Disruption of PML Subnuclear Domains by the Acidic IE1 Protein of Human Cytomegalovirus Is Mediated through Interaction with PML and May Modulate a RING Finger-Dependent Cryptic Transactivator Function of PML

Abstract

Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.Several herpesvirus nuclear regulatory proteins expressed at immediate-ea...

Nuclear Domain 10 Components Promyelocytic Leukemia Protein and hDaxx Independently Contribute to an Intrinsic Antiviral Defense against Human Cytomegalovirus Infection

“…Consistent with this observation, a pp71 knockout virus inefficiently enters productive infection (7), and depletion of hDaxx annihilates the impaired growth phenotype associated with this virus (8,45). Furthermore, the IE regulatory protein IE1 targets to ND10 to induce a dispersal of the whole structure by abrogating the SUMOylation (SUMO refers to the small ubiquitin-related modifier) of the major component PML (2,3,29,32,49). This kind of posttranslational modification of PML was shown to be essential for ND10 formation, since only SUMOylated PML is capable of organizing ND10 accumulations (25,53).…”

Transcriptional regulation in acute promyelocytic leukemia

“…PML shows intrinsic repression activity when tethered to a promoter through fusion with the Gal4 DBD (Ahn et al, 1998;Vallian et al, 1997). Consistent with its repression activity, overexpression of PML led to repression of various promoters, including the MDR and EGF-receptor (EGF-R) promoters (Mu et al, 1994).…”

DNA viruses and viral proteins that interact with PML nuclear bodies

“…Parallel studies on HCMV quickly established that PML NBs are disrupted early during infection, and that regulatory protein ie1 is necessary and sucient for this eect (Ahn et al, 1998;Ahn and Hayward, 1997;Kelly et al, 1995;Korioth et al, 1996;Wilkinson et al, 1998). It is intriguing that HCMV mutants that do not express ie1 have a phenotype similar in many ways to that of HSV-1 ICP0 mutants ± they enter the lytic cycle ineciently, but the defect can be overcome by using a high dose of virus (Mocarski et al, 1996).…”