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.

ACKNOWLEDGMENTS

This study was funded by Public Health Service research grant RO1 AI24576 to G.S.H. from the National Institute for Allergy and Infectious Diseases.

We thank Peter O’Hare, James J.-D. Hsieh, and Yilong Wang for gifts of plasmids. We also thank Dolores Ciufo for plasmids and rabbit PAbs against PML. Generous gifts of yeast strains and plasmids from Stephen J. Elledge (Baylor College of Medicine, Waco, Tex.), plasmids for PML from Ronald M. Evans (The Salk Institute, San Diego, Calif.), the Ad-IE1 vector from Gavin Wilkinson (University of Wales, College of Medicine, Cardiff, United Kingdom), and MAb 5E10 from K. van der Krann (Universiteit van Amsterdam) are greatly acknowledged. We are also grateful to Edward S. Mocarski (Standford University, Calif.) for a gift of samples of the pair of IE1-deleted CR208 virus and its parent HCMV(Towne) virus. We also thank Mike Delannoy (Department of Cell Biology, Johns Hopkins School of Medicine) for assistance with the confocal microscopy analysis and Sarah Heaggans for help in preparation of the manuscript.