title: Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.
abstract: We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, 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 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular 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 3'X 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 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X 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 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.
authors.affiliation: Department of Molecular Virology, Tokyo Medical and Dental University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tsuchihara
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Hijikata
authors.forename: S
authors.initials: S
authors.lastname: Kuge
authors.forename: H
authors.initials: H
authors.lastname: Toyoda
authors.forename: A
authors.initials: A
authors.lastname: Nomoto
authors.forename: N
authors.initials: N
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Shimotohno
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D038941:Polypyrimidine Tract-Binding Protein; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D038941:Polypyrimidine Tract-Binding Protein