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. 2020 Apr 17;16(4):e1008509. doi: 10.1371/journal.ppat.1008509

Fig 5. NS1 bound to MMP9 and facilitated K63-linked polyubiquitination of MMP9.

Fig 5

(A) HEK293T cells were cotransfected with empty vector, plasmid pcDNA3.1(+)-Flag-NS1 expressing Flag-tagged NS1 and plasmid pCAGGS-HA-MMP9 expressing HA-tagged MMP9. 48h later, cells were treated with 40μg/mL CHX for 0, 9h, and then cells were lysed, and subjected to western blot with indicated antibodies. (B) HEK293T cells were cotransfected with empty vector/ Flag-NS1 and HA-MMP9 for 30h. Cells were treated with 40μg/mL CHX for 0, 9h, then immunostained with anti-Flag (red) and anti-HA (green) antibodies, and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy. Scale bar, 10μm. (C-D) HEK293T cells were cotransfected with empty vector, Flag-NS1 and HA-MMP9, cells lysates were immunoprecipitated with anti-HA (C) or anti-Flag (D) antibodies. The immunoprecipitates and whole-cell lysates (WCLs) were analyzed by western blotting with anti-Flag and anti-HA antibodies. (E) HEK293T cells were cotransfected with plasmid Flag-NS1 and HA-MMP9. Cells were lysed, and cell lysates were immunoprecipitated with anti-Flag and anti-mouse immunoglobulin G (IgG) antibodies. The immunoprecipitates and whole-cell lysates (WCLs) were analyzed by western blotting with anti-Flag and anti-HA antibodies. (F) Primary MEFs were infected with ZIKV at an MOI of 1 for 30h, and cell lysates were immunoprecipitated with anti-MMP9 and anti-goat IgG antibodies. The immunoprecipitates and WCLs were analyzed by western blotting with anti-MMP9 and anti-NS1 antibodies. HeLa (G) and HEK293T cells (H) were cotransfected with Flag-NS1 and HA-MMP9 for 24h. Cells were immunostained with anti-Flag (red) and anti-HA (green) antibodies, and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy. Scale bar, 10μm. (I) Immunoblot analysis of extracts of HEK293T cells transfected with plasmid for HA-MMP9 and Flag-NS1 and treated with dimethylsulfoxide (DMSO) or MG132. (J) HEK293T cells were cotransfected with pGFP-MMP9, pHA-Ub, and pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-HA antibodies. (K) HEK293T cells were cotransfected with pGFP-MMP9, pHA-K63, and pHA-K48, together with pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with anti-HA antibodies. (L) HEK293T cells were cotransfected with pGFP-MMP9 and pHA-K63R or pHA-K48R, together with pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-HA antibodies.