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Biochemical Analyses of the Interactions Between Viral Polymerases and RNAs

  • Young-Chan Kim
  • C. Cheng Kao
Part of the Methods in Molecular Biology™ book series (MIMB, volume 451)

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.

Keywords

Brome mosaic virus Hepatitis C virus RNA replication RNA-dependent RNA polymerase Template competition assay Fluorescence spectroscopy Reversible crosslinking Mass spectrometry 

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Young-Chan Kim
    • 1
  • C. Cheng Kao
    • 1
  1. 1.Department of Biochemistry & Biophysics103 Biochemistry/Biophysics Building, Texas A&M UniversityTXUSA

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