Dicer is cleaved by the Leader protease encoded by foot-and-mouth disease virus to promote infection in mammalian cells

Figures

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  Fig. 1.. Dicer is cleaved during FMDV infection by Lpro. (A) Schematic representation of Dicer showing the putative Lpro target motif conserved across different species including FMDV hosts. (B) IBRS-2 cells (1 × 106) were transfected with 2 μg of DDK-Dicer plasmid and 24 hours later infected with FMDV (O1BFS isolate) at an MOI of 5. Cells were lysed at different times after transfection and analyzed by immunoblot for detection of the indicated proteins. (C) HEK293 cells (1 × 106) were transfected with 1 μg of plasmids encoding LbWT, LbC51A or an empty vector (EV) and lysed 24 hours later for analysis of the indicated proteins by immunoblot. (D) IBRS-2 cells were infected with FMDV WT or FMDV-∆Lb at an MOI of 5 and lysed at different times after infection for immunoblot analysis. The N-terminal and C-terminal cleavage products of Dicer are indicated by white and gray arrows, respectively. The 110-kDa cleavage product of eIF4G is also depicted. Data shown are representative of independent biological replicates (n = 2 to 4).

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  Fig. 2.. Lpro specifically cleaves Dicer at the KGRAR motif. (A) HEK293 cells were cotransfected with 2 μg of DDK-Dicer plasmid and increasing amounts of LbWT (0.2, 2, 20, 200, and 2000 ng), 1 μg of EV, or 1 μg of LbC51A plasmids. Cells were lysed 24 hours later for detection of the indicated proteins by immunoblot. (B) HEK293 cells were cotransfected with 2 μg of DDK-Dicer and 300 ng of LbWT plasmids in the presence of zVAD (20 μM), MG132 (10 μM), or chloroquine (CQ) (50 μM). In control cells, apoptosis was induced with puromycin (20 μM). Cells were lysed 24 hours later and analyzed by immunoblot for detection of the indicated proteins using the specified antibodies. (C) HEK293 cells were cotransfected with 1 μg of DDK-Dicer or DDK-EV and 1 μg of LbWT or LbC51A plasmids. Cells were lysed 24 hours later, and IP was performed using an anti-DDK monoclonal antibody. IP fractions and lysates were analyzed by immunoblot. (D) HEK293 cells were cotransfected with 1 μg of DDK-Dicer or DDK-Dicer-TM (K550E, R552E, and R554E) and 20 ng of LbWT plasmids. Lysates were collected 24 hours later and analyzed by immunoblot. The specific residues modified in DDK-Dicer are shown. The N-terminal and…

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  Fig. 3.. The ability of FMDV to cleave Dicer correlates with replication efficiency. (A) IBRS-2 cells were transfected with 100 nM specific Dicer siRNAs (si-Dicer-1) or a scramble siRNA and 24 hours later transfected with 500 ng of DDK-Dicer, DDK-Dicer-TM or an EV. After 24 hours, cells were infected with FMDV WT or FMDV-∆Lb at an MOI of 5. At 6 hours after infection, supernatants were collected and cells lysed for protein analysis and RNA extraction. (B) Western blot analysis of Dicer levels and integrity at 6 hours after infection. The knockdown efficiency prior to infection was analyzed 24 hours after transfection with the EV (knockdown lanes). The C-terminal cleavage product of Dicer is indicated by a gray arrow. (C to D) Virus titration of supernatants by plaque assay in BHK-21 cells and quantification of viral RNA by RT-qPCR of cells infected with FMDV WT (C) or FMDV-∆Lb (D). Data shown in (C) and (D) are means ± SD of three independent experiments (n = 3). Statistical analyses were performed by one-way ANOVA test with Tukey’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Representative results from a single experiment are shown in (B).

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  Fig. 4.. FMDV Lpro suppresses the shRNA-induced RNAi mediated by Dicer in swine cells. (A to C) IBRS-2 cells were cotransfected with plasmids encoding EGFP (0.3 μg) and a specific EGFP shRNA (5 μg) together with an EV (100 ng) or a plasmid encoding LbWT or LbC51A (0.2, 2, or 20 ng). At 48 hours after transfection, cells were directly observed by fluorescence and bright-field microscopy and images were taken with a ×10 magnification (A) and then lysed for detection of the indicated proteins by Western blot (B); the C-terminal cleavage product of Dicer is indicated by a gray arrow. The levels of EGFP, Lpro, and GAPDH mRNAs were examined by RT-PCR after RNA extraction from the same lysates (C). Data shown are representative of independent biological replicates (n = 2 to 4).

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  Fig. 5.. Deletion of Lbpro impairs the suppression of shRNA-induced RNAi phenotype of FMDV in swine cells. IBRS-2 cells were cotransfected with plasmids encoding EGFP (0.3 μg) and a specific EGFP shRNA (5 μg) for 24 hours and then mock-infected or infected with FMDV WT or FMDV-∆Lb at an MOI of 0.001 (A) or an MOI of 1 (C). EGFP fluorescence and bright-field microscopy images were taken at 20 hours (A) or 12 hours (C) after infection with a ×10 magnification and then lysed for detection of EGFP, Dicer, FMDV Lpro, FMDV 3C, or tubulin by Western blot (B and D). All results are representative of at least two independent experiments.

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  Fig. 6.. Analysis of the shRNA-induced RNAi mediated by Dicer-∆Nt and Dicer-TM in HEK293T NoDice cells. (A and B) HEK293T NoDice cells were cotransfected with plasmids encoding EGFP (0.1 μg) and a specific EGFP shRNA (5 μg) together with 0.5 μg of plasmids encoding Dicer or Dicer-∆Nt, LbWT (2 ng), LbC51A (2 ng), or IAV NS1 (0.3 μg), and EGFP fluorescence and bright-field microscopy images were taken at 24 hours after transfection with a ×10 magnification (A). Cells were then lysed for protein analysis by Western blot (B). (C and D) HEK293T NoDice cells were cotransfected with plasmids encoding EGFP (0.1 μg); EGFP shRNA (5 μg) together with 0.5 μg of a plasmid encoding Dicer or Dicer-TM and LbWT, LbC51A (0.2 or 2 ng); or EV (2 ng). EGFP fluorescence and bright-field microscopy images were taken at 24 hours after transfection with a ×10 magnification (C), and cells were then lysed for detection of the indicated proteins by Western blot (D). All results are representative of at least two independent experiments.

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  Fig. 7.. Production of FMDV-derived small RNAs in swine cells. IBRS-2 cells were infected with FMDV WT or FMDV-∆Lb at an MOI of 5 or mock-infected. RNA was extracted at 4 hours pi and subjected to deep sequencing. (A) Size distribution and abundance of vsRNAs (positive-stranded in blue and negative-stranded in red) shown as counts mapped to the viral genome. (B) Presence of pairs of 22-nt reads with 2-nt 3′-overhangs (−2 peak), defined as canonical vsiRNAs. (C) Distribution of 21- to 23-nt vsRNAs in the FMDV WT and FMDV-∆Lb genomes. The relative abundances of positive- and negative-stranded vsRNAs are indicated (coverage per nucleotide of the viral genome). The different proteins encoded by the FMDV RNA and the flanking 5′ and 3′ NCRs are shown with arrows. (D) Abundance of 18- to 28-nt RNAs (cellular and viral small RNAs) in IBRS-2 cells infected with FMDV WT–, FMDV-∆Lb– or mock-infected shown as counts per million of total reads (CPM). Data represent one independent experiment.