Biochemical analyses of the interactions between viral polymerases and RNAs.
The interaction between viral polymerases and their cognate RNAs is vital to regulate the timing and abundance of viral replication products. Despite this, only minimal detailed information is available for the interaction between viral polymerases and cognate RNAs. We study the biochemical interactions using two viral polymerases that could serve as models for other plus-strand RNA viruses: the replicase from the tripartite brome mosaic virus (BMV), and the recombinant RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV). Replicase binding sites in the BMV RNAs were mapped using a template competition assay. The minimal length of RNA required for RNA binding by the HCV RdRp was determined using fluorescence spectroscopy. Lastly, regions of the HCV RdRp that contact the RNA were determined by a method coupling reversible protein-RNA crosslinking, affinity purification, and mass spectrometry. These analyses of RdRp-RNA interaction will be presented as three topics in this chapter.
Kim
Young-Chan
YC
Department of Biochemistry & Biophysics, 103 Biochemistry/Biophysics Building, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.
Cheng Kao
C
C
D000595:Amino Acid Sequence; D017795:Bromovirus / Q000201:enzymology / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012328:RNA Viruses / Q000201:enzymology* / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry
D003432:Cross-Linking Reagents; D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases