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

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Human Retroviruses

Part of the book series: Methods in Molecular Biology ((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.

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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.

Author information

Authors and Affiliations

  1. Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

    Karin Moelling

  2. Max Planck Institute for Molecular Genetics, Berlin, Germany

    Karin Moelling

  3. Institute for Advanced Study, Princeton, NJ, USA

    Karin Moelling

  4. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

    Felix Broecker

  5. The Cancer Institute of New Jersey, New Brunswick, NJ, USA

    John E. Kerrigan

Authors
  1. Karin Moelling
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  2. Felix Broecker
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  3. John E. Kerrigan
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Editors and Affiliations

  1. Div. Immunology, Transplantation, and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy

    Elisa Vicenzi

  2. Div. Immunology, Transplantation, and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy

    Guido Poli

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Moelling, K., Broecker, F., Kerrigan, J.E. (2014). RNase H: Specificity, Mechanisms of Action, and Antiviral Target. In: Vicenzi, E., Poli, G. (eds) Human Retroviruses. Methods in Molecular Biology, vol 1087. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-670-2_7

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  • DOI: https://doi.org/10.1007/978-1-62703-670-2_7

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-669-6

  • Online ISBN: 978-1-62703-670-2

  • eBook Packages: Springer Protocols

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