Cleavage and Sub-Cellular Redistribution of Nuclear Pore Protein 98 by Coxsackievirus B3 Protease 2A Impairs Cardioprotection

Figures

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  Figure 1. NUP98 is cleaved by viral protease 2A during CVB3 infection. (A) NUP98 cleavage in CVB3-infected HeLa cells. HeLa cells were infected with CVB3 or sham-infected with PBS. At the indicated time points post-infection (pi), cell lysates were harvested for western blot analysis of NUP98 cleavage products. VP1, a viral capsid protein, was used to represent viral replication. β-actin was used as a loading control. Molecular weight markers in kDa are indicated. (B) In vitro cleavage assay. Non-infected HeLa cells lysates were incubated overnight with purified recombinant viral protease 2A, 3C, 2Amut , or 3Cmut (mut indicates mutated, inactive catalytic sites). Lysates were subjected to western blot analysis of NUP98 cleavage products. CVB3-infected cell lysate harvested at 7 h pi was used as a positive control. β-actin was used as a loading control.

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  Figure 2. CVB3 infection induces the sub-cellular redistribution of NUP98 in cardiomyocytes. Mouse HL-1 cardiomyocytes were sham-infected with PBS or infected with CVB3 for 2–6 h. Cells were fixed and immunostained using a NUP98 specific antibody. Blue represents the nuclei stained with DAPI and green shows NUP98 protein. Beginning at 4 hpi, NUP98 was relocalized to the cytoplasm in punctate structures, becoming most apparent at 6 hpi. Cells were observed at each time-point by confocal microscopy at 20× magnification. Scale bars = 20 μm.

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  Figure 3. Ectopic expression of viral protease 2A or 3C results in differential sub-cellular redistribution of NUP98. HeLa cells were transfected with plasmid expressing viral protease 2A or 3C or with empty vector for 24 h. Cells were fixed and immunostained as described in Figure 2. Cells were visualized by confocal microscopy, images are shown at 60× magnification. Blue represents the nuclei stained with DAPI and green shows NUP98 protein. Scale bars = 5 μm. 2A transfected cells show NUP98 redistributed to the cytoplasm, indicating cleavage.

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  Figure 4. siRNA knockdown or overexpression of NUP98 differentially regulates NRG1, ERBB4, PSEN1, and VP-1 expression. (A) HeLa cells were transfected with scrambled or NUP98 siRNA for 48 h. Cells were subsequently infected with CVB3 or sham-infected with PBS. Cell lysates were harvested at the indicated time-points for western blot analysis using the indicated antibodies. β-actin was used as a loading control. (B) Quantification of protein expression. Densitometry was performed on the western blot images using the NIH ImageJ program. The data were normalized against the loading control (n = 3, *p < 0.05) and displayed graphically. Each scrambled siRNA transfected protein was compared to the corresponding timepoint of siNUP98 transfected cells. (C) HeLa cells were transfected with GFP-NUP98 plasmid or vector only for 48 h and then infected with CVB3 or sham-infected (PBS). Cell lystates were harvested at the indicated time-points for western blot analysis. (D) Quantification of protein expression. Densitometry and data normalization were performed as described for (B) (n = 3, *p < 0.05) and displayed graphically.

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  Figure 5. siRNA knockdown of NUP98 enhances viral particle formation and reduces cell viability. (A) HeLa cells were transfected with scrambled or NUP98 siRNA for 48 h. Cells were subsequently infected with CVB3 or sham-infected with PBS for 5 or 7 h. Supernatants were collected and used to measure viral particle release by viral plaque assay. Data were subjected to statistical analysis, n = 3, **p < 0.01. (B) Cell viability assay. HeLa cells were transfected with scrambled siRNA or siNUP98 for 48 h and then infected with CVB3 or sham-infected with PBS for 5 or 7 h. MTS viability assay was performed. Sham infected control cell viability was set to 100% survival and other data were converted to percentage of the control, n = 3, *p < 0.05.

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  Figure 6. Nup98, Nrg1, and erbB4 are upregulated at early phase of infection and were downregulated at later phase in mice. Four-weeks-old A/J mice were infected with CVB3 at 105 PFU or sham-infected with PBS. Mouse heart tissues were harvested at the indicated days post-infection. (A) Tissue lysates was subjected to western blot analysis of NUP98 cleavage products and its downstream gene expression using the indicated antibodies. (B) Quantification of protein expression levels. Densitometry was performed on western blot bands using the NIH ImageJ program as described in Figure 4B (n = 3, *p < 0.05). (C) H&E staining. Tissues were fixed in formalin and paraffin embedded at time of harvest corresponding to dpi. Images shown are at 10× magnification. Arrows indicate inflammatory infiltration. (D) Tissue was fixed in formalin and paraffin embedded. Immunohistochemistry was performed using the indicated antibodies. Red staining indicates the protein of interest. The Images were taken at 10× magnification. Scale bar = 200 μm. (E) Quantification of the expression levels of Nup98, Nrg1, and erbB4 was conducted using the Aperio ImageScope software. Expression for sham-infected control was set to 1…