FIGURE 5.

Arm/07/CBM/c2 and NH/P68 infection triggers proteasomal-dependent STAT2 degradation. STAT1 and STAT2 mRNA detection by qRT-PCR (A). PAMs were mock infected or infected with attenuated NH/P68 or with virulent Arm/07/CBM/c2 ASFV strains (1 PFU/cell). Cells were collected at 3 hpi for a qRT-PCR analysis of STAT1 and STAT2 mRNA levels. As a control of infection, viral p32 mRNA (CP204L) was measured. STAT2 (B) and STAT1 levels (C) were analyzed in presence of the proteasome inhibitor MG132 or the lysosome/autophagosome inhibitor chloroquine (CQ) by Western blot analysis. PAMs were mock infected (M) or infected with Arm/07/CBM/c2 (A) or NH/P68 (N) ASFV strains (3 PFU/cell) and treated with MG132 (20 μM) or chloroquine (50 μM) at 12 hpi. At 16 hpi, cells were collected and lysed for the Western blot analysis. The Western blot bands corresponding to STAT1 (91k Da) and STAT2 (113 kDa) were quantified according with their actin levels and relativized with the corresponding mock control using ImageJ. Immunoprecipitation of STAT2 in mock-infected or infected COS-1 cells (D). COS-1 cells were co-transfected either with pCI-His-hUbiquitin vector and pCAGGS empty vector or hSTAT2-FLAG vector. At 6 h post-transfection cells were infected with Arm/07/CBM/c2 strain (2 PFU/cell). Cells were collected at 16 hpi and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2 and anti-actin antibodies is shown. Ubiquitinated STAT2 is indicated with an arrowhead in the figure.