Cellular hnRNP A1 Interacts with Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus and Impairs Viral Replication

Figures

• 

  Figure 1. Confirmation of expression of hnRNP A1 by western blot. Analysis of hnRNP A1 expression level in the jejunum tissues of the control, YN13-infected and YN144-infected groups. The WB-ratio (western blotting ratio, PEDV infected/mock) was calculated based on the relative intensity of each band in PEDV-infected group to the corresponding band in control. β-actin was used as a loading control. The MS-ratio (infection/control) obtained by MS analysis are shown on the right.

• 

  Figure 2. Co-localization of N protein with hnRNP A1. CCL-81 cells were infected with different PEDV strains. At 12 hpi, the localization of PEDV N protein and hnRNP A1 in cells was examined by immunofluorescence staining with anti-PEDV N protein and anti-hnRNP A1 antibodies. The nucleus is indicated by DAPI (blue) staining. PEDV N protein is indicated in green and the hnRNP A1 in red.

• 

  Figure 3. Interaction of hnRNP A1 and PEDV N protein. HEK293T cells were transfected with a vector expressing PEDV N protein with a flag tag (flag-PEDV-N) or empty vectors (flag-pCAGGS) and the whole-cell lysates obtained at 24 hpt were immunoprecipitated with anti-Flag mAb. After separation by SDS-PAGE, proteins were detected by immunoblotting with the indicated antibodies (IP: Flag). Cell lysates were also applied to confirm the expression of proteins (Input).

• 

  Figure 4. Silencing hnRNP A1 by siRNAs. CCL81 cells were transfected with no siRNA (Mock), negative control siRNA (NC), SiRNA-1, SiRNA-2 or SiRNA-3 at a concentration of 20 nM. Cells were collected at 60 hpt to analyze the expression of hnRNP A1 by western blot. HnRNP A1 protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin.

• 

  Figure 5. Knockdown of hnRNP A1 expression inhibits YN144 replication. CCL-81 cells were transfected without siRNA (a) or with NC (b) or siRNA-1 (c). At 60 hpt, cells were infected with YN144 Strain at a moi of 0.001 or mock infected (d). Virus suspensions were harvested at 12 hpi to calculate the virus titer by real-time RT-PCR. The virus copies per mL suspension was calculated (A). Cells were harvested at 12 hpi to analyze the expression of PEDV N protein by Western blot (B) and the numbers of cells infected with PEDV by IFA (Original magnification 100×) (C). PEDV N protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. Data are shown as means ± the standard errors of the mean (SEM) of at least three independent experiments, with the error bars representing the standard deviations.

• 

  Figure 6. Knockdown of hnRNP A1 expression inhibits YN13 replication. CCL-81 cells were transfected without siRNA (a) or with NC (b) or siRNA-1 (c). At 60 hpt, cells were infected with YN13 Strain at a moi of 0.001 or mock infected (d). Virus suspensions were harvested at 24 hpi to calculate the virus titer by real-time RT-PCR. The virus copies per mL suspension was calculated (A). Cells were harvested at 24 hpi to analyze the expression of PEDV N protein by Western blot (B) and the numbers of cells infected with PEDV by IFA (Original magnification 100×) (C). PEDV N protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. Data are shown as means ± SEM of at least three independent experiments, with the error bars representing the standard deviations.

• 

  Figure 7. Knockdown of hnRNP A1 expression inhibits CV777 replication. CCL-81 cells were transfected without siRNA (a) or with NC (b) or siRNA-1 (c). At 60 hpt, cells were infected with CV777 Strain at a moi of 0.001 or mock infected (d). Virus suspensions were harvested at 24 hpi to calculate the virus titer by real-time RT-PCR. The virus copies per mL suspension was calculated (A). Cells were harvested at 24 hpi to analyze the expression of PEDV N protein by western blot (B) and the numbers of cells infected with PEDV by IFA (Original magnification 100×) (C). PEDV N protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. Data are shown as means ± the SEM of at least three independent experiments, with the error bars representing the standard deviations.