title: Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.
abstract: 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.
authors.affiliation: Division of Immunity and Infection, Hepatitis C Research Group, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Farquhar
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Harris
authors.forename: Mandy
authors.initials: M
authors.lastname: Diskar
authors.forename: Sarah
authors.initials: S
authors.lastname: Jones
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Mee
authors.forename: Søren U
authors.initials: SU
authors.lastname: Nielsen
authors.forename: Claire L
authors.initials: CL
authors.lastname: Brimacombe
authors.forename: Sonia
authors.initials: S
authors.lastname: Molina
authors.forename: Geoffrey L
authors.initials: GL
authors.lastname: Toms
authors.forename: Patrick
authors.initials: P
authors.lastname: Maurel
authors.forename: John
authors.initials: J
authors.lastname: Howl
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.forename: Sven C D
authors.initials: SC
authors.lastname: van Ijzendoorn
authors.forename: Peter
authors.initials: P
authors.lastname: Balfe
authors.forename: Jane A
authors.initials: JA
authors.lastname: McKeating
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D062445:Claudin-1; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D010957:Plasmids; D011991:Receptors, Virus / Q000032:analysis / Q000502:physiology; D051122:Scavenger Receptors, Class B / Q000032:analysis; D014162:Transfection; D053586:Virus Internalization
chemical_list: D015703:Antigens, CD; C568108:CLDN1 protein, human; D062445:Claudin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D011991:Receptors, Virus; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases