Mecanismos de iniciación de la traducción de los mRNAS del virus Sindbis
Author
García Moreno, ManuelEntity
UAM. Departamento de Biología Molecular; Centro de Biología Molecular Severo Ochoa (CBM)Date
2015-04-20Funded by
El trabajo presentado en esta tesis doctoral ha sido realizado mediante la concesión de la beca FPI BES-2010- 039571 del Ministerio de Economía y Competitividad. Ha sido financiado por el proyecto BFU2009-07352 otorgado por la Dirección General de Investigación Científica y Técnica, Ministerio de Economía y CompetitividadSubjects
Alphavirus - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-04-2015Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Alphaviruses are responsible for a variety of diseases, including some fatal encephalitis, in humans
and other vertebrates. Sindbis virus (SINV) is a representative member of alphaviruses, which contain a
positive single-stranded RNA as genome. In the early phase after infection, this genomic RNA is translated
following a canonical mechanism, while a subgenomic mRNA (sgmRNA) directs the synthesis of viral
structural proteins during the late phase of infection, when the translation of host cellular mRNAs has been
shut-off. The mechanism by which this sgmRNA is translated under these conditions has been the object of
intensive research during the past few years.
In the present work we demonstrate that the eukaryotic initiation factor (eIF) 4A is not required to
translate SINV sgmRNA in mammalian infected cells. However, eIF4A is required for efficient translation in
cells transfected with in vitro synthesized sgmRNA. Therefore, this viral mRNA exhibits a dual mechanism
for its translation. In addition, other eIFs including eIF4E or PABP may not participate in this process in
infected cells. Furthermore, in this viral mRNA exists a hairpin structure (DLP) that confers eIF2, but not
eIF4A independence. In sharp contrast to what is observed in mammalian cells, active eIF2 is necessary to
translate SINV sgmRNA in insect cells, and the DLP does not confer any translational advantage.
Nevertheless, eIF4A is dispensable for SINV sgmRNA translation in mosquito cells. These findings indicate
that SINV sgmRNA translation requires different initiation factors in vertebrate and insect cells. Besides, we
show that the translation mechanism of this mRNA involves the scanning of the 5’ untranslated region (5’-
UTR) by the preinitiation complex in both cell lines. Moreover, we have constructed some viral mRNAs
bearing two AUG initiation codons that respond differentially to eIF2 inactivation, indicating that AUG
selection is dependent on the cellular context, the phosphorylation state of eIF2α and the presence of
regulatory elements such as DLP. Collectively, these results suggest that translation of alphavirus sgmRNA
follows a novel scanning mechanism to initiate translation and to select the correct AUG initiation codon,
without the participation of crucial eIFs.
Finally, we found a new motif, constituted by three repeated sequences, at the 3’-UTR that are
necessary for the translation of both SINV genomic and subgenomic mRNAs in insect, but not in
mammalian systems. Complementary sequences at the 5’-UTR also regulate SINV mRNA translation and
transcription. Thus, these SINV repeated regions constitute the first example of a motif present at the 3’-
UTR that provides translatability to mRNAs from an animal virus in a cell specific manner. In addition, these
findings constitute a clue to better understand, at the molecular level, the evolution of alphaviruses and other
arboviruses.
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