Hepatitis C virus (HCV) RNA contains a highly conserved 5'-noncoding region (5'NCR) which may be important in viral multiplication. To study the possible mechanisms of the cellular proteins involved in HCV replication and pathogenesis, a gel mobility shift assay and competition analysis were performed with the HCV 5'NCR. Two specific complexes were formed between the 341-nucleotide RNA of the HCV 5'NCR and proteins of mammalian cells. The specific RNA-protein complexes were maintained in the region of the 5'NCR from nucleotides 131 to 253. Nevertheless, the slower migrating RNA-protein complex failed to form when a polypyrimidine tract sequence (191-UCCUUUCUU-199) in the stem-loop III structure of HCV 5'NCR was changed to 191-UCCUUUggU-199. A uv cross-linking assay further identified two cellular proteins, p87 and p120, that specifically bound to the stem-loop III structure. Mutations at the polypyrimidine tract sequence inhibited the binding of p87, but maintained the ability of the mutant HCV RNA to interact with p120. Translation competition assay demonstrated that the 5'NCR from nt 131 to 253 within the stem-loop III structure is important for the translation of HCV core protein. In addition, p120 and unidentified cellular proteins are likely to be involved in the translation of HCV polyprotein, whereas p87 may play important roles in HCV multiplication other than translation.
