We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3X, as the extreme 3-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3 end of positive-strand viral RNA is the initiation site of RNA replication, the 3X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular

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... proteins have been shown to interact with the 3-end regions of other viral genomes. In this study, we found that both 38-and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5-terminal 19 nt of the 3X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3X-binding p57 is identical to PTB. The 3X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3X RNA. In addition, p57 bound solely to the 3-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3X-PTB interaction is involved in the specific initiation of HCV genome replication.