Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers
Fig 5
Interaction of ratIE1 with ratPML followed by ratPML deSUMOylation and dispersion.
(A) Schematic overview of full-length ratPML and deletion mutants. (B) Efficient interaction of ratIE1CORE with ratPML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged ratIE1 or ratIE1core (residues 1–392) and Myc-tagged ratPML variants. After cell lysis, immunoprecipitation was performed with an anti-FLAG antibody. Co-precipitated ratPML proteins (IP), precipitated ratIE1 proteins, and proteins within the cell lysate (input) were analyzed by Western blotting as indicated. (C) Binding of ratIE1CORE to ratPML requires both the coiled-coil and the RING domain. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged ratPML variants and Myc-tagged ratIE1CORE (residues 1–382) as indicated. Upper two panels: Western blot detection of ratIE1 and ratPML after immunoprecipitation using an anti-FLAG antibody. Lower two panels: detection of ratIE1 and ratPML in cell lysates before precipitation (input). (D) Inhibition of ratPML SUMOylation by ratIE1 expression. HEK293T cells were transfected with expression plasmids encoding Myc-ratPML, HA-SUMO2 and FLAG-ratIE1 as indicated. After cell harvest, ratPML and SUMOylated ratPML were visualized by Western blotting using anti-Myc and anti-HA antibodies, respectively. Expression of IE1 was analyzed with an anti-FLAG antibody and β-actin was included as internal control. (E) Impact of RCMV infection on ratPML SUMOylation. Rat embryonic fibroblast (REF) cells were infected with RCMV at an MOI of 1.5 or mock infected, and were harvested at indicated times for Western Blot analysis of ratPML (upper panel), ratIE1 (middle panel), and β-actin (lower panel) as loading control. (F) Impact of RCMV infection on ratPML-NB integrity. REF cells were infected with RCMV at an MOI of 0.7 or mock infected, and were harvested at indicated times for immunofluorescence analysis of ratIE1 (left panel) or ratPML (right panel). Cell nuclei were stained with DAPI. F, FLAG; M, Myc; R, RING domain; B, B-boxes; CC, coiled-coil domain.