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. 2001 Nov;75(22):10683-95.
doi: 10.1128/JVI.75.22.10683-10695.2001.

Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PML-mediated transcriptional repression

Y Xu  1 , J H Ahn, M Cheng, C M apRhys, C J Chiou, J Zong, M J Matunis, G S Hayward
Affiliations

Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PML-mediated transcriptional repression

Y Xu et al. J Virol. 2001 Nov.

Abstract

Human cytomegalovirus (HCMV) major immediate-early protein IE1 is an abundant 72-kDa nuclear phosphoprotein that is thought to play an important role in efficient triggering of the lytic cycle, especially at low multiplicity of infection. The best-known properties of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML oncogenic domains [PODs] or nuclear domain 10 [ND10]), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes. Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1. In this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells, as well as in in vitro assays, with up to 30% of the protein representing the covalently conjugated 90-kDa form in stable U373/IE1 cell lines. Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs. Surprisingly, unlike PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast two-hybrid assays, suggesting that some additional unknown intranuclear cofactors must play a role in IE1 sumoylation. Interestingly, stable expression of either exogenous PML or exogenous Flag-SUMO-1 in U373 cell lines greatly enhanced both the levels and rate of in vivo IE1 sumoylation during HCMV infection. Unlike the disruption of PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the disruption of PODs by HCMV IE1 proved not to involve proteasome-dependent degradation of PML. We also demonstrate here that the 560-amino-acid PML1 isoform functions as a transcriptional repressor when fused to the GAL4 DNA-binding domain and that wild-type IE1 inhibits the repressor function of PML1 in transient cotransfection assays. Furthermore, both IE1(1-346) and IE1(L174P) mutants, which are defective in displacing PML from PODs, failed to inhibit the repression activity of PML1, whereas the sumoylation-negative IE1(K450R) mutant derepressed as efficiently as wild-type IE1. Taken together, our results suggest that proteasome-independent disruption of PODs, but not IE1 sumoylation, is required for efficient IE1 inhibition of PML-mediated transcriptional repression.

