Hepatitis C virus 3'X 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.
Wood
J
J
MRC Virology Unit, Institute of Virology, Glasgow G11 5JR, United Kingdom.
Frederickson
R M
RM
Fields
S
S
Patel
A H
AH
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D012367:RNA, Viral / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D020413:3' Untranslated Regions; D012367:RNA, Viral; D012269:Ribosomal Proteins