Interaction of IE1 with PIAS1.

<p>(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 <i>in vitro</i>-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).</p

SUMOylation patterns of IE1 and IE2 during HCMV infection.

<p>(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.</p

HCMV ORFs that contain possible SUMOylation sites.

a<p>ORFs are listed by score with the SUMOsp program.</p>b<p>UL89 exon 2. For the full-length UL89 protein, one lysine residue (K315) was predicted as a SUMO attachment site with the same score from both SUMOplot and SUMOsp.</p

Inhibition of the PIAS1-mediated IE1 SUMOylation by IE2 <i>in vitro</i>.

<p>(A) <i>In vitro</i> SUMOylation reactions for IE1 were conducted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103308#pone-0103308-g005" target="_blank">Fig. 5C</a> using His-IE1, GST-SAE2/1, His-Ubc9, GST-SUMO-1_GG, 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 <i>in vitro</i> SUMOylation reactions were detected by immunoblotting with an anti-GST antibody.</p

Enhancement of IE1 SUMOylation by PIAS1.

<p>(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) <i>In vitro</i> SUMOylation reactions were conducted with bacterially purified His-IE1, GST-SAE2/1, His-Ubc9, and GST-SUMO-1_GG proteins, and immunoprecipitated flag-PIAS1 proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103308#s2" target="_blank">Materials and Methods</a>). 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.</p

IE2 reverses the SUMOylation-dependent inhibition of IE1 activity to downregulate ISRE activation.

<p>(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 <i>t</i>-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.</p

Evaluation of SUMOylation in cotransfection and <i>in vitro</i> assays.

<p>(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) <i>In vitro</i> SUMOylation reactions. Myc-UL123(IE1) and myc-US34A produced by <i>in vitro</i> transcription/translation were incubated with GST-SAE2/1, His-Ubc9, and His-SUMO-1_GG or GST-SUMO-1_GG 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 (<a href="http://iupred.enzim.hu" target="_blank">http://iupred.enzim.hu</a>). The lysine residues modified by SUMO (for IE1 and IE2) or predicted to be SUMOylation sites (for US34A) are indicated.</p

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators

<div><p>Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.</p></div

Effects of IE1 on ISG15 transcription and protein ISGylation.

<p>(A-B) HF cells were mock-infected (M) or infected with wild-type HCMV, UV-HCMV, or CR208 virus at an MOI of 3. Total RNAs were prepared at the indicated time points and the levels of ISG15 and β-actin transcripts were determined by RT-PCR (A). Cell lysates were also prepared and analyzed by immunoblotting as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005850#ppat.1005850.g001" target="_blank">Fig 1A</a> (B). (C) Control and IE1-expressing HF cells produced by retroviral vectors were mock-infected or infected with wild-type HCMV, UV-HCMV, or CR208 virus at an MOI of 3, or treated with IFNβ (1,000 U/ml) for 48 h. Immunoblotting was performed with antibodies for ISG15, IE1/IE2, p52 (encoded by UL44) and β-actin. (D) 293T cells were co-transfected with plasmids expressing myc-ISG15 (wild-type, ISG15_GG, or ISG15_AA), HA-UBE1L (E1), Flag-UbcH8 (E2), or HA-Herc5 (E3) as indicated. At 48 h after transfection, cell lysates were prepared by boiling the cell pellets in sodium dodecyl sulfate (SDS) loading buffer and immunoblotted with anti-myc and anti-β-actin (a loading control) antibodies. (E and F) Co-transfection/ISGylation assays were performed in 293T cells with or without increasing amounts of plasmid expressing IE1 (E) or in control and IE1-expressing HF cells (F). Immunoblotting was performed with anti-myc, anti-IE1, and anti-β-actin antibodies.</p

Roles of UL26 ISGylation in viral growth.

<p>(A) Three lysine residues of UL26-p21 from Towne strain and their conservation in other human (Toledo, Ad169, and Merlin) and primate CMVs (chimpanzee CMV, ChCMV; rhesus CMV, RhCMV; simian CMV, SCMV) are shown. (B) 293T cells were co-transfected with plasmid expressing SRT-UL26-p21 (wild-type or lysine to arginine mutants), myc-ISG15 (with GG or AA terminus), HA-UBE1L, Flag-UbcH8, or HA-Herc5 as indicated. At 48 h after transfection, cell lysates were immunoprecipitated with anti-SRT antibody. Immunoprecipitated samples and whole cell lysates were detected by immunoblotting with anti-myc and anti-SRT antibodies. (C) Control HF cells or cells expressing UL26-p21 (wild-type or K136/359R mutant) were produced by retroviral vectors. Cell lysates were prepared and immunoblotted with antibodies for UL26 and β-actin (a loading control). (D-F) Control HF cells or cells expressing UL26-p21(K136/169R) or UL26-p21(K136/169R)-ISG15_AA were produced by retroviral transduction. To determine the expression levels of UL26 proteins, cell lysates were immunoblotted with antibodies for IE1/IE2, UL26, and β-actin (D). Cells were treated or not with 50 ng/ml of TNFα for the indicated times. Immunoblotting was performed to detect the levels of p65 and its phosphorylated form. The levels of UL26-p21, its ISG15_AA-fused form, and β-actin were also shown (E). Cells were infected with the UL26-deleted mutant virus (AD169) at an MOI of 0.2. At 72 h after infection, virus titers in the culture supernatants were determined by infectious center assays (F).</p