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Journal of Virology, September 2008, p. 8797-8811, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00592-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity{triangledown}

Michelle J. Farquhar,1 Helen J. Harris,1 Mandy Diskar,2 Sarah Jones,3 Christopher J. Mee,1 Søren U. Nielsen,4 Claire L. Brimacombe,1 Sonia Molina,5 Geoffrey L. Toms,4 Patrick Maurel,5 John Howl,3 Friedrich W. Herberg,2 Sven C. D. van IJzendoorn,6 Peter Balfe,1* and Jane A. McKeating1

Hepatitis C Research Group, Division of Immunity and Infection, University of Birmingham, Vincent Drive, Birmingham, United Kingdom,1 University of Kassel, Department of Biochemistry, Heinrich Plett Str. 40, D-34132 Kassel, Germany,2 Molecular Pharmacology Research Group, Research Institute in Healthcare Science, University of Wolverhampton, Wulfruna Street, Wolverhampton, United Kingdom,3 Liver Research Group, School of Clinical Medical Sciences, The Medical School, Newcastle upon Tyne, United Kingdom,4 Inserm U632, Hepatic Physiopathology, 191 Route de Mende, 34293 Montpellier Cedex 5, France,5 Department of Cell Biology/Membrane Cell Biology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands6

Received 17 March 2008/ Accepted 16 June 2008

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

* Corresponding author. Mailing address: Hepatitis C Research Group, Division of Immunity and Infection, University of Birmingham, Vincent Drive, Birmingham B15 2TT, United Kingdom. Phone: (44) 121 414 8174. Fax: (44) 121 414 3599. E-mail: p.balfe{at}bham.ac.uk

{triangledown} Published ahead of print on 25 June 2008.

Journal of Virology, September 2008, p. 8797-8811, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00592-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mee, C. J., Harris, H. J., Farquhar, M. J., Wilson, G., Reynolds, G., Davis, C., van IJzendoorn, S. C. D., Balfe, P., McKeating, J. A. (2009). Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells. J. Virol. 83: 6211-6221 [Abstract] [Full Text]  


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