3C protease of enterovirus 71 cleaves promyelocytic leukemia protein and impairs PML-NBs production

Figures

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  Fig. 1. EV71 3C protease cleaves PML. A Structure of the PML isoforms. The diagram presents the exon composition of different mRNA variants coding seven PML isoforms (PMLI–VIIb) and the protein length. Light color is used to highlight the difference of their 3′-terminus. The domains, the motifs, and amino acid (aa) positions relative to the PMLI are shown, including RBCC motif, NLS, NES, SIM, two breakpoints in APL, and three major (K65, K160, and K490) SUMOylation sites. B Co-transfection analysis of six nuclear isoforms of PML and 3C or 3C (E71A) mutant. Cell lysates of HEK293T were tested by Western blotting after 36 h of transfection

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  Fig. 2. EV71 3C protease cleaves PML specifically. A, B HEK293T cells were co-transfected with Flag-PMLIII (A) or Flag-PMLIV (B) and increasing amounts of Myc-3C (0, 0.2, 0.5, and 0.8 μg, respectively) or Myc-3C (E71A) (0.5 μg). After 36 h, cell lysates were harvested and subjected to Western blotting in which anti-Flag, anti-Myc, and tubulin antibodies were used. C, D Co-transfection analysis of Flag-PMLIII (C) or Flag-PMLIV (D) and different 3C variants (3C, E71A, R84Q, and V154Q). After 36 h of transfection, cell lysates were tested by Western blotting. E HEK293T cells were treated in the presence and absence of 5 μM MG-132 for 5 h after 32 h of co-transfection with Flag-PMLIV and 3C (lane 2, 5, 8) or 3C (E71A) (lane 3, 6, 9) or vectors (lane 1, 4, 7). Cell lysates were immunoblotted with anti-Flag, anti-Myc, or β-actin antibodies. F HEK293T cells were treated in the presence and absence of 100 μM Chloroquine (CQ) for 5 h after 32 h of co-transfection with Flag-PMLIV and 3C (lane 2, 5, 8) or 3C (E71A) (lane 3, 6, 9) or vectors (lane 1, 4, 7). Cell lysates were subjected to Western blotting. G, H HEK293T cells (4 × 106) were co-transfected with empty vector or Flag-PMLIV and Myc-3C (G)…

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  Fig. 3. Q430–A431 and Q444–S445 are the sites of PMLIV cleavage by 3C. A The amino acids sequence of PMLIV protein (NCBI Reference Sequence: NP_002666.1). The predicted cleavage sites of EV71 3C protease were marked in red. The Hash sign represents the terminator. B Co-transfection analysis of PMLIV variants and 3C (lane 2, 5, 8) or 3C (E71A) (lane 3, 6, 9) or vectors (lane 1, 4, 7) in HEK293T cells. After 36 h, lysates were subjected to Western blotting. C Co-transfection analysis of PMLIV (Q430A/Q444A) dual-mutation variant and 3C (lane 2, 5) or 3C (E71A) (lane 3, 6) or vectors (lane 1, 4) in HEK293T cells. After 36 h, lysates were tested by Western blotting. D Schematic demonstration of the identified cleavage sites of 3C in PMLIV. E HEK293T cells were transfected with Myc-3C and/or Flag-PMLIV (Q430A/Q444A) as indicated for 36 h. The cell lysates were subjected to Flag IP, and samples of both cell lysates and immunoprecipitates were tested with the indicated antibodies

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  Fig. 4. EV71 3C protease cleaves PML to maintain virus replication. The control (HeLa-ScrambledKO), and the stable PML-knockout cells (HeLa-PMLKO) were cultured in 12-well plates to 60% confluency and transfected with Flag-PMLIV (lane 2, 5), Flag-PMLIV (Q430A/Q444A) (lane 3, 6), or vectors (lane 1, 4). After 24 h, all cells were infected with EV71 at the multiplicity of infection (MOI) of 1 for 8 h. EV71 capsid protein VP1, and PMLIV or its variant were analyzed by Western blotting. Tubulin was used as the protein loading control

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  Fig. 5. The morphological change of PML-NBs caused by EV71 3C cleavage. HeLa cells were transfected with Flag-PMLIV, Flag-PMLIV (Q430A/Q444A), Myc-3C, Myc-3C (E71A), or co-transfected with their combinations, respectively, for 48 h. Cells were subjected to immunofluorescence staining using an anti-Flag antibody (FITC) or anti-Myc antibody (TRITC), and the cell nuclei were stained with DAPI. Fluorescence images were taken by a Leica TCS SP5 Confocal system