Abstract
Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis.
Highlights
3% of the world’s population is chronically infected with the hepatitis C virus (HCV),2 and a significant percentage of these individuals will progress to life-threatening liver cirrhosis and hepatocellular carcinomas [1, 2]
HCV RNA-dependent RNA polymerase (RdRp) has an unusual -hairpin loop that protrudes into the active site that helps position the 3Ј-end of the RNA template for proper initiation of RNA synthesis and decreases the ability to extend from a primed template [10, 11]
The minimal length of RNA required for stable binding of HCV RdRp was determined by fluorescence anisotropy analyses of different lengths of RNAs, and the HCV RdRp regions that could be cross-linked to the 7-nt RNA template were identified by reversible cross-linking and mass spectrometry (MS) analyses
Summary
Materials—All RNAs used were synthesized chemically by Dharmacon Research (Boulder, CO). 5Ј-radiolabeled 7-nt RNA (3Ј-CAUAUGC-P*-5Ј, named 7CP) were incubated in 20 mM Hepes (pH 7.5), 4 mM MgCl2, 1 mM dithiothreitol at room temperature for 5 min by the addition of a final concentration of 0.1% formaldehyde in a 20-l reaction. The cross-linking reaction was quenched by the addition of final concentration of 0.2 M glycine, and aliquots (10 l) of the reaction products were mixed with SDS sample buffer and loaded onto 4 –12% gradient NuPAGE gel (Invitrogen). Preparative cross-linking was performed in a 100-l reaction with a final concentration of 2 M ⌬21 and 4 M 7CB RNA in 20 mM Hepes (pH 7.5), 4 mM MgCl2, 1 mM dithiothreitol. Subgenomic HCV replicon RNAs were transcribed in vitro using a T7 Ampliscibe kit (Epicenter, Madison, WI) from the WT and mutant DNA constructs after linearizing with ScaI. After 3 weeks of selection with G418 sulfate, cell colonies in culture dishes were stained with 0.01% Coomassie Blue
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