Abstract
Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.
Highlights
Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors
To measure the translation capability of such an altered riboproteome, we used in vitro– transcribed mRNA from which translation is driven by several 5ЈUTRs upstream of the luciferase reporter gene; the cricket paralysis virus (CrPV) IRES, which does not require eukaryotic translation initiation factor 3 (eIF3) for translation, was translated in parallel as a positive control
It has been clearly shown that eIF3 binds the IIIabc stem–loop domain of this RNA motif, and mutations in this sequence lead to complete inactivation of the IRES
Summary
Several cryo-EM studies have led to an anthropomorphic model of the eIF3 general organization (Fig. 1A). From what was observed with the constructs derived from cellular RNAs, the activity of the HCV IRES-driven translation was not decreased when the eIF3eKD riboproteome was used, despite the fact that this sequence has been shown to bind the eIF3 complex (Fig. 2C) This was true whether or not a 5Ј cap and/or a poly(A) tail was added to the reporter mRNA. Whereas under HCV infection conditions, eIF3e silencing led to a decrease in several cellular translation initiation factors such as eIF4E, PABP, eIFA1, and eIF2␣, possibly due to the marked decrease in eIF3e as a consequence of the combined effects of infection and siRNA transfection, none of the viral protein levels were reduced (Fig. 7B) This absence of effect of eIF3eKD on viral expression was observed by flow cytometry monitoring the expression level of the NS5A protein (Fig. 7, C and D). In agreement with the in vitro data, eIF3e silencing did not impact HCV replication and protein expression
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