The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells

Abstract

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts

“…Furthermore, it is able to interfere with several cellular processes, including gene regulation, cell cycle progression, signal transduction, apoptosis, interaction with specific nuclear domains, such as promielocytic leukemia (PML) bodies [Margolis et al, 1995;Zhu et al, 1995;Wilkinson et al, 1998;McElroy et al, 2000;Castillo and Kowalik, 2002]. Specifically, IEp72 was previously reported to accumulate in PML bodies and to trigger their disruption about 4 h after infection [Kelly et al, 1995;Ahn and Hayward, 1997;Muller and Dejean, 1999]. Disruption of PML bodies coincides with an organization of new nuclear compartments, i.e., the viral replication centers [Ahn et al, 1999].…”

Mediation of Epstein–Barr virus EBNA-LP transcriptional coactivation by Sp100

“…Herpesviruses interact with and modify PML NBs. Human cytomegalovirus (CMV) genomes initially localize to PML NBs and the CMV Immediate-Early protein 1 (IE1) displaces Sp100 and PML from these structures (Korioth et al, 1996;Ahn and Hayward, 1997). Herpes simplex virus (HSV)-1 Immediate-Early Protein Zero (ICP0) disrupts PML NBs by inducing proteosome-dependent degradation of PML and Sp100 (Everett, 2001;Hagglund and Roizman, 2004).…”

Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0

“…Therefore, the mechanisms that ICP0 uses to degrade PML are likely more intricate than previously thought.Cytomegalovirus (CMV) infection can also disrupt ND10, but the mechanism underlying the dispersion is different from that by HSV-1. IE1 protein has been identified for CMV to disperse ND10, but PML is not degraded (24)(25)(26). It has been demonstrated that human CMV (HCMV) IE1 induces deSUMOylation of PML and Sp100, and one amino acid mutation (L174P) abolishes its function of deSUMOylation of PML and Sp100 (27).…”