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RNase H: Specificity, Mechanisms of Action, and Antiviral Target

  • Karin Moelling
  • Felix Broecker
  • John E. Kerrigan
Part of the Methods in Molecular Biology book series (MIMB, volume 1087)

Abstract

The Ribonuclease (RNase) H is one of the four enzymes encoded by all retroviruses, including HIV. Its main activity is the hydrolysis of the RNA moiety in RNA–DNA hybrids. The RNase H ribonuclease is essential in the retroviral life cycle, since it generates and removes primers needed by the Reverse Transcriptase (RT) for initiation of DNA synthesis. Retroviruses lacking RNase H activity are noninfectious. Despite its importance, RNase H is the only enzyme of HIV not yet targeted by antiretroviral therapy.

Here, we describe functions and mechanisms of RNase H during the HIV life cycle and describe a cleavage assay, which is suitable to determine RNase H activity in samples of various kinds. In this assay, an artificial, fluorescence-labeled RNA–DNA hybrid is cleaved in vitro by an RT/RNase H enzyme. Cleavage products are analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). This assay may be used to detect the RNase H, assess the effect of inhibitors, or even activators, of the RNase H, as we have described, as candidates for novel antiretroviral agents.

Key words

RNase H Cellular RNases H Polypurine tract (PPT) Reverse Transcriptase Argonaute Inhibitors Activators Validated target Microbicide Cleavage assay 

Notes

Acknowledgements

KM gratefully acknowledges the support of this work by the Institute for Advanced Study at Princeton, USA. FB would like to thank Prof. Peter H. Seeberger (Max Planck Institute of Colloids and Interfaces) for his generous support.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Karin Moelling
    • 1
    • 2
    • 3
  • Felix Broecker
    • 4
  • John E. Kerrigan
    • 5
  1. 1.Institute of Medical MicrobiologyUniversity of ZurichZurichSwitzerland
  2. 2.Max Planck Institute for Molecular GeneticsBerlinGermany
  3. 3.Institute for Advanced StudyPrincetonUSA
  4. 4.Max Planck Institute of Colloids and InterfacesPotsdamGermany
  5. 5.The Cancer Institute of New JerseyNew BrunswickUSA

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