Hepatitis C Virus 39X Region Interacts with Human Ribosomal Proteins

To identify proteins that can bind the 3* untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3*-terminal 98 nucleotides (3*X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3*X region and these proteins by a three-hybrid mating assay using mutant 3*X sequences. We have further characterized the interaction between 3*X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3*X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3*X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase [GST] fusion protein) together with a 3*X riboprobe. The 3*X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3*X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.