Abstract
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.
Highlights
Mutations in the mAb 7G8 Epitope Exert Differential Effect on the Primer-dependent HCV NS5B RdRp Activity—We evaluated the efficiency of purified mutant RdRps in our in vitro replication assays
The rational design of polymerase substrate analogues resulted in the identification of different nucleoside analogue inhibitors competing for the catalytic site and interfering with the elongation of nascent RNA synthesized by NS5B
The binding sites for NNI molecules are dispersed in the palm and thumb subdomains of the HCV polymerase. These circumstances validate the approach of using mAbs as molecular probes for studies aimed at the elucidation of the HCV RdRp fingers subdomain function
Summary
Materials—[␣-32P]GTP, [␣-32P]CTP, and poly(rC), were from Amersham Biosciences; [␥-32P]ATP was from PerkinElmer Life Sciences; nucleoside 5Ј-triphosphates, RNase inhibitor, phage T7 RNA polymerase, and RQ1 DNase were from Promega; (rG)[12] oligonucleotide was from Proligo; heparin was from Sigma; and peptides were from JPT Peptide Technologies. Primer-dependent RdRp assays containing 0.12 M HCV NS5B, 0.4 g of poly(rC) pre-annealed with 4 pmol of (rG)[12] primer, and various mAbs concentrations (0.048 –1.2 M) were performed in RP buffer (10 mM Tris, pH 7.5, 12.5 mM KCl, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EGTA, pH 7.5, 10 units of RNasin (Promega)) in a total volume of 25 l. In primer-dependent RdRp reactions the NS5B and mAbs were preincubated at 4 °C for 10 min, after that RNA was added and incubation was extended for another 10 min at 4 °C. After NS5B, mAb, and RNA preincubations, the RdRp reactions were initiated by the addition of NTPs GTP, UTP, and ATP at a final concentration of 0.5 mM each and 10 Ci of [␣-32P]CTP complemented with unlabeled 10 M CTP per reaction. Total protein content in the lysate was measured in Bradford assay (Bio-Rad) for normalization of the replication signals
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