Regulation of eukaryotic protein synthesis: Selective influenza viral mRNAtranslation is mediated by the cellular RNA-binding protein GRSF-1

To better understand regulation of eukaryotic protein synthesis, we studied cellular and viral mRNA translation in influenza virus infected cells, Inf luenza virus infection results in a dramatic shut-off of cellular protein s ynthesis that is concomitant with selective viral mRNA translation. Earlier work showed that these events are mediated by viral and/or cellular factor s binding to the 5' untranslated region (5' UTR) of viral mRNAs, To identif y trans-acting cellular proteins responsible for selective viral protein sy nthesis, we employed the yeast three-hybrid system. Using the 5' UTR of the influenza virus nucleocapsid protein (NP) mRNA as bait, we identified the cellular RNA-recognition motif containing RNA-binding protein G-rich sequen ce factor 1 (GRSF-1) as a positive-acting translational regulatory factor. The in vivo yeast assay revealed GRSF-1 specifically bound to the NP 5' UTR but not select NP 5' UTR mutants or cellular RNA 5' UTRs, These data were confirmed by gel shift assays using recombinant GRSF-1, Importantly, recomb inant GRSF-1 specifically stimulated translation of a NP 5' UTR-driven temp late in cell-free translation systems. Furthermore, translation efficiency of NP 5' UTR-driven templates was reduced markedly in GRSF-1-depleted HeLa cell extracts, but restored in GRSF-1-reconstituted extracts. GRSF-1 also s timulated translation of an NP 5' UTR-driven template in HeLa cell extracts that were depleted of essential factors by addition of RNA oligonucleotide s representing the viral 5' UTR RNA. Taken together, these data document th e functional demonstration of a cellular protein binding to influenza virus RNAs and, importantly, suggest that influenza virus may recruit GRSF-1 to the 5' UTR to ensure preferential translation of viral mRNAs in infected ce lls.