ALV-miRNA-p19-01 Promotes Viral Replication via Targeting Dual Specificity Phosphatase 6

Figures

• 

  Figure 1. Identification of ALV-J-encoded miRNAs by deep sequencing. (A) Cluster analysis showing the differentially expressed miRNAs between ALV-J-infected DF-1 cells (ALV-J) and normal DF-1 cells (Mock). (B) Schematic diagram of the ALV-miRNA-p19-01 location on the ALV-J SCAU1903 genome. (C) Secondary-structure prediction of ALV-miRNA-p19-01. (D) Conserved analysis of ALV-miRNA-p19-01 among different ALV-J strains.

• 

  Figure 2. Experimental validation of ALV-miRNA-p19-01. (A,B) DF-1 cells were infected with ALV-J SCAU1903, normal cells as a negative control. (A) Mature ALV-miRNA-p19-01 were examined by stem-loop-RT-PCR in DF-1 cells. (B) ALV-miRNA-p19-01 was detected with Northern blotting. The probe was the biotin-conjugated antisense strand of ALV-miRNA-p19-01. Left: 30 μg of small RNA from normal DF-1 cells; Right: 30 μg of small RNA from DF-1 cells infected with ALV-J SCAU1903. U6 was used as a control. (C) The ALV-miRNA-p19-01 expression levels were measured by RT-PCR in different tissues of ALV-J-positive chickens. (D) Dicer and Drosha were suppressed with siRNAs in DF-1 cells, and ALV-miRNA-p19-01 expression was detected by RT-PCR. NC, normal control. (E) DF-1 cells were infected with ALV-J SCAU1903 and then treated with α-amanitin. ALV-miRNA-p19-01 expression was detected by RT-PCR. Statistically significant values are indicated by ** p < 0.01.

• 

  Figure 3. ALV-miRNA-p19-01 promotes ALV-J SCAU1903 replication. (A–C) DF-1 cells respectively transfected with ALV-miRNA-p19-01 mimic and miR-NC were infected with ALV-J SCAU1903 at 4 hpt. (A) The expression of p27 protein were examined by Western Blot. (B) The transcriptional level of viral particles in supernatants were measured by RT-PCR. (C) The supernatants from infected cells were titrated in DF-1 cells by ELISA for TCID50. (D) ALV-miRNA-p19-01 mimic was transfected into DF-1 cells and miR-NC was used as a control. DF-1 cells were infected with ALV-J SCAU1903 at 4 hpt and processed for transmission electron microscopy as described in Materials and Methods. (E–G) DF-1 cells respectively transfected with ASO-ALV-miRNA-p19-01 and ASO-NC were infected with ALV-J SCAU1903 at 4 hpt. (E) The expression of p27 protein were examined by Western Blot. (F) The transcriptional level of viral particles in supernatants were measured by RT-PCR. (G) The supernatants from infected cells were titrated in DF-1 cells by ELISA for TCID50. Statistically significant values are indicated by ** p < 0.01.

• 

  Figure 4. ALV-miRNA-p19-01 targets DUSP6 in DF-1 cells. (A) Putative candidate genes enriched from those ALV-miRNA-p19-01 binding genes obtained from RNA-pull down and RNA-sequence. (B) Luciferase reporter assays. Bioinformatics predictions of binding sites in DUSP6 3′UTR region. Wild-type (wt) and mutant-type (mut) sequences are indicated. DF-1 cells were co-transfected with wt or mut DUSP6 3′-UTR and ALV-miRNA-p19-01 mimic, nonspecific mimic (NC) was used as a control. The data are expressed as relative firefly luciferase activity normalized to Renilla luciferase activity. The data are presented as mean ± standard deviation values from 3 independent experiments. (C) DF-1 cells were transfected with ALV-miRNA-p19-01 mimic or miR-NC. The mRNA level of DUSP6 was measured by qRT-PCR. (D) DF-1 cells were transfected with ALV-miRNA-p19-01 mimic or miR-NC or infected with ALV-J SCAU1903. The protein level of DUSP6 was measured by Western Blot. β-actin was used as a control. Statistically significant values are indicated by ** p < 0.01.

• 

  Figure 5. DUSP6/ERK2 pathway inhibits ALV-J replication. (A–C) DF-1 cells respectively transfected with pRK5-flag-DUSP6 plasmid and pRK5-flag plasmid were infected with ALV-J rSCAU1903-mut at 4 hpt. (A) The expression of p27 protein were examined by Western Blot. (B) The transcriptional level of viral particles in supernatants were measured by RT-PCR. (C) The supernatants from infected cells were titrated in DF-1 cells by ELISA for TCID50. (D–F) DF-1 cells respectively transfected with si-DUSP6 and si-NC were infected with ALV-J rSCAU1903-mut at 4 hpt. (D) The expression of p27 protein were examined by Western Blot. (E) The transcriptional level of viral particles in supernatants were measured by RT-PCR. (F) The supernatants from infected cells were titrated in DF-1 cells by ELISA for TCID50. (G) Si-DUSP6 was transfected into DF-1 cells and si-NC was used as a control. DF-1 cells were infected with ALV-J rSCAU1903-mut at 4 hpt and processed for transmission electron microscopy as described in Materials and Methods. Statistically significant values are indicated by ** p < 0.01.

• 

  Figure 6. ALV-miR-p19-01 promotes viral replication via activating DUSP6/ERK2 pathway. (A) DF-1 cells respectively transfected with ALV-miRNA-p19-01 mimic and miR-NC were infected with ALV-J rSCAU1903-mut at 4 hpt. (B) DF-1 cells respectively transfected with ASO-ALV-miRNA-p19-01 mimic and ASO-NC were infected with ALV-J SCAU1903 at 4 hpt. (C) DF-1 cells were co-transfected with ALV-miRNA-p19-01 mimic and pRK5-flag-DUSP6, and then DF-1 cells were infected with ALV-J rSCAU1903-mut at 4 hpt. The expressions of p27, DUSP6, ERK2, p-ERK2 and β-actin were detected by Western Blot.