Table 1.
Primers used for plasmids construction.
Table 2.
Real-time PCR primers used to measure the mRNA levels of IFN downstream antiviral genes.
Fig 1.
STAT1 expression in NDV infected or V-expressing plasmids transfected cells.
(A) No STAT1 reduction was observed in Vero cells infected with NDV ZJ1, 9a5b or LaSota at MOI 3 at 6, 12 or 24 hpi. (B) Over-expression of ZJ1, 9a5b and LaSota V protein did not effect on STAT1 expression in Vero cells transfected with V-expressing plasmids. (C) STAT1 was reduced at 48 h post-transfection in NDV-infected Vero cells (MOI = 3) transfected in advance with V-expressing plasmids. (D) STAT1 expression in A549 cells transfected with V expressing plasmids or infected with ZJ1 or LaSota was detected at 48 h post-transfection or 12 h post-infection by indirect fluorescence assay. These infected A549 cells were fixed and detected for P/V/W proteins with a mixture of anti-serum Pab-V1 and Pab-V2; while the presence of STAT1 was determined by anti-STAT1 antibody (ab31369). Cellular nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).
Fig 2.
Newcastle disease virus infection impaired IFN-α-induced STAT1 phosphorylation.
(A) IFN-α-induced phosphorylated STAT1 degradation in NDV-infected A549 and Vero cells. A549 or Vero cells were infected with NDV strains ZJ1 at MOI 3. At indicated time points post infection, A549 and Vero cells were stimulated with 500 U/ml human IFN-α or IFN-γ in 1 ml DMEM at 37°C for 15 min. Uninfected cells were stimulated with IFN as negative controls (IFNα+/infection-). IFN-α-induced phospho-STAT1 was observed (IFNα+/infection+) for reduction in total STAT1 proteins. IFN-γ-induced phospho-STAT1 proteins were not reduced. (B) Phospho-STAT1 in A549 cells transfected with V-expressing plasmids after stimulation with IFN-α. (C) Expression level of STAT1 and phospho-STAT1 decreased in V-expressing Vero cells after IFN-α stimulation. Vero cells were mock transfected with pCI-neo plasmids. Cells on glass coverlips were transfected with pCI-V/ZJ1 plasmids. At 48 h post-transfection, cells were treated with IFN-α for 15 min prior to fixation as in “Material and methods”. V protein was detected by a mixture of anti-serum Pab-V1 and Pab-V2; STAT1 and phosphorylation were determined by anti-STAT1 antibody (ab31369) and anti-phospho-STAT1 antibody (ab30645). Cellular nuclei were stained with DAPI.
Fig 3.
The reduction of total and phosphorylated STAT1 in NDV-infected cells was inhibited after treatment with Ub E1 inhibitor PYR-41 at different time points.
(A) STAT1 and phospho-STAT1 expression levels in PYR-41 treated Vero cells at 6 hpi. (B) Phospho-STAT1 levels in PYR-41 treated Vero cells at 4, 6, 8 hpi. (C) Exogenous mutant STAT1 lacking 701aa phosphorylation site were not degraded in the course of NDV infection. One microgram pFlag-STAT1 or pFlag-Y701F was transfected into A549 cells cultured in 6-well plates. At 12 h post transfection, the cells were subsequently infected with NDVs at a MOI of 3. The cells were harvested at 24 hpi. (D) The expression levels of IFN-responsive genes in V-expressing A549 cells after stimulation of IFN-α. A549 cells in 6-well plates were transfected with 3 μg pCI-V or pCI-neo for each well as above-described. At 4 h and 8 h post-transfection, the cells were harvested following the treatment with 500 U/ml IFN-α for 30 min.
Fig 4.
Recovery of V-deficient recombinant NDV from an infectious clone.
(A) Schematic representation of a V-deficient gene mutation in a full-length cDNA clone. To selectively block expression of V, modification was performed after the RNA-editing site of the full-length cDNA clone pTVT-ZJ1 to introduce a stop codon in the ORF with +1 frameshift. (B) Mutation in the genome of recovered rZJ1-VS was confirmed by sequencing. Total viral RNA was extracted and RT-PCR-amplified for sequencing. The genome of rZJ1-VS was identical to the wt ZJ1 except for AT for TA after the RNA editing-site. (C) Expression of V protein was examined in DF-1 cells infected with rZJ1-VS or ZJ1 at MOI 3. The V protein was undetectable in the rZJ1-VS infected cells; by contrast, ZJ1 expressed V protein in the infected cells at the same point. (D) Growth curves of rZJ1-VS and the wt ZJ1 in DF1 and Vero cells. Replication of rZJ1-VS was compared to ZJ1 in early infection. (E) Cytopathic effect (CPE) caused by rZJ1-VS or ZJ1 in DF1 cells infected with rZJ1-VS or ZJ1 at MOI 0.01.
Table 3.
Cytopathic effects and virus titers of NDV-infected cells.
Table 4.
Determination of virus virulence.
Fig 5.
Infection with rZJ1-VS did not result in degradation of phospho-STAT1.
A549 cells were infected with rZJ1-VS or wt ZJ1 at a MOI of 1 or 3. At indicated time points, cells were stimulated with 500 U/mL IFN-α for 15 min and harvested for Western blot. Phospho-STAT1 and STAT1 was observed in A549 cells infected with ZJ1 or rZJ1-VS, which expressed incomplete V protein.
Fig 6.
The expression levels of IFN-β and IFN-responsive genes in NDV-infected A549 and DF1 cells.
(A) The IFN-β protein levels in A549 cells stimulated by NDV were assayed by ELISA so as to determine its effect to IFN signaling. (B) The IFN-responsive genes IFIT1, ISG15, Mx1, and IFN-β were further quantified by Real-time PCR in V-deficient rZJ1-VS and wt ZJ-1-infected A549 cells. (C) IFN-responsive genes in rZJ1-VS or ZJ1 infected DF1 cells. Gene expression was compared between DF1 cells infected with rZJ1-VS and wt virus ZJ1 at a MOI 3. *Gene expressions were enhanced significantly in rZJ1-VS-infected cells than those in rZJ1-infected cells (P < 0.01). (D) The V protein expression levels in NDV-infected A549 and DF1 cells. The P and V protein were detected in WB assay at different time points post infection with NDV strain ZJ1.