Mecanismos de iniciación de la traducción de los mRNAS del virus Sindbis

Resumo

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 eIF2¿ 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.