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Protein Kinase A dependent step(s) in Hepatitis C virus entry and infectivity

 

Farquhar M.J., H.J. Harris, M. Diskar, S. Jones, C.J. Mee, S.U. Nielsen, C.L. Brimacombe, S. Molina, G.L. Toms, P. Maurel, J. Howl, F.W. Herberg, S.C. van IJzendoorn, P. Balfe and J.A. McKeating J Virol 82:8797-8811

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Synopsis:

In this study we investigated a role for protein kinase signaling in HCV infection by examining the effect of kinase inhibitors and antagonists on viral entry, replication and the release of infectious particles. Inhibition of PKA led to a redistribution of CLDN1 from the plasma membrane and a concomitant reduction in viral entry, confirming the importance of CLDN1 localization at the plasma membrane for viral receptor activity. In addition, we reveal a role for PKA in regulating the infectivity of cell-free virus particles and demonstrate increased levels of cAMP and PKA substrates in HCV infected cells, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.


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. Inhibition of PKA with H89, cAMP antagonists or the protein kinase inhibitor peptide (PKI) reduced HCV entry into Huh-7.5 hepatoma cells. BRET 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 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 FRET analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to 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 specific infectivity was reduced. Viral particles released from H89 treated cells displayed the same range of buoyant densities as 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.


Farquhar figure 1

Inhibition of PKA attenuates HCV infection. (A-B) Dose-dependent reduction of HCVcc infection by inhibition of PKA. Huh-7.5 cells were incubated for 1h with increasing concentrations of the PKA inhibitors H89 (A) or myrPKI (B) and infected with J6/JFH (☐) or JFH-1 (△). (C-D) Dose-dependent reduction of HCVpp entry by inhbition of PKA. Huh-7.5 cells were incubated for 1h with increasing concentrations of the PKA inhibitors H89 (C) or myrPKI (D) and infected with HCVpp-JFH-1 (white bars) or MLVpp (black bars). Infectivity is expressed relative to untreated control cells and represents the mean of three replicate infections.


Farquhar figure 2

Inhibition of PKA disrupts CLDN1 localization in Huh-7.5 cells. Huh-7.5 cells were seeded onto glass coverslips and incubated with DMSO (control), H89 (PKA inhibitor-10 μM), FK (PKA activator-10 μM), Rp-cAMPS (PKAII-inhibitor 500 μM) or Rp-8-Br-cAMPS (PKAI inhibitor 500 μM). Cells were fixed and stained for CLDN1 or CD81. Nuclei were visualized using DAPI. Laser scanning confocal microscopic images were obtained using a 63x 1.2NA objective (scale bar represents 10 μm).


Farquhar figure 3

Dose-dependent reduction in extracellular HCV infectivity by PKA inhibition. J6/JFH (☐) and JFH-1 (△) infected Huh-7.5 cells were seeded in 48 well plates and the following day incubated with increasing concentrations H89 (A) or myrPKI (B). Cells were extensively washed, supernatant collected after 1h and the infectivity quantified. Virus infectivity was determined by infection of naïve Huh-7.5 cells and NS5A positive cells enumerated. Infectivity is expressed relative to control untreated cells and represents the mean of three replicate infections.


Farquhar figure 4

HCV infection increases cAMP levels and PKA activity. (A) cAMP levels were measured in uninfected, J6/JFH and JFH-1 infected Huh-7.5 cells 72h post infection. As a control, Huh-7.5 cells were incubated with Forskolin (FK, 10 µM) a compound known to activate adenyl cyclase and increase cAMP levels. cAMP levels are shown relative to control untreated cells and represent data from the mean of three replicate wells. (B) PKA activity was assessed by measuring the reactivity of an anti-PKA substrate specific antibody (p-PKAs) with 10 μg of total protein separated by SDS PAGE from control (lane 1), FK (10 µM) stimulated (lane 2) and JFH-1 infected (72h post infection) (lane 3) Huh-7.5 cells. (C) Uninfected and (D) JFH-1 infected (72h post infection) Huh-7.5 cells were incubated with FK or H89 for 1h (H89 > FK indicates a 1h pre-incubation with H89 prior to FK treatment), fixed and stained with the PKA substrate specific antibody (p-PKAs) (green). JFH-1 infected cells were visualised by staining for NS5A (red) and nuclei counter stained with DAPI (blue). Laser scanning confocal microscopic images were obtained using a 63x 1.2NA objective (scale bar represents 10 μm).


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