Proteolytic Activities of Enterovirus 2A Do Not Depend on Its Interaction with SETD3

Figures

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  Figure 1. Schematic representation of EMCV-Lzn viral genome encoding CVB 2Apro or inactivated CVB3 2Am. The EMCV leader protein (L) was inactivated by mutating its Zn-finger domain. The CVB3 2Apro (wt or inactive mutant 2Am) coding sequence was introduced upstream of the inactivated L protein. A 3CD cleavage site (VFETQ/G) was introduced between 2Apro and L to allow efficient release of 2Apro.

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  Figure 2. Recombinant EMCV-2Apro replicates well in WT and SETD3KO Hela cells. (a) Virus titers from WT Hela cells. (b) Virus titers from SETD3KO Hela cells. Cells were infected with indicated viruses at an MOI of 5. Then cells were lysed at 2, 4, 6, and 8 hpi, respectively. Virus titers were determined by endpoint titration on BHK-21 cells. Three biological replicates were performed in each panel. One representative of two experiments is shown.

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  Figure 3. EIF4GI cleavage and 2A expression in WT and SETD3KO cells. Cells were infected with EMCV-2Apro viruses (MOI = 10). At 2, 4, 6, and 8 hpi, cells were collected and lysed in 100 μL lysis buffer. Proteins from both mock cells and cells infected with EMCV-2Am were collected at 6 hpi. Cell lysates were subjected to SDS-PAGE and analyzed by Western blot analysis. Three independent experiments were performed.

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  Figure 4. SETD3 protein is not required for NCTD induction by CVB3 2Apro. WT and SETD3KO Hela cells were infected with indicated viruses at an MOI of 50. At 2, 4, 6, and 8 hpi, cells were fixed. Subsequently, IFA assays were performed with antibodies against hnRNP K, as a marker for NCTD, and against dsRNA, as a marker for viral RNA replication. The uninfected mock was collected from 6 hpi. Scale bar represents 10 μm, all images are in the same scale. Three independent experiments were performed.

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  Figure 5. SETD3 protein is not required for the ability of CVB3 2Apro to suppress SG formation. (a) WT and SETD3KO Hela cells were infected with indicated viruses at an MOI of 10. At 2, 4, 6, and 8 hpi, cells were fixed. Subsequently, IFA assays were performed with antibodies against dsRNA, as a marker for viral RNA replication, and against eIF3 and G3BP2 as markers for SGs. Three independent experiments were performed. Scale bar represents 10 μm, all images are in the same scale. (b) Quantification of (a). In total, 250 cells were quantified in each sample. Graphs depict the ratio of SG-positive cells. The solid columns represent the ratio from WT cells and the diagonal columns represent the ratio from SETD3KO cells.

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  Figure 6. SETD3 protein is not required for the ability of CVB3 2Apro to suppress IFN induction. WT and SETD3KO Hela cells were infected with the indicated viruses at an MOI of 10. Cells were lysed at 2, 4, 6, and 8 hpi. Total RNA was extracted and used for RT-qPCR analysis for IFN-β and actin mRNA. The IFN-β levels are depicted as a fold induction compared to levels in uninfected mock cells, after correction for actin mRNA levels. The solid columns represent the levels from SETD3KO cells, and the diagonal columns represent the levels from WT cells. Error bars depict the standard deviations. Three biological replicates were performed in each panel.

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  Figure 7. EMCV-2Am infection induces NCTD in WT and SETD3KO Hela cells. WT and SETD3KO Hela cells were infected with EMCV-2Am virus at an MOI of 50. At 2, 4, 6, and 8 hpi, cells were fixed. Subsequently, IFA assays were performed. Staining with hnRNP K antibody was performed to monitor NCTD and staining with dsRNA antibody was performed to monitor viral RNA replication. The uninfected mock was collected from 6hpi. Three independent experiments were performed.

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  Figure 8. EMCV-2Am infection induces NCTD in Hela-GFP-3NLS cells. Hela-GFP-3NLS cells were infected with indicated viruses at an MOI of 50. At 2, 4, 6, and 8 hpi, cells were fixed. Subsequently, IFA assays were performed with corresponding antibodies. The uninfected mock was collected from 6 hpi. DsRNA staining was performed as a viral replication marker. GFP fluorescence was observed and hnRNP K staining was performed as the NCTD marker. Three independent experiments were performed.