Factores de iniciación implicados en la traducción de mRNAS virales. Importancia de los motivos estructurales en el RNA

Abstract

Alphaviruses are positive polarity single-stranded RNA viruses responsible for several human and other animal diseases, causing usually arthrosis or encephalitis that continue to be a worldwide health threat. They are usually transmitted by blood-sucking arthropods to vertebrate hosts. Sindbis virus (SINV) is a well-characterized member of this group and is usually employed as a model system in molecular virology. SINV RNA genome contains two open reading frames (ORFs) that correspond to the translation of the genomic RNA (gRNA) and subgenomic RNA (sgRNA), translated at early or late times post-infection, respectively. SINV infection profoundly blocks cellular protein synthesis while sgRNA translation occurs, which follows a non-canonical mechanism, independent of several eukaryotic initiation factors (eIFs), which involves the scanning of 5’-UTR. In this work, we have studied the requirement of different eIFs for SINV mRNAs translation. We have examined the role of the RNA helicase eIF4A using the selective inhibitor pateamine A. Translation of SINV gRNA involves the participation of eIF4A, while translation of sgRNA is independent of this factor in infected cells but dependent in transfected cells or in cell-free systems, indicating a dual mechanism of translation. Moreover, sgRNA translation does not require eIF2, and the identification of substitute factors has been the object of intensive research in the past few years. eIF2A and eIF2D were proposed as candidates by several studies. However, we have demonstrated that eIF2A and eIF2D are not required for the translation of SINV mRNAs, even when eIF2α is phosphorylated. In addition, eIF2A and eIF2D do not participate in the translation of SINV sgRNA bearing non-AUG codons. The initiation on non-AUG codons for sgRNA translation is highly dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region of capsid protein. Furthermore, in collaboration with Dr. A. Castelló (University of Oxford), we have demonstrated that the loss of cellular mRNAs and the emergence of viral RNA induced by SINV infection alters the dynamics and activity of the compendium of RNA-biding proteins (RBPs) of the host cell. Many of the RBPs whose activity is stimulated by SINV redistribute to viral replication factories and regulate the capacity of the virus to infect. Finally, we examined the involvement of several eIFs in hepatitis C virus (HCV) IRES-driven translation in human cells in a comparative analysis with mRNAs bearing the encephalomyocarditis virus or the Cricket paralysis virus IRES elements. We concluded that eIF2, eIF2A, eIF2D, eIF4A and eIF4G are not involved in the initiation of translation driven by HCV IRES.