CELLULAR PROTEINS SPECIFICALLY BIND TO THE 5'-NONCODING REGION OF HEPATITIS-C VIRUS-RNA

Hepatitis C virus (HCV) RNA contains a highly conserved 5'-noncoding r egion (5'NCR) which may be important in viral multiplication. To study the possible mechanisms of the cellular proteins involved in HCV repl ication and pathogenesis, a gel mobility shift assay and competition a nalysis were performed with the HCV 5'NCR. Two specific complexes were formed between the 341-nucleotide RNA of the HCV 5'NCR and proteins o f 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 polypyr imidine tract sequence (191-UCCUUUCUU-199) in the stem-loop III struct ure of HCV 5'NCR was changed to 191-UCCUUUggU-199. A uv cross-linking assay further identified two cellular proteins, p87 and p120, that spe cifically bound to the stem-loop III structure. Mutations at the polyp yrimidine tract sequence inhibited the binding of p87, but maintained the ability of the mutant HCV RNA to interact with p120. Translation c ompetition assay demonstrated that the 5'NCR from nt 131 to 253 within the stem-loop III structure is important for the translation of HCV c ore protein. in addition, p120 and unidentified cellular proteins are likely to be involved in the translation of HCV polyprotein, whereas p 87 may play important roles in HCV multiplication other than translati on. (C) 1995 Academic Press, Inc.