An Internal Polypyrimidine-Tract-Binding Protein-Binding Site in the Hepatitis C Virus RNA Attenuates Translation, Which Is Relieved by the 3′-Untranslated Sequence

AbstractHepatitis C virus (HCV) RNA binds to several cellular proteins, which may regulate translation or replication of viral RNA. One of these is polypyrimidine tract-binding protein (PTB), which binds to the 5′-untranslated region (UTR) and the 3′-end 98 nucleotides (nt) (X region) of HCV RNA. Both of these PTB-binding sites regulate HCV translation. In this study, we further investigated the nature of PTB binding on HCV RNA. UV cross-linking studies using HeLa cell extracts and a recombinant PTB showed that the PTB–5′-UTR binding was much weaker than the PTB–3′-UTR binding. Unexpectedly, we found an even stronger PTB-binding site in the core-protein-coding region of HCV RNA. The binding domain was mapped to the 3′-end of this region, which contains a pyrimidine-rich sequence highly conserved among HCV isolates. Using a set of synthetic HCV RNAs with or without this sequence inin vitrotranslation studies, we showed that the PTB-binding sequence in the core-coding region strongly inhibited translation of HCV RNA. This inhibition was relieved by the presence of the X region at the 3′-end. Furthermore, the previously reported translational enhancement by the HCV 3′-UTR was more pronounced when this PTB-binding site was present in the RNA. These results suggest that PTB binding to an internal site of HCV RNA provides another mechanism for regulation of HCV translation