Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 59 and 39 termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein.HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 59-39 interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 59untranslated region (59UTR) and structured sequence elements within the 39UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 59cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 39UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 59UTR and 39UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 59cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 59UTR and HCV 39UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 59UTR and/or HCV 39UTR, recruits eIF3 and enhances HCV IRES-mediated translation.

The modifier of mdg4, mod(mdg4), locus in Drosophila melanogaster represents a new type of complex gene in which functional diversity is resolved by mRNA trans-splicing. A protein family of Ͼ30 transcriptional regulators, which are supposed to be involved in higher-order chromatin structure, is encoded by both DNA strands of this locus. Mutations in mod(mdg4) have been identified independently in a number of genetic screens involving position-effect variegation, modulation of chromatin insulators, apoptosis, pathfinding of nerve cells, and chromosome pairing, indicating pleiotropic effects. The unusual gene structure and mRNA trans-splicing are evolutionary conserved in the distantly related species Drosophila virilis. Chimeric mod(mdg4) transcripts encoded from nonhomologous chromosomes containing the splice donor from D. virilis and the acceptor from D. melanogaster are produced in transgenic flies. We demonstrate that a significant amount of protein can be produced from these chimeric mRNAs. The evolutionary and functional conservation of mod(mdg4) and mRNA trans-splicing in both Drosophila species is furthermore demonstrated by the ability of D. virilis mod(mdg4) transgenes to rescue recessive lethality of mod(mdg4) mutant alleles in D. melanogaster.T HE majority of genes in higher eukaryotes repreencoded by the four 5Ј-exons but differ in their C-terminal region encoded by alternative 3Ј-exons. This kind sents monocistronic units where noncoding intron regions interrupt the protein-coding exon sequences.of trans-splicing clearly differs from splice leader transsplicing that predominates in Caenorhabditis and TryThe resulting mature mRNA usually encodes a unique panosomes where polycistronic transcripts are resolved polypeptide. Recent advances in genome analysis of sevby addition of noncoding leader sequences (Blumeneral model organisms and the molecular characterizathal 1998). Mutational dissection and differential bindtion of a large number of genes revealed that alternative ing of Mod(mdg4) isoforms on polytene chromosomes pre-mRNA splicing is one of the main mechanisms gensuggest that the variable C-terminal regions encoded by erating a highly expanded proteome diversity.