Table 1.
Primers used in this study.
Table 2.
Predicted SUMOylation sites in the nonstructural and structural proteins of PRRSV.
Fig 1.
Screening of PRRSV proteins interacting with Ubc9 by yeast two-hybrid.
Yeast Y2HGold cells co-transformed with various bait and prey plasmids were selected on QDO/X/ABA plates. Shown are the blue colonies presenting the interaction of PRRSV Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9, and white colonies without interaction of the rest of PRRSV proteins with Ubc9. p53 (Gal4 binding domain fused to the murine p53) and T (Gal4 activation domain fused to the SV40 large T-antigen), Lam (Gal4 binding domain fused to nuclear lamina protein) and T were served as positive control and negative control, respectively. The three spots in the same line mean the replicates of one analysis and each spot represents a different colony.
Fig 2.
The interaction of PRRSV Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9.
(A) The interaction of Nsp1β, Nsp4, Nsp9, Nps10 and N protein with exogenous Ubc9 by using a Co-IP assay. HEK293 cells were co-transfected with the Myc-Ubc9-expressing plasmid and the HA-Nsp1β-, HA-Nsp4-, HA-Nsp9-, HA-Nsp10- and HA-N-expressing plasmid, respectively. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb and anti-Myc PAb. The left panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates and the right panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates. The asterisk (★) indicates the IgG light chain band with 26 KDa, and the solid triangle (▲) represents the target protein Myc-Ubc9. (B) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 by using a GST pull-down assay. The cell lysates containing Nsp1β, Nsp4, Nsp9, Nsp10 and N protein individually were pulled down with prokaryotic expressed and purified GST-Ubc9 protein with an anti-GST mAb and probed with anti-HA and anti-GST mAb. (C) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9. MARC-145 cells were transduced with the lentiviruses that were expressing GFP, Nsp1β, Nsp4, Nsp9, Nsp10, or N individually. The cell lysates were immunoprecipitated with an anti-GFP mAb and followed by Western blot analysis with anti-Ubc9 and anti-GFP antibodies. The left panel indicates the identification of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP expressed in cell lysates, while the right panel shows the Co-IP analyses of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP from cell lysates.
Fig 3.
Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9.
(A) Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 in HEK293 cells. HEK293 cells were co-transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N with pCMV-Myc-Ubc9, respectively. The cells were fixed at 36 h post-transfection and processed by immunostaining with a mouse anti-HA mAb or rabbit anti-Myc PAb and TRITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG, and were then examined by confocal microscopy (600×magnification). Nuclei were stained with DAPI. Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9 in mock-infected MARC-145 cells and PRRSV-infected MARC-145 cells (B), mock-infected PAMs and PRRSV-infected PAMs (C). The mock- or PRRSV-infected cells were fixed at 24 h post-infection and processed by immunostaining with a rabbit anti-Ubc9 PAb or mouse anti-Nsp1β, anti-Nsp4, anti-Nsp9, anti-Nsp10 and anti-N mAb, respectively, and then immunostained with TRITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI.
Fig 4.
Inhibition of PRRSV JXwn06 replication by Ubc9.
(A) PRRSV titers in Ubc9-overexpressed MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction, and the virus titers were then assayed by a microtitration infectivity assay at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (**p<0.01). (B) PRRSV RNA replication in Ubc9-overexpressing MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction and collected at the indicated time points post-infection. The total cellular RNA was extracted and the mRNA levels of PRRSV N gene were determined by quantitative RT-PCR. Data are shown as means ± SD of three independent experiments (***p<0.001; ns, no significant). (C) SiRNA-mediated knockdown of endogenous Ubc9. MARC-145 cells were transfected with siRNA (siUbc9-1, siUbc9-2, siUbc9-3) and control siRNA (siCon). The cells were harvested at 48 h post-transfection and the cell lysates were probed with an anti-ubc9 antibody. The optical density ratios of Ubc9/β-actin in Ubc9 gene-silenced MARC-145 cells are shown with graphs. Data are shown as means ± SD of three independent experiments (***p<0.001). (D) The optical density ratios of Ubc9/β-actin in Ubc9 gene-silenced MARC-145 cells are shown with graphs. Data are shown as means ± SD of three independent experiments (***p<0.001). (E) PRRSV titers in Ubc9 gene-silenced MARC-145 cells. MARC-145 cells transfected with the siRNA (siUbc9-1) or control siRNA (siCon) for 48 h were infected with PRRSV JXwn06 at MOI of 0.01, and the virus titers were examined at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (*p<0.05; ***p<0.001; ns, no significant). (F) PRRSV RNA replication in Ubc9-silenced MARC-145 cells. MARC-145 cells transfected with the siRNA (siUbc9-1) or control siRNA (siCon) for 48 h were infected with PRRSV JXwn06 at MOI of 0.01 and collected at the indicated time points post-infection. The total cellular RNA was extracted and the mRNA levels of PRRSV N gene were determined by quantitative RT-PCR. Data are shown as means ± SD of three independent experiments (***p<0.001; ns, no significant). (G) PRRSV growth in the MARC-145 cells treated with GA. Data are shown as means ± SD of three independent experiments (***p<0.001; **p<0.01; ns, no significant).
Fig 5.
SUMOylation of PRRSV N protein.
(A and B) The expression of Nsp1β, Nsp4, Nsp9, Nsp10 and N proteins in HEK293 cells using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N, separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with an anti-HA mAb. The left panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates and the right panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates. (C) The expression of N and mutated N proteins (C23S and C23A) in HEK293 cells by using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-N, pCMV-HA-N (C23S), pCMV-HA-N (C23A), separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb. The left panel indicated the identification of HA-N, HA-N (C23S) and HA-N (C23A) expressed in cell lysates and the right panel showed the Co-IP analyses of HA-N, HA-N (C23S) and HA-N (C23A) from cell lysates. (D) The expression of N protein in MARC-145 cells following PRRSV infection using a Co-IP assay. MARC-145 cells were infected with PRRSV JXwn06 at a MOI of 0.1. At 36 h the cell lysates were immunoprecipitated with an anti-N mAb, anti-SUMO1 or anti-SUMO2/3 mAb and probed with these mAb, separately.