Detection of cellular proteins and viral core protein interacting with the 5′ untranslated region of hepatitis C virus RNA

Abstract

Hepatitis C virus (HCV) is a pesti- and flavi-like virus, which contains a highly conserved 5′-untranslated region (UTR). This region is implicated in the regulation of both translation and RNA replication. To examine the possible cellular factors involved in HCV replication, we performed UV cross-linking experiments to detect cellular proteins binding to 5′-UTR of HCV RNA. No cytoplasmic proteins were found to cross-link to 5′-UTR. Surprisingly, when nuclear extracts were used for UV cross-linking, a major protein of 110 kD and several other minor proteins were detected. Competition assays confirmed that the binding of the 110-kD protein was specific to the 5′-UTR. The protein-binding site was mapped within the 78-nt region between nucleotides 199 and 277 from the 5′ end of the viral RNA. This protein was present in several different cell lines tested. No cellular proteins specifically bound to the complementary strands of the 5′-UTR. We have also shown by an RNA-protein blotting assay that 5′-UTR bound to the HCV core protein, which can be translocated to the nuclei. These findings suggest that HCV RNA may enter nuclei by complexing with the viral core protein and interact with nuclear proteins that are involved in the regulation of RNA replication or translation. It is thus possible that HCV employs a replication strategy distinct from its related pestiviruses or flaviviruses.