Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis
- Adriaan H. de Wildea,
- Yanhua Lib,
- Yvonne van der Meera,
- Grégoire Vuagniauxc,
- Robert Lysekd,
- Ying Fangb,e,
- Eric J. Snijdera and
- Martijn J. van Hemerta
- aMolecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
- bDepartment of Veterinary and Biomedical Science, South Dakota State University, Brookings, South Dakota, USA
- cDebiopharm S.A., Lausanne, Switzerland
- dDebio R.P., Martigny, Switzerland
- eDepartment of Biology/Microbiology, South Dakota State University, Brookings, South Dakota, USA
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Fig 1Cyclosporine treatment blocks EAV-GFP and PRRSV-GFP replication. (A) BHK-21 cells were infected with EAV-GFP at an MOI of 5, and at 1 h p.i., the inoculum was replaced by medium containing different concentrations of CsA, as indicated on the x axis. Cells were fixed at 18 h p.i., and GFP reporter expression was quantified and normalized to the GFP signal of control cells (100%) treated with DMSO (the solvent concentration was equal to that in cultures that received 4 μM CsA). (B) The effect of CsA on cell viability compared to the viability of untreated control cells (100%) was determined using a CellTiter 96 AQueous nonradioactive cell proliferation assay (Promega). Graphs show the results (average and SD) of a representative experiment performed in quadruplicate. (C) MARC-145 cells were infected with PRRSV-GFP at an MOI of 0.1, and at 1 h p.i., the inoculum was replaced by medium with CsA. Cells were fixed at 24 h p.i., and GFP reporter expression was quantified and normalized to the signal in solvent-treated control cells (100%). (D) Effect of CsA on the viability of the MARC-145 cells compared to the viability of untreated control cells (100%). Graphs show the results (average and SD) of a representative experiment (n = 8). All experiments were repeated at least twice.
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Fig 2The CsA analog Debio-064 blocks EAV-GFP and PRRSV-GFP replication. (A) EAV-GFP-infected BHK-21 cells (MOI, 5) were incubated with various concentrations of Debio-064 from 1 h p.i. onwards. Cells were fixed at 18 h p.i., and GFP reporter expression was quantified and normalized to the GFP signal of control cells (100%) that were treated with DMSO (the solvent concentration was equal to that in medium containing 4 μM Debio-064). (B) Effect of Debio-064 on BHK-21 cell viability compared to the viability of untreated control cells (100%). (C) PRRSV-GFP-infected MARC-145 cells (MOI, 0.1) were incubated with various concentrations of Debio-064 from 1 h p.i. onwards. Cells were fixed at 24 h p.i., and GFP reporter expression was quantified and normalized to the GFP signal of control cells (100%) that were treated with DMSO. (D) Effect of Debio-064 on MARC-145 cell viability compared to the viability of control cells (100%). Results (average and SD) of a representative experiment performed in quadruplicate are shown, and all experiments were repeated at least twice.
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Fig 3CsA and Debio-064 treatment block viral protein expression in cells infected with wild-type EAV. BHK-21 cells were infected with EAV (MOI, 5) and treated from 1 h p.i. on with CsA (A) or Debio-064 (B) at the concentration indicated above each lane. Cells were lysed at 6 h p.i., and viral protein expression was analyzed by SDS-PAGE and Western blotting with antibodies against nsp9 and nsp5-8, the M protein, and the N protein. β-Actin was used as a loading control. For immunofluorescence microscopy, mock-infected or EAV-infected and CsA-treated (C) or Debio-064-treated (D) cells were fixed at 6 h p.i. and stained with an anti-nsp3 antiserum. The drug concentrations used are indicated in each panel. Bars, 50 μm.
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Fig 4Treatment of infected cells with cyclophilin inhibitors strongly reduces arterivirus yields. (A) EAV-infected BHK-21 cells (MOI, 5) were treated with various concentrations of CsA (gray bars) or Debio-064 (white bars) from 1 h p.i. onwards, and virus titers in the culture medium at 12 h p.i were determined by plaque assay. (B) MARC-145 cells infected with PRRSV-GFP (MOI, 0.1) were treated from 1 h p.i. onwards with CsA (gray bars) or Debio-064 (white bars) at the concentrations indicated below the x axis, and virus titers in the medium at 24 h p.i. were determined by fluorescent focus assay.
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Fig 5The in vitro and in vivo RNA-synthesizing activity of EAV RTCs can be blocked by CsA or Debio-064 treatment. (A) Metabolic labeling of EAV-infected cells with [3H]uridine at between 5.5 and 6.5 h p.i. in the presence or absence of 4 μM CsA. Total RNA was isolated, analyzed in denaturing agarose gels, and detected by fluorography. The amount of [3H]uridine that was incorporated into viral genomic RNA was quantified and normalized to that in EAV-infected, untreated control cells (100%). 28S rRNA detected by hybridization with a 32P-labeled probe (bottom) was used as a control to correct for variations in loading during viral RNA quantification. (B and C) Semipurified RTCs isolated from EAV-infected BHK-21 cells at 6 h p.i. were used in an in vitro RNA synthesis assay in which [32P]CTP was incorporated into viral RNA. Reactions, performed in the presence of various concentrations of CsA (B) or Debio-064 (C), as indicated above the lanes, were terminated after 100 min. RNA was isolated, and reaction products were analyzed in denaturing formaldehyde agarose gels. The positions of the genomic RNA (position 1) and subgenomic RNAs (positions 2 to 7) are indicated next to the gels.
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Fig 6RNAi-mediated knockdown of CypA, but not CypB, strongly affects EAV replication. 293/ACE2 cells were transfected with a nontargeting control siRNA or siRNAs targeting CypA or CypB. (A) Knockdown of CypA and CypB levels was monitored by Western blotting with CypA- and CypB-specific antisera. β-Actin was used as a loading control. (B) Viability of cells at 48 h posttransfection with the various siRNAs normalized to that of cells transfected with the nontargeting control siRNA (100%). (C) GFP reporter expression of cells transfected with the siRNAs indicated below the graph and infected at 48 h posttransfection with EAV-GFP at an MOI of 5. Cells were fixed at 24 h p.i., and GFP fluorescence was quantified and normalized to that in infected cells transfected with nontargeting siRNA. (D) Virus titers at 32 h p.i. in the culture medium of cells transfected with the siRNAs indicated below the graph and infected at 48 h posttransfection with wt EAV at an MOI of 0.01.
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Fig 7Cosedimentation of CypA with EAV RTCs in density gradients. The postnuclear supernatant of EAV- and mock-infected Vero E6 cells was fractionated using a continuous 0 to 30% OptiPrep density gradient. (A) Densities of the fractions were determined with a refractometer. (B) Distribution of CypA, CypB, the cytosolic marker protein GAPDH, and the ER marker protein calnexin in density gradient fractions of mock-infected cell lysates analyzed by Western blotting. (C) Distribution of CypA, CypB, the cytosolic marker protein GAPDH, the ER marker protein calnexin, and EAV RdRp nsp9 in density gradient fractions of EAV-infected cells analyzed by Western blotting. (D) Before loading on a 0 to 30% OptiPrep gradient, PNS was pretreated with 12 μM CsA for 30 min on ice. Western blot analysis was used to determine the sedimentation of CypA, the ER marker protein calnexin, and the cytosolic marker protein GAPDH in nsp9-containing membrane fractions of CsA-treated and untreated PNSs and a fraction with the same density prepared from mock-infected cells.
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