Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.

TLDR: The interaction between a partially purified recombinant NS5B protein and a 3′ viral genomic RNA with or without the conserved 98-nucleotide tail is demonstrated.

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 Hepatitis C virus (HCV) is an enveloped virus that possesses a single-stranded positive-sense RNA genome encoding a polyprotein of approximately 3,000 amino acid residues (7, 20, 29) The conserved 59 noncoding region (59NCR) of the HCV genome (4, 13) is highly structured and contributes to the internal ribosome entry site important for the translation initiation of HCV RNA (14, 15, 25, 34, 35, 39) The 39 noncoding region (39NCR) consists of a short genotype-specific region and a poly(U)-C(U)n repeat stretch of variable length followed by a highly conserved tail of 98 nucleotides (nt) (12, 21, 30, 37) Studies to understand the molecular mechanism of HCV replication have been restricted by the lack of a well-established cell culture system, but studies from other positive-sense RNA viruses may provide some clues Upon flavivirus infection, translation of incoming viral genomic RNA occurs, and replication of the viral RNA begins with the synthesis of minus-strand RNA which then serves as the template for the synthesis of progeny genomic RNA The replication appears to take place at the perinuclear endoplasmic reticulum and requires virus-encoded proteins NS3 (proteinase/helicase) and NS5 (polymerase) as components of the presumed replicative complex (36) In addition, the 39 terminus of the flavivirus genomic RNA forms a conserved pseudoknot structure (26) It is generally believed that conserved sequences and structures at the 39 terminus of viral genomic RNA function as cis-acting signals that interact with viral and cellular proteins to initiate the synthesis of minus-strand RNA during viral replication The NS5B protein of HCV is a membrane-associated phosphoprotein (16) that possesses the conserved GDD motif of RNA-dependent RNA polymerase (RdRp) (19) RdRp activity of the HCV NS5B has been demonstrated in vitro, and