sites on a bulge of M¿5TE.Mutational analyses of Ma5TE revealed residues involved in (inside the bulge associated with loss of) binding to eIF4F. Further structural and functional analyses of M¿5TE:eIF4F are in progress. References: Miras, M., Sempere, R.N., Kraft, J., Miller, A.W., Aranda, M.A. and Truniger, V. 2014. New Phytologist 202: 233-246. Nieto, C., Rodriguez-Moreno, L., Rodriguez-Hernández, A.M., Aranda., M.A. and Truniger, V. 2011. The Plant Journal, 66, 492-501. Truniger, V., Nieto, C., González-Ibeas, D. and Aranda, M.A. 2008. The Plant Journal 56:716-727
DescripciónPóster presentado en el 35th Annual Meeting American Society for Virology, celebrado del 11 al 15 de julio de 2015 en London, Ontario (Canadá)
URIhttp://hdl.handle.net/10261/129714
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