Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71

Figures

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  Figure 1. Stau1 binds to 5′ untranslated region of EV-A71 RNA. (A) Stau1 binds to EV-A71 viral RNA in an in vitro RNA pulldown assay. The recombinant GST-tagged Stau1 protein was expressed and purified from Escherichia Coli. The biotin-labeled EV-A71 viral RNA was used for the RNA pulldown analysis. Lanes 1 and 2: Input of the recombinant GST or GST-Stau1 protein; Lane 3: GST-tagged Stau1 protein only; Lane 4: GST-tagged Stau1 protein interacted with non-biotin-labeled RNA (competitive RNA); Lane 5–7: GST-tagged Stau1 protein interacted with biotin-labeled RNA without or with competitive RNAs. (B) Stau1 binds to 5′-UTR but not 3′-UTR of EV-A71 RNA genome. GSI-tagged Stau1 protein was expressed for the in vitro RNA pulldown assay as described above. The GST-Stau1 (RBD2-3) is a truncated protein containing RNA-binding domain 2 and 3 as described in Section 3.2.

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  Figure 2. Colocalization of Stau1 with viral RNA in EV-A71-infected cells. RD cells were infected without or with EV-A71 at an MOI of 1 for 6, 12, and 24 h, followed by fixation and coimmunostaining with anti-Stau1 (green) and anti-dsRNA (red) antibodies. Bound primary antibodies were detected using secondary antibodies conjugated to Texas Red (for dsRNA) or fluorescein isothiocyanate (for Stau1). The images represent three independent experiments that used three independent infections. The merged images of anti-Stau1 (green) and anti-dsRNA (red) are shown in panels c, f, i and l, respectively. Panels m and n are enlarged images of panels i and l, respectively. Bar = 10 µm.

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  Figure 3. Stau1 interacts with EV-A71 viral RNA through its RBD2-3. (A) Domain structure of Stau1. The regions RBD2-3 and RBD4-5 are indicated with superscript numbers (nt). (B) Pulldown experiments using streptavidin beads directed against biotin-labeled RNA. Binding of an RNA-specific protein was determined using an anti-GST antibody (Lane 3). The non-biotin-labeled RNA was used as the competitive RNA (Lane 4–7). The input of the GST-Stau1 recombinant protein is shown as the control (Lane 2). The “+” indicates the addition of recombinant GST-Stau1 protein; the “−“ indicates no biotin-RNA/competitive RNA.

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  Figure 4. Reduction of EV-A71 viral RNA accumulation and viral protein production in the Stau1-knockout cells. (A) The Stau1 knockout cell line was generated using the CRISPR/Cas9 system as indicated in the Materials and Methods section. No expression of Stau1 was confirmed in the cells upon EV-A71 infection. (B) Reduction in EV-A71 viral RNA was determined using RT-qPCR analysis in the EV-A71-infected normal cells and Stau1 knockout cells. (C) Decreased production of EV-A71 viral proteins in the EV-A71-infected normal cells and Stau1 knockout cells. The right panels are the relative densities of protein bands measured using ImageJ software. (D) Ectopic expression of Stau1 restored the EV-A71 viral RNA accumulation in the Stau1 knockout cells. Overexpression of Flag-tagged Stau1 protein was detected during the Western blotting using anti-Flag- and anti-Stau1-specific antibodies in normal cells and Stau1 knockout cells (right panel). Star sign * indicates significant difference in comparisons * p < 0.05; ** p < 0.01; *** p < 0.001. OE: Overexpression; M: Transfection with plasmid only.

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  Figure 5. Stau1 positively regulates the EV-A71 IRES activity. (A) EV-A71 viral IRES genome structure. (B) The effect of Stau1 on the IRES activity was evaluated with IRES domain II–IV. The IRES domain II–IV was subcloned into pGL4.17 (containing Firefly luciferase gene) plasmid followed by cotransfection with pCDNA3.1-CMV (containing Renilla luciferase gene) in normal cells and Stau1 knockout cells. A dual luciferase reporter assay was performed to measure the IRES activity. Overexpression of recombinant Stau1 RBD2-3 protein was used for the restored experimental assay.

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  Figure 6. Investigation of polysome profile in the Stau1 knockout cells. The normal and Stau1 knockout RD cells were infected with EV17 at an MOI of 1. At 24 h postinfection, the cell lysates were harvested and subjected to sucrose density fractionation to obtain the polysome profile. Each fraction was harvested for RNA isolation and quantitation. (a) EV-A71-infected normal RD cells. (b) EV-A71-infected Stau1 knockout RD cells. The representative of different ribosomal RNAs and ribosome-bound mRNA (between two vertical dot lines) is shown in the enlarged panel (The wave line is referred to the different fractions).

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  Figure 7. Stau1 improves the stability of 5′-UTR ofEV-A71 viral RNA. The 5′-UTR of EV-A71 RNA was transfected in the normal, Stau1 knockout, and recombinant Stau1-overexpressed RD cells for the viral RNA stability assay. The transfectant cell lysates were then harvested at 30 min posttransfection for the RNA stability assay. RNase inhibitor was added for the negative control as shown in * (a). The 5′-UTR of GAPDH RNA was used as the experimental control (b).