Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

Figures

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  FIG 1. Structural analysis of FMDV Lpro and ISG15 binding interface. (A) Superimposition of Lpro (PDB number 1QBB) and USP18 cocrystalized with ISG15 (PDB number 5CHV) using catalytic residues as tether points highlights structural homology. (B) Simulated surface of Lpro shows potential binding cleft for ISG15 based on superimposition with USP18. (C) Image of peptide docking results showing putative binding mechanism between ISG15 C-terminal peptide and Lpro binding pocket rendered as ribbons. (D) Close-up interactions between aromatic amino acid Trp/W 105 in red and ISG15 C-terminal peptide, rendered as a stick. Other hydrophobic interactions are highlighted in magenta, namely Pro/P 99 and Leu/L 102. All images were rendered in Discovery Studio Visualizer.

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  FIG 2. Lpro deISGylation in vitro activity. (A) Recombinant human proISG15 treated with bacterial extracts expressing WT or indicated mutant forms of Lpro. Lpro-dependent cleavage of proISG15 is detected as a visible 2-kDa band shift. (B) Immunoblot of samples treated with bacterial extracts expressing WT or Lpro mutants. Bacterial extracts prepared from IPTG-induced cultures were mixed with SK6 lysates, followed by incubation at 4°C to allow for Lpro-dependent enzymatic digestion of host proteins. Cleavage of eIF4G (220 kDa) generates a fragment of approximately 120 kDa (CP). For each figure, one representative blot is shown out of three independent experiments.

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  FIG 3. Plaque phenotype and viral growth kinetics. (A) Plaque phenotypes for FMDV A12 WT, A12 LLV, and A12 LproW105A were evaluated in BHK-21 cells. Cell monolayers were infected for 48 h in semisolid medium, followed by staining with crystal violet. (B) Kinetics of growth in multiple cell lines; BHK-21, LFPKαVβ6, and SK6 cells were infected at an MOI of 5 with WT, LLV, or LproW105A FMDV, and at the indicated times, virus titer was measured in BHK-21 cells. FMDV yield was determined by the endpoint dilution method on BHK-21 cells 24 h after infection. Titers are expressed as log10 50% tissue culture infective dose (TCID50) per milliliter. The values are presented as the mean ± standard deviation of three independent experiments. (C) Fluorescent microscopy images from LFPKαVβ6 cells infected with WT or LproW105A FMDV at an MOI of 10 and fixed at 4 h postinfection. Samples were stained with anti-Lpro (green) and with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar represents 10 μm. Statistical analysis was performed using Student’s t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001; black asterisks represent statistical significance between LLV and WT; red asterisks…

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  FIG 4. Lpro deISGylase activity during FMDV infection of porcine cells. (A) LFPKαVβ6 cells were mock transfected or transfected with either GFP or the ISG15 machinery (DDK-ISG15, E1 [UBE1L], E2 [UbcH8], and E3 [HERC5]). At 24 h posttransfection, proteins were resolved by SDS-PAGE and Western blot was applied to detect DDK-tagged free or conjugated ISG15 and UbcH8. (B) LFPKαVβ6 transfected as indicated in A were infected with FMDV A12WT, LLV, or LproW105A. At 6 hpi, cell lysates were prepared, and proteins were resolved in 3% to 8% SDS-PAGE, followed by Western blotting to detect DDK-tagged free or conjugated ISG15 and UbcH8, FMDV VP1, eIF4G, and GAPDH. (C) DUB activity of A12-WT, A12-LLV, and A12 Lpro W105A virus. LFPKαVβ6 cells were transfected with an HA-Ub expression plasmid, and 24 h posttransfection, cells were infected with the indicated viruses. Cells were lysed 4.5 h postinfection and analyzed by Western blotting to detect HA-tagged Ub conjugates, FMDV VP1, and tubulin. (D) LFPKαVβ6 cells were mock infected or infected with either A12-WT, A12-LLV, or A12-LproW105A virus, and lysates were collected at 2, 4, 6, and 24 hpi. Proteins were resolved by SDS-PAGE, and Western blot was…

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  FIG 5. FMDV replication in cells overexpressing ISG15. (A) Viral titers were examined in LFPKαVβ6 previously transfected with ISG15 and infected at an MOI of 0.1 with WT, LLV, or LproW105A. FMDV yield was determined by plaque assay in BHK-21 cells. Plaques were counted 24 hpi, and titers were expressed as plaque forming units (PFUs) per ml. Values are presented as the mean ± standard deviation from three independent experiments. (B) Schematic representation of the replication-defective Ad5-pISG15 plasmid. HA-tagged pISG15 was cloned in the Ad5-Blue plasmid using ClaI and XbaI restriction enzyme sites. (C) Western blot analysis of protein ISGylation in mock or Ad5-GFP/Ad5-HA-pISG15-transduced LFPKαVβ6 cells in the presence or absence of increasing amounts of porcine IFN-β (5 to 40 U). Eighteen hours after IFN-β treatment, protein ISGylation was detected by using anti-HA antibodies. (D) Viral titers were examined in LFPKαVβ6 previously transduced with Ad5-HA-pISG15 and infected at an MOI of 0.1 with WT virus. Virus yield were determined by plaque assay in BHK-21 cells. The values are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was…

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  FIG 6. Expression of IFN-stimulated genes in response to WT, LLV, and W105 FMDV infection. Porcine SK6 cells were infected at an MOI of 0.1 with A12 WT, A12 LLV, or A12 LproW105A virus. Total RNA was isolated at 6 hpi, and different set of genes involved in type I IFN (A), antiviral activity (B), ISG15 machinery (C), and RNA sensing (D) were analyzed by qPCR using specific primers. Porcine GAPDH was used as an internal control. The results are expressed as the relative fold increase in gene expression with respect to mock cells. Results represent the mean from three independent experiments. Statistical analysis was performed using Student’s t test. *, P < 0.05; **, P < 0.005.

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  FIG 7. Virulence of A12-LproW105A in infected mice. Six- to 7-week-old female C57BL/6 mice (n = 6/group) were subcutaneously inoculated in the footpad with the indicated doses of A12-WT or A12-LproW105A mutant FMDV. (A) Clinical disease was followed for 7 days after inoculation, and percent survival was calculated as (number of surviving animals/number of animals per group) × 100, daily. (B) Serum samples collected at the indicated times after inoculation were assayed for the presence of virus by plaque assay on BHK-21 cells. (C) FMDV serum neutralizing antibody titers were determined by endpoint dilution and expressed as log TCID50/ml (48). Statistical analysis was performed using Student’s t test. *, P < 0.05; **, P < 0.005.