SPECIFIC INTERACTION OF POLYPYRIMIDINE TRACT-BINDING PROTEIN WITH THEEXTREME 3'-TERMINAL STRUCTURE OF THE HEPATITIS-C VIRUS GENOME, THE 3'X

We previously identified a highly conserved 98-nucleotide (nt) sequenc e, the 3'X, as the extreme 3'-terminal structure of the hepatitis C vi rus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Bi ochem. Biophys. Res. Commun. 215:744-749, 1995), Since the 3' end of p ositive-strand viral RNA is the initiation site of RNA replication, th e 3'X should contribute to HCV negative-strand RNA synthesis, Cellular factors mag also be involved in this replication mechanism, since sev eral cellular proteins have been shown to interact with the 3'-end reg ions 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 cr oss-linking assay, The 57-kDa protein (p57), which had higher affiniti es to RNA probes, recognized a 26-nt sequence including the 5'-termina l 19 nt of the 3'X and 7 flanking nt, designated the transitional regi on. This sequence contains pyrimidine-rich motifs and show's similarit y to the consensus binding sequence of the polypyrimidine tract-bindin g protein (PTB), which has been implicated in alternative pre-mRNA spl icing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB hound to the 3'X RNA. In addit ion, p57 bound solely to the 3'-end region of positive-strand RNA not to this region of negative-strand RNA, We suggest that 3'X-PTB interac tion is involved in the specific initiation of HCV genome replication.