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Figures

FIG. 1
FIG. 1
Covalent modification of HCMV IE1 by SUMO-1 in DNA-transfected cells. (A) Detection of higher-molecular-weight IE1 and IE2 isoforms in U373 cell lines stably expressing IE1 (U373-A3) or IE1 plus IE2 (U373-A45). Lysate from a cell line carrying the NeoR selection marker (U373-AN) was used as a negative control. (B and C) Detection of SUMO-1-conjugated IE1 in transfected 293T cells. Immunoblot analysis was carried out with MAb CH810 (A and C) or 6E1 (B).
FIG. 2
FIG. 2
Covalent modification of IE1 by SUMO-1 in HCMV-infected cells. (A and B) Detection of the 90-kDa covalently modified isoform of IE1 in U373-MG cells infected with HCMV(Towne). Immunoblot analysis was carried out with mouse anti-IE1 MAb 6E1 (A) or mouse anti-IE1 and -IE2 MAb CH810 (B). (C) Mock-infected and HCMV-infected whole-cell lysates were immunoprecipitated (IP) with anti-SUMO-1 PAb FL101 and subjected to immunoblot analysis with MAb CH810 (lanes 1 and 2). The same blot was then stripped and reprobed with MAb 6E1 (lanes 3 and 4).
FIG. 3
FIG. 3
Identification of lysine residues in IE1 that are conjugated by SUMO-1. (A) Total extracts were prepared from 293T cells transfected with 2 μg of vector alone (lane 11) or 2 μg of pJHA303 encoding wild-type (WT) IE1 (lane 1), pYX108 encoding IE1(K163R) (lane 3), pYX109 encoding IE1(K197R) (lane 5), pYX102 encoding IE1(K331R) (lane 7), or pYX118 encoding IE1(K450R) (lane 9), or the same five plasmids plus 2 μg of pJHA312 encoding Flag-SUMO-1 (lanes 2, 4, 6, 8, and 10). Cell extracts were subjected to SDS–8% polyacrylamide gel electrophoresis (PAGE), and immunoblot analysis was carried out with MAb CH810. (B) In vitro-translated [35S]Met-labeled wild-type IE1 and K450R mutant IE1 were incubated at 37°C for 2 h in the absence or presence of reaction mixtures (Rxn Mix) containing ATP-regenerating buffer, GST-Ubc9, His-tagged SUMO-1, and a HeLa cell fraction with E1 activity for SUMO-1 conjugation. For reaction mixtures containing GST-PML-L, its approximate final concentration was 1 μg/μl. Reaction products were separated by SDS–8% PAGE and visualized by autoradiography.
FIG. 4
FIG. 4
HCMV IE1 does not interact with SUMO-1 or Ubc9 in yeast two-hybrid assays. Plasmids encoding GAL4-DB fusions and GAL4-A fusions were introduced together into Y190 cells. Transformants were selected on plates lacking Trp and Leu, and β-galactosidase (β-Gal) activities of the transformants were measured by the standard ONPG method as the interaction readout. The unit of β-Gal activity was defined as 1,000(A420 − 1.75A550)/(A600× t × v) (t, reaction time in minutes; v, reaction volume in milliliters). Values are means of duplicate assays; error ranges are indicated.
FIG. 5
FIG. 5
Intracellular localization patterns of wild-type IE1 and the sumoylation-negative IE1(K450R) mutant. HF cells were transfected with expression plasmids encoding wild-type IE1 (A and B) or IE1(K450R) (C and D). At 24 h after transfection, the cells were fixed with methanol and double-label IFA was carried out with mouse MAb 6E1 for IE1 (A and C) and rabbit PAb PML(C) for endogenous PML (B and D). Fluorescein isothiocyanate-labeled anti-mouse immunoglobulin G (IgG) and rhodamine-coupled anti-rabbit IgG were used for visualization. Insets, transfected cells with transient punctate colocalization of IE1 and PML.
FIG. 6
FIG. 6
Overexpressed PML or SUMO-1 leads to up-regulated IE1 sumoylation during HCMV infection. U373-MG cells were stably transfected with pCMX-PML1 or pJHA312 encoding Flag-SUMO-1 or an empty vector carrying a Neo resistance marker to establish U373-PML, U373-SUMO-1, and U373-Neo cell lines. (A) Comparison of IE1 sumoylation in U373-PML and U373-Neo cells during HCMV infection. (B) Comparison of IE1 sumoylation in U373-SUMO-1 and U373-Neo cells during HCMV infection. Cells were either mock infected (M) or infected with HCMV(Towne) at an MOI of 5, and cell lysates were harvested at the indicated time points after infection (hours postinfection, hpi). Immunoblot analyses were performed with mouse MAb CH810 recognizing both HCMV IE1 and IE2. After enhanced chemiluminescence visualization of the immunoblots, densitometric quantitation of the 72-kDa unmodified IE1 and the 90-kDa sumoylated IE1 was acquired from X-ray films with a Fluorchem 8000 digital imaging system. The intensities were then used for the calculation of the abundance of the 90-kDa IE1 isoform relative to the total amount of IE1 protein at different time points after infection.
FIG. 7
FIG. 7
Proteasome-independent disruption of PODs during HCMV infection. HF cells were infected with HCMV(Towne) at an MOI of 5 in the absence (A and B) or presence (C and D) of 5 μM MG132 and stained for IE1 (A and C) and PML (B and D) at 6 h after infection. As a positive control for MG132 activity, Vero cells were infected with HSV-1(KOS) at an MOI of 20 in the absence (E and F) or presence (G and H) of MG132 (5 μM) and stained for IE110 (E and G) and PML (F and H) 4 h after infection.
FIG. 8
FIG. 8
The HCMV IE1 protein specifically inhibits PML1-mediated transcriptional repression. HeLa cells were transiently cotransfected with the (GAL4)5-TK-CAT target gene and the indicated effector genes. Cell lysates were then harvested 48 h after transfection and assayed for CAT activity. Shown are mean values for the percentage of CAT activity in duplicate assays and error ranges. Basal CAT activity upon cotransfection with the control GAL4-DB gene is defined as 100%, which corresponds to 81.7% acetylation in one experiment and 75.2% acetylation in another.
FIG. 9
FIG. 9
The inhibitory effect of IE1 on PML-mediated repression requires its ability to displace PML from PODs. HeLa cells were transiently cotransfected with either the (GAL4)5-TK-CAT (top) or the (GAL4)5-TK-LUC (bottom) target gene and the indicated effector genes. Cell lysates were then harvested at 48 h after transfection and assayed for CAT activity or luciferase activity, respectively. (Top) Relative values of CAT activity. Basal CAT activity upon cotransfection with GAL4-DB is defined as 100%, which corresponds to 90.2% acetylation in one experiment. (Bottom) Mean values for relative light units (RLU) in duplicate assays, with error ranges indicated.
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