An internal polypyrimidine-tract-binding protein-binding site in the hepatitis C virus RNA attenuates translation, which is relieved by the 3′-untranslated sequence

Hepatitis 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 in in vitro translation 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, (C) 1999 Academic Press.