Specific Interaction between the Hepatitis C Virus NS5B RNA Polymerase and the 3′ End of the Viral RNA

Abstract

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3′ viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5′ noncoding region and very poorly by the 3′ conserved 98-nucleotide tail. A 3′ coding region with conserved stem-loop structures rather than the 3′ noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3′ coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.

cis -Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

“…Similar studies by Kim et al (29) have indicated that although NS5B binds to its own C-terminal coding RNA sequence, it does so in a sequence-nonspecific manner. In contrast, Cheng et al (8) observed that partially purified NS5B specifically interacts with an RNA fragment that con-tains 295 nt of the C-terminal NS5B coding sequence and 45 nt downstream from the stop codon. To determine whether the two stem-loops (SL-V and SL-VI) are important for replication function as cis-replicating elements by virtue of their ability to bind the RNA polymerase specifically, we have carried out gel shift analyses with purified NS5B protein and 32 P- labeled transcript RNAs of the stem-loops as probes.…”

“…7C, lanes 6 to 8), indicating the specificity of the RNA-protein interaction. Cheng et al (8) have previously reported that NS5B has the ability to weakly interact with the 3Ј NTR but not with the 5Ј NTR. In agreement with these findings, we observed that transcripts of the HCV 3Ј NTR exhibited weak competition of the NS5B-NS5BCR interaction (Fig.…”

Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase

“…With results derived from EMSA and competition experiments, we demonstrate that purified recombinant RdRp could bind specifically to the 3Ј UTR of BaMV RNA without any host factor from plants or other virus-encoded polypeptides. Hepatitis C virus NS5B and encephalomyocarditis virus 3Dpol are two other cases in which overexpressed RdRp was demonstrated to bind the conserved stem-loop structures of the viral 3Ј end RNA (6) and both the 3Ј UTR and poly(A) tail (7), respectively, without any other factor being involved. This differs from phage Q␤, in which the phage-encoded RNA polymerase subunit must form a complex with at least three host proteins to attain specificity for binding to Q␤ RNA and related RNAs (2).…”

“…The results of the in vivo protoplast assay involving full-length RdRp and the in vitro footprinting and gel shifting analyses with the truncated form of RdRp are correlated. Functional analysis of the tertiary structure of the 3Ј UTR of BaMV RNA revealed that maintaining the integrity of the terminal pseudoknot is important, and the deletion of the ABC domain seriously impaired virus accumulation in protoplasts (6,32). It is possible that these regions could also interact with either a host-or virus-encoded factor(s).…”

“…There are several lines of evidence from studying the replication of turnip yellow mosaic virus (9,25,26), brome mosaic virus (4,29), turnip crinkle virus (3,27), and the picornaviruses (14,20,21,22,23) showing that the specific sequence or the structure at the 3Ј UTR is important for de novo minus-strand RNA synthesis. However, there are only a few cases, including encephalomyocarditis virus (7,8) and hepatitis C virus (6), demonstrating that the viral RNA polymerase interacts directly with the 3Ј UTR of its own genome.…”