Abstracts Biological Sciences

Selective translation of influenza viral messenger RNAs mediated by trans-acting factor(s) through an interaction with the sequence element in the 5'-untranslated region

by Youngwoo Park

In cells infected by influenza virus, cellular mRNAs are subject to the translation shutoff while viral mRNAs are preferentially translated. Such preferential viral mRNA translation has been shown to be directed by the 5 ' untranslated region (UTR) of viral mRNAs. In an effort to elucidate the molecular mechanism underlying the selective translation, we attempted to identify trans-acting factors using in vitro protein-RNA binding analyses.We found that the viral 5' UTR interacted with several cellular proteins. To further characterize these RNA-binding proteins, their cDNAs were screened in a HeLa cDNA library using a yeast three-hybrid system. The yeast three-hybrid system, which has a major drawback of the frequent occurrence of false positives due to bait RNA-independent activation of the reporter gene, was modified to easily identify false positives. Using this system, ∼230 RNA-dependent positive clones were selected and, in a subsequent counterscreen, one showed a specific interaction with the NP 5' UTR. This cDNA encodes a cellular RNA binding protein, GRSF-1, which has the three RNA recognition motifs. The binding specificity of GRSF-1 was confirmed in vitro. Moreover, we found that GRSF-1 preferentially bound to the AGGGU pentamer sequence present in the 5'UTRs of viral mRNAs.More importantly, we demonstrated that GRSF-1 mediates the selective translation of the viral mRNAs using a cell-free translation system. Recombinant GRSF-1 enhanced translation of the viral 5'UTR-driven template. Moreover, the depletion of GRSF-1 from HeLa cell extract compromised the viral 5'UTR-driven translation, which was subsequently restored in HeLa extracts reconstituted with recombinant GRSF-1. The functional role of GRSF-1 was further substantiated in HeLa cell extract treated with excess amounts of ribooligonucleotides. Viral 5'UTR-driven translation was also compromised in HeLa extracts treated with ribooligonucleotides containing the GRSF-1-binding site, but this translational lesion could be recovered by recombinant GRSF-1. These results clearly demonstrate that GRSF-1 directs the preferential translation of the viral mRNA through an interaction with the viral 5'UTR. Interestingly, GRSF-1 appears to associate with the cytoskeleton as a RNP complex in cells. Several potential models for the selective translation of the viral mRNAs are proposed.