Figure 1.
NSDV replication has no obvious effect on the ERGIC and Golgi.
Vero cells were infected with the NSDVi isolate at a MOI of 0.3 TCID50. After 16 h, cells were fixed using 3% PFA followed by ice-cold methanol. Cells were co-stained with mouse anti-ERGIC53 (ERGIC; a–c), mouse anti-GM130 (cisGolgi; d–f) or rat anti-p102 (transGolgi; g–i) and rabbit antiserum against the viral N protein. Proteins were visualised by co-staining with Alexa Fluor 488 goat anti-mouse or anti-rat IgG (green), and Alexa Fluor 568 goat anti-rabbit IgG (red). Nuclei were counterstained using DAPI (blue). Bars correspond to 20 μm.
Figure 2.
NSDV-infected cells show reduced level of PDI and ERp57 in the ER.
The experiment was carried out as for Figure 1, but cells were co-stained with mouse anti-PDI (clone 1D3; a–c), mouse anti-PDI (clone RL90; d–f), rabbit anti-ERp57 (g–i) or mouse anti-calnexin (j–l), and rabbit antiserum against the viral N protein. Proteins were subsequently visualised by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red) (a–f and j–l), except for g–i, where rabbit anti-ERp57 IgGs were labelled with Zenon Alexa Fluor 488 rabbit IgG labelling reagent (green) and rabbit anti-N IgGs were labelled with Zenon Alexa Fluor 594 rabbit IgG labelling reagent (red). Bars correspond to 40 μm.
Figure 3.
PDI and ERp57 appear not to be degraded in NSDV-infected cells.
Vero cells were infected with the NSDVi isolate at a MOI of 5 TCID50 or left uninfected. After 16 h, cells were harvested by lysis and proteins separated on an acrylamide SDS-PAGE gel; proteins were detected by Western blot using antibodies specific to PDI (clone RL90), ERp57, calnexin (CNX), cis Golgi (GM130), trans Golgi (p102), PCNA and the NSDV N protein.
Figure 4.
PDI and ERp57 appear at the surface of NSDV-infected cells.
A. Samples were prepared as for Figure 1 except that, after fixing with 3% PFA, cells were labelled with mouse anti-PDI (clone 1D3; a–c), mouse anti-PDI (clone RL90; d–f) or rabbit anti-ERp57 (g–i). Then cells were again fixed with 3% PFA, opened with ice-cold methanol, and stained with rabbit antiserum against the viral N protein (a–f) or with mouse anti-PreGn antibody (g–i). Proteins were visualised by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). B. Vero cells were infected with the NSDVi isolate at a MOI of 6 TCID50 or left uninfected. After 16 h, cells were fixed using 3% PFA and left non-permeabilised. Cells were incubated with mouse anti-PDI (clone 1D3) antibody followed by Alexa Fluor 488 goat anti-mouse IgG (green). Nuclei were counterstained using DAPI (blue). Bars correspond to 40 μm.
Figure 5.
Effect of NSDV infection on the distribution of PDI in a caprine endothelial cell line (CJE).
CJE cells (a caprine endothelial cell line) were infected with the NSDVi isolate at a MOI of 6 TCID50. After 72 h, cells were fixed with 3% PFA followed by ice cold methanol (a–c) or with 3% PFA only (d–f). Cells were co-stained with mouse anti-PDI (clone 1D3) antibody and rabbit antiserum against the viral N protein (a–c), or stained only with mouse anti-PDI antibody, then again fixed with 3% PFA followed by treatment with ice-cold methanol and stained with rabbit antiserum against the N protein (d–f). Proteins were visualised by Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). Nuclei were counterstained using DAPI (blue). Bars correspond to 40 μm; arrows in “a” and “d” indicate infected cells.
Figure 6.
Time course of changes to PDI in NSDV-infected cells.
Vero cells were infected with the NSDVi isolate at a MOI of 6 TCID50 and fixed at 8, 12 and 16 hpi. For internal staining (a–i) cells were fixed using 3% PFA followed by ice cold methanol. Cells were co-stained with mouse anti-PDI (clone 1D3) antibody and rabbit antiserum against the viral N protein. For surface PDI staining (j–l), cells were fixed with 3% PFA followed by staining with mouse anti-PDI (clone 1D3) antibody. Then cells were again fixed with 3% PFA, opened with ice-cold methanol, and virus was detected by rabbit antiserum against the N protein. Proteins were visualised with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). Nuclei were counterstained using DAPI (blue). Bars correspond to 16 μm.
Figure 7.
The replication of NSDV induces secretion of PDI and ERp57 from infected cells.
Vero cells were infected with the NSDVi isolate at a MOI of 0.1 TCID50 or left uninfected. After 48 h, supernatants and cells were harvested; proteins were separated on acrylamide SDS-PAGE gels and cellular and viral proteins were detected by Western blotting using specific antibodies to PDI (clone RL90), ERp57, the viral N protein, calnexin (CNX) or α-tubulin.
Figure 8.
Viral glycoprotein association with PDI and ERp57.
A. Samples were prepared as for Figure 1 except that cells were co-stained with rabbit anti-ERp57 and mouse anti-PreGn antibodies followed by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). Bar corresponds to 40 μm. B. Vero cells were infected with the NSDVi isolate at a MOI of 0.1 TCID50 or left uninfected. After 48 h, cells were harvested and proteins were immunoprecipitated (IP) from cell lysates using mouse anti-PDI (clone RL90) antibody and protein G-agarose beads. Proteins were separated on acrylamide SDS-PAGE gels and detected by Western blotting (WB) using anti-PreGn, anti-PDI or anti-N antibodies.
Figure 9.
The effect of NSDV glycoprotein expression on PDI.
Vero cells were transfected with 1 μg of pCAGGs_MCSII_PreGn_V5 (a–c and j–l), pCAGGs_MCSII_PreGc_V5 (d–f) or pCAGGs_MCSII_NSM_V5 (g–i). After 24 h, cells were fixed with 3% PFA followed by ice-cold methanol, and were stained using mouse anti-PDI (a–i) or anti-calnexin (j–l) antibodies followed by Alexa Fluor 568 goat anti-mouse IgG (red). Plasmid-expressed proteins were visualised with mouse anti-V5 antibody conjugated to Alexa Fluor 488 (green). Nuclei were counterstained using DAPI (blue). Bars correspond to 40 μm. Arrows in a–b and j–k indicate cells expressing PreGn.