The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Sindbis virus (SINV) contains an RNA genome of positive polarity with two open reading frames (ORFs). The first ORF is translated from the genomic RNA (gRNA), rendering the viral non-structural proteins, whereas the second ORF is translated from a subgenomic mRNA (sgRNA), which directs the synthesis of viral structural proteins. SINV infection strongly inhibits host cell translation through a variety of different mechanisms, including the phosphorylation of the eukaryotic initiation factor eIF2α and the redistribution of cellular proteins from the nucleus to the cytoplasm. A number of motifs have been identified in SINV sgRNA, including a hairpin downstream of the AUG initiation codon, which is involved in the translatability of the viral sgRNA when eIF2 is inactivated. Moreover, a 3′-UTR motif containing three stem-loop structures is involved in the enhancement of translation in insect cells, but not in mammalian cells. Accordingly, SINV sgRNA has evolved several structures to efficiently compete for the cellular translational machinery. Mechanistically, sgRNA translation involves scanning of the 5′-UTR following a non-canonical mode and without the requirement for several initiation factors. Indeed, sgRNA-directed polypeptide synthesis occurs even after eIF4G cleavage or inactivation of eIF4A by selective inhibitors. Remarkably, eIF2α phosphorylation does not hamper sgRNA translation during the late phase of SINV infection. SINV sgRNA thus constitutes a unique model of a capped viral mRNA that is efficiently translated in the absence of several canonical initiation factors. The present review will mainly focus in the non-canonical mechanism of translation of SINV sgRNA.

Animal viruses have evolved a variety of strategies to ensure the efficient translation of their mRNAs. One such strategy is the use of internal ribosome entry site (IRES) elements, which circumvent the requirement for some eukaryotic initiation factors (eIFs). Much effort has been directed to unravel the precise mechanism of translation initiation by hepatitis C virus (HCV) mRNA. In the present study, we examined the involvement of several eIFs in HCV IRES-driven translation in human cells in a comparative analysis with mRNAs bearing the encephalomyocarditis virus or the Cricket paralysis virus IRES element. Consistent with previous findings, several inhibitors of eIF2 activity, including sodium arsenite, thapsigargin, tunicamycin, and salubrinal, had no inhibitory effect on the translation of an mRNA bearing the HCV IRES, and all induced the phosphorylation of eIF2α. In addition, hippuristanol and pateamine A, two known inhibitors of eIF4A, failed to block HCV IRES-directed translation. To test the release of nuclear proteins to the cytoplasm and to analyze the formation of stress granules, the location of the nuclear protein TIA1 was tested by immunocytochemistry. Both arsenite and pateamine A could efficiently induce the formation of stress granules containing TIA1 and eIF4G, whereas eIF3 and eIF2 failed to localize to these cytoplasmic bodies. The finding of eIF4A and eIF4G in stress granules suggests that they do not participate in mRNA translation. Human HAP1 cells depleted for eIF2A, eIF2D, or both factors, were able to synthesize luciferase from an mRNA bearing the HCV IRES even when eIF2α was phosphorylated. Overall, these results demonstrate that neither eIF2A nor eIF2D does not participate in the translation directed by HCV IRES. We conclude that eIF2, eIF4A, eIF2A, and eIF2D do not participate in the initiation of translation of HCV mRNA.

Translation of Sindbis virus subgenomic mRNA (sgmRNA) can occur after inactivation of eIF2 by phosphorylation in mammalian cells. Several studies have suggested that eIF2 can be replaced by eIF2A or eIF2D. HAP1 human cell lines knocked-out for eIF2A, eIF2D or both by CRISPR/Cas9 genome engineering were compared with wild-type (WT) cells to test the potential role of eIF2A and eIF2D in translation. Sindbis virus infection was comparable between the four cell lines. Moreover, synthesis of viral proteins during late stage infection was similar in all four cell lines despite the fact that eIF2α became phosphorylated. These findings demonstrate that eIF2A and eIF2D are not required for the translation of sgmRNA when eIF2α is phosphorylated. Moreover, silencing of eIF2A or eIF2D by transfection of the corresponding siRNAs in HAP1 WT, HAP1-eIF2A⁻ and HAP1-eIF2D⁻ cells had little effect on the synthesis of viral proteins late in infection. Modification of AUG_i to other codons in sgmRNA failed to abrogate translation. Sindbis virus replicons containing these sgmRNA variants could still direct the synthesis of viral proteins. No significant differences were found between the cell lines assayed, suggesting that neither eIF2A nor eIF2D are involved in the translation of this sgmRNA bearing non-AUG codons.

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.