Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

Abstract

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

“…The essential components of the cap-dependent translation initiation machinery, EIF4A1 and EIF4E, were not stimulated by the infection in spite of the activation of their protein partner EIF4G1 (Table S1). This agrees with previous data showing that these two initiation factors do not participate in SINV sgRNA translation (Carrasco et al, 2018; Castello et al, 2006; Garcia-Moreno et al, 2015; Garcia-Moreno et al, 2013). A recent report showed that EIF3D is a cap-binding protein that substitutes EIF4F in the translation of specific mRNA pools (Lee et al, 2016).…”

A viral RNA motif involved in signaling the initiation of translation on non-AUG codons

“…Of note, there was a substantial inhibition in the synthesis of C protein in insect cells (∼70%) with replicons bearing AUG or CUG (Fig. 7C,E), likely because melting of this DSH variant is hampered by insect ribosomes as was described previously (Garcia-Moreno et al 2015). After 7 h, cells were collected in loading buffer and extracted proteins were analyzed by western blotting with an anti-C antibody.…”

“…This aminoacyl-tRNA is then translocated to the P site mediated by eEF2 in a GTP-dependent process. We have previously advanced the idea that some similarities exist between the mechanism of initiation directed by IGR CrPV and the alphavirus sgRNA (Garcia-Moreno et al 2015;Sanz et al 2017;Carrasco et al 2018).…”

“…In the case of the ternary complex aminoacyl-tRNA-eEF1A-GTP, it has to interact with the A site on the 80S ribosomes, since elongation factors have to bind to the GTPase center located at the major ribosomal subunit. We have previously suggested that this A site should be open if the P site is occupied by the DSH element (Garcia-Moreno et al 2015;Carrasco et al 2018). Our present findings are in good agreement with this idea since bruceantin had no effect on sgRNA C + luc (CUG) translation in RRL, and the blockade of viral translation by bruceantin was poorer than that for cellular protein synthesis in BHK cells infected with SINV-CUG.…”

“…Interestingly, alterations in the DSH structure result in leaky scanning, such that initiation of capsid (C) protein synthesis is observed at downstream AUG codons (Frolov and Schlesinger 1996;Sanz et al 2009). It was recently demonstrated that the initiation of protein synthesis directed by sgRNA occurs following the classical scanning model (Garcia-Moreno et al 2015); however, scanning can occur in the absence of some crucial eIFs, such as eIF4G, eIF4A, and eIF2 (Castello et al 2006;Garcia-Moreno et al 2013;Gonzalez-Almela et al 2015). The current model for translation initiation on sgRNA is that preinitiation complexes interact with the cap structure present at the 5 ′ -end, perhaps promoted by eIF3D, and without the participation of the eIF4F complex (Lee et al 2016).…”