Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase.
Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the Δ1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.
The Biochemistry Interdisciplinary Program, Indiana University, Bloomington, Indiana 47405, USA.
Robert
R
Vaughan
Baochang
B
Fan
Jin-Sam
JS
You
C Cheng
CC
Kao
D016174:Hepacivirus / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D010446:Peptide Fragments; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase