Table 1.
HCMV ORFs that contain possible SUMOylation sites.
Figure 1.
SUMOylation analysis of HCMV proteins in bacteria.
E. coli (BL21) cells were transformed with plasmids expressing GST-HCMV ORF (ampicillin-resistant) or cotransformed with plasmids expressing GST-HCMV ORF and pT-E1E2S1 (chloramphenicol-resistant). One milliliter of bacterial cell culture was induced with 0.4 mM IPTG for 5 h at 30°C. Total cell lysates were prepared by boiling the cell pellet in 200 µl of 1× protein loading dye. Clarified cell lysates were separated by SDS-PAGE, and immunoblot analysis was performed with anti-GST antibody. HCMV ORFs fused to GST and the ORF sizes (number of amino acids in parenthesis) are indicated. The SUMO-modified forms of UL123 (IE1), UL122 (IE2), and US34A are indicated as open arrowheads.
Figure 2.
Evaluation of SUMOylation in cotransfection and in vitro assays.
(A) 293T cells in six-well plates were cotransfected with 0.5 µg of plasmid expressing myc-US34A, myc-UL150, flag-SUMO-1, or flag-SUMO-2 as indicated. At 48 h, total cell lysates were prepared and immunoblotted with an anti-myc antibody. The bands corresponding to unmodified and SUMO-modified forms of myc-US34A and unmodified myc-UL150 are indicated. NS, non-specific bands. (B) In vitro SUMOylation reactions. Myc-UL123(IE1) and myc-US34A produced by in vitro transcription/translation were incubated with GST-SAE2/1, His-Ubc9, and His-SUMO-1GG or GST-SUMO-1GG as indicated. The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with a myc-IE1 antibody. Unmodified and SUMO-modified forms of IE1 and US34A are indicated. (C) The disorder in UL122 (IE2), UL123 (IE1), and US34A was predicted with the IUPred program (http://iupred.enzim.hu). The lysine residues modified by SUMO (for IE1 and IE2) or predicted to be SUMOylation sites (for US34A) are indicated.
Figure 3.
SUMOylation patterns of IE1 and IE2 during HCMV infection.
(A) HF cells were mock-infected or infected with HCMV at an MOI of 5. Total cell lysates were prepared at indicated time points and immunoblotting was performed with antibodies that recognize IE1 (6E1), IE2 (12E2), or both IE1 and IE2 (8131). The β-actin levels are shown as a loading control. The bands indicated as open circles appear to be non-specific or represent other modified forms of IE1 and IE2. (B) 293T cells in six-well plates were cotransfected with plasmids expressing IE1 (1 µg), flag-SUMO-1 (1 µg), and increasing amounts of IE2 (0.3, 1, and 3 µg), as indicated. At 48 h, total cell lysates were prepared and immunoblot assays were performed with anti-IE1/IE2 antibody.
Figure 4.
Interaction of IE1 with PIAS1.
(A) 293T cells in a 100-mm dish were cotransfected with 5 µg of plasmids expressing myc-PIAS1 and HA-IE1 or HA-UL53, as indicated. At 48 h, total cell lysates were prepared and immunoprecipitated with an anti-myc antibody, followed by immunoblotting with an anti-HA antibody. The levels of HA-IE1, HA-UL53, and myc-PIAS1 in whole cell lysates (WCL) were also shown by immunoblotting. (B) The GST and GST-IE1 proteins purified from bacteria were used in GST pull-down assays. Five micrograms of GST and GST-IE1 proteins were immobilized on glutathione-Sepharose beads and were incubated with in vitro-translated and [35S]-methionine–labeled PIAS1. Input PIAS1 (5%) and the GST pull-down samples were separated by SDS-PAGE and visualized by autoradiography (upper panels). The purified GST and GST-IE1 used in pull-down assays are shown by SDS-PAGE and Coomassie Brilliant Blue staining (lower panel).
Figure 5.
Enhancement of IE1 SUMOylation by PIAS1.
(A) 293T cells in six-well plates were cotransfected with plasmids expressing IE1 (1 µg), flag-SUMO-1 (1 µg), and increasing amounts of SRT-PIAS1 (0.3, 1, and 3 µg), as indicated. At 48 h, total cell lysates were prepared and immunoblotted with an anti-IE1 antibody. (B) 293T cells were cotransfected with plasmids expressing IE1 (1 µg), flag-SUMO-1 (1 µg), and wild-type or C351S mutant SRT-PIAS1 (0.5 µg), as indicated. At 48 h, total cell lysates were prepared and immunoblotted with anti-IE1 or anti-SRT antibodies. (C) In vitro SUMOylation reactions were conducted with bacterially purified His-IE1, GST-SAE2/1, His-Ubc9, and GST-SUMO-1GG proteins, and immunoprecipitated flag-PIAS1 proteins (see Materials and Methods). The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with anti-IE1 antibody. The amounts of flag-PIAS1 protein used were also shown by immunoblotting with anti-flag antibody.
Figure 6.
Inhibition of the PIAS1-mediated IE1 SUMOylation by IE2 in vitro.
(A) In vitro SUMOylation reactions for IE1 were conducted as in Fig. 5C using His-IE1, GST-SAE2/1, His-Ubc9, GST-SUMO-1GG, and increasing amounts (0.1, 0.5, and 1 µg) of GST or GST-IE2(346–542) with (left panel) or without (right panel) immunoprecipitated flag-PIAS1. The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with anti-IE1 antibody. (B) The GST and GST-IE2(346–542) proteins added to in vitro SUMOylation reactions were detected by immunoblotting with an anti-GST antibody.
Figure 7.
IE2 reverses the SUMOylation-dependent inhibition of IE1 activity to downregulate ISRE activation.
(A and B) The reporter assays using the ISG54 ISRE-luciferase construct. 293T cells in 12-well plates were cotransfected with 0.5 µg of the ISG54 ISRE-luciferase reporter construct and plasmids expressing HA-IE1, flag-SUMO-1, HA-PIAS1, and myc-IE2(346–542) as indicated. The total amount of plasmid was adjusted with empty vectors. At 24 h, cells were untreated or treated with IFNβ (1,000 units/ml) for 8 h, and luciferase reporter assays were performed. The results shown are the mean values and standard errors of three independent experiments. Statistical significance between samples was determined using Student's t-test (values of *P<0.0005). The expression levels of IE1, IE2, and β-actin proteins in cell lysates were determined by immunoblotting with specific antibodies. (C) A hypothetical model showing that expression of IE2 and its SUMOylation regulates the PIAS1-mediated IE1 SUMOylation, enhancing IE1 activity to downregulate type I IFN-stimulated gene (ISG) expression. ISRE, interferon stimulated response element.