Biochemical analyses of the interactions between viral polymerases and RNAs

Methods Mol Biol. 2008:451:185-200. doi: 10.1007/978-1-59745-102-4_13.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Bromovirus / enzymology
  • Bromovirus / genetics
  • Chromatography, Affinity / methods
  • Cross-Linking Reagents
  • DNA-Directed RNA Polymerases / metabolism*
  • Fluorescence Polarization / methods
  • Kinetics
  • Mass Spectrometry / methods
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • RNA Viruses / enzymology*
  • RNA Viruses / genetics
  • RNA, Viral / genetics
  • Viral Proteins / chemistry

Substances

  • Cross-Linking Reagents
  • RNA, Viral
  • Viral Proteins
  • DNA-Directed RNA Polymerases