The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers

Figures

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  FIG 1. FMDV genome and expression constructs. (A) Representation of the FMDV genome, showing mature protein products in boxes, noncoding regions (NCR), and viral protein genome-linked (VPg), represented with an oval. (B) Subgenomic replicon based upon the O1K FMDV sequence encoding renilla luciferase in place of the majority of the capsid. (C) pET28b plasmid expression constructs encoding 3C protease with C95K/C142A solubility mutations (50). (D) Plasmid expression constructs encoding full-length capsid precursor (P1-2A), capsid precursor with only the cleavage recognition sequence at the start of 2A (P1-Δ2A), and P1-Δ2A encoding a G2A mutation in VP4 to prevent myristoylation (P1-Δ2A G2A). All capsid precursor constructs were under the control of a T7 promoter in the pBG200 backbone (11). Protein processing key: ↑, cleavages performed by 3Cprotease; ●, ribosomal skip; ■, autocatalytic cleavage by Lprotease; ◆, maturation cleavage.

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  FIG 2. 17-DMAG reduces the growth of FMDV in cell culture without affecting translation or replication of the RNA. (A) BHK21 cells were treated with 17-DMAG at the concentrations indicated or mock treated before infection with FMDV strain A22 Iraq. Cells were lysed 8 h postinfection and infectious virus titrated by TCID50 assay. Replicates represent independent experiments. (B) IB-RS-2 cells were treated with 17-DMAG (0.5 μM) or GuHCl (3 mM) for 30 min at 37°C before being transfected with FMDV replicon RNA. Luciferase production was measured at half-hour intervals and expressed as relative light units (RLU). Error bars represent the standard deviations from triplicate samples. (C) 17-DMAG cytotoxicity. Cultures of IB-RS-2 and BHK21 cells were treated with a dilution series of 17-DMAG for 8 h at 37°C. Cell viability was determined by quantitating cellular ATP using a luminescence assay (Promega ToxGlo) and luminometer (Hidex Chameleon). Values were converted to percent relative light units (RLU) obtained from mock-treated normal healthy cells. Error bars represent standard deviations from quadruplicate samples.

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  FIG 3. 17-DMAG inhibition of virus growth is dose dependent. BHK-21 cells were treated with a 2.5-fold dilution series of 17-DMAG (A) or GuHCl (B). The cells were infected with FMDV at an MOI of 0.01 (□) or mock infected (■) and incubated for 72 h at 37°C. Cell viability was determined by quantitating cellular ATP using a luminescence assay (Promega ToxGlo) and luminometer (Hidex Chameleon). Values were converted to percent RLU obtained from healthy cells. Error bars represent standard deviations from quadruplicate samples.

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  FIG 4. Validation of the cell-free assay for FMDV capsid precursor processing and pentamer assembly. (A) P1-2A processing assay validation. P1-Δ2A capsid precursor generated from expression in rabbit reticulocyte lysates was mixed with a range of concentrations of purified 3Cpro for 1 h at 37°C, and the proteins were resolved on 12% SDS-PAGE gels, followed by fluorography. (B) Marker proteins with known sedimentation were separated through 5 to 30% sucrose density gradients, with linear regression analysis used to extrapolate and interpolate unknowns. (C and E) Sucrose density gradient analysis of pentamer assembly reactions with P1-Δ2A-wt (C) and -G2A mutant (E) expression constructs showing mock (●) and 3Cpro (○) treatments. (D) Cell-free expression of P1-Δ2A-wt and G2A expression constructs in the presence of [3H]myristic acid. (F) Capsid precursor processing assay using P1-Δ2A-wt and -G2A expression constructs labeled with [S35]methionine.

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  FIG 5. 17-DMAG inhibition of hsp90 reduces P1-2A processing and pentamer assembly in a dose-dependent manner. Cell-free expression reactions were pretreated with the concentrations of 17-DMAG indicated prior to expression of radiolabeled P1-2A and processing by addition of purified 3Cpro. (A) The majority of the samples were sedimented through 5 to 30% (wt/vol) sucrose density gradients and material in gradient fractions detected by scintillation counting. (B) Samples were also removed before sedimentation, resolved through 12% SDS-PAGE gels, and analyzed by fluorography.

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  FIG 6. Inhibition of hsp90 reduces the rate of P1-2A processing. (A) Cell-free expression reaction mixtures were pretreated with 10 μM 17-DMAG or mock treated prior to expression of radiolabeled P1-2A and processing by addition of purified 3Cpro for the times indicated. Samples were resolved through 12% SDS-PAGE gels and analyzed by fluorography. A reduced concentration of 3Cpro was used (0.25 μM compared with 1 μM in a standard assay). Relative band intensity was performed on scanned film images for each cleavage product using NIH ImageJ analysis software (v1.50). (B) Densitometric analysis using ImageJ software was used to generate relative band intensity of the image shown in panel A, and this was plotted for the mock- and drug-treated conditions over time.

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  FIG 7. Luminespib inhibits virus growth and reduces P1-2A processing is a dose-dependent manner. (A) BHK-21 cells were treated with a 2.5-fold dilution series of luminespib and then infected with FMDV at an MOI of 0.01 (□) or mock infected (■) and incubated for 72 h at 37°C. Cell viability was determined by quantitating cellular ATP using a luminescence assay (Promega ToxGlo) and luminometer (Hidex Chameleon). (B) Cell-free expression reactions were pretreated with luminespib at the concentrations indicated prior to expression of radiolabeled P1-2A and processing by addition of purified 3Cpro. The samples were resolved through 12% SDS-PAGE gels and analyzed by fluorography.