Initiation of cytomegalovirus infection at ND10.

As a large double-stranded DNA virus, CMV replicates in the nucleus, a highly structured environment. Diffusional and solid phases exist as interdependent sets of interactions between many components that determine either replicative success of an infecting virus or the defensive success of the host cell. In their extremes, cell death may be part of the lytic release of viral particles, or, in defense terms, the ultimate sacrifice preventing virus release. Between these extremes exists an evolutionarily derived standoff between virus and cell. Exogenous shifts in homeostasis can disturb this balance, diminishing the cell's defensive powers and reactivating the silenced viral genome. Many of the solid-phase aspects of this process can be seen in situ and analyzed. This review evaluates structural information derived from CMV-infected cells in situ at very early times of infection and the conceptional advances derived from them, mostly centering on the major immediate early gene products, specifically IE1. A scientific basis for considering the major immediate early proteins as potential targets in suppressing CMV disease is discussed.

Regulation of c-met expression by transcription repressor Daxx.

The protooncogene c-met encodes the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF). While overexpression of c-met is documented in many types of tumors, the mechanism of c-met regulation remains elusive. Here, we demonstrate Daxx as a repressor of c-met transcription. The expression of c-met is elevated in Daxx knockout mouse cells and is reversed by Daxx reconstitution. C-met promoter analysis of Daxx-/- cells reveled changes in chromatin acetylation, but not in DNA methylation. Daxx binds to the mouse c-met promoter and Daxx-binding region is sufficient for transcription repression, while HDAC2 is associated with c-met promoter mostly in Daxx+/+ cells, pointing to Daxx-dependent HDAC2 recruitment as a potential mechanism of c-met repression. HGF-induced cell mobility and invasion confirmed augmented activity of c-Met/HGF pathway in Daxx-/- cells. Finally, inverse correlation between Daxx and c-Met in cancer cell lines and in metastatic breast cancer specimens suggests potential function of Daxx as a c-met repressor during cancer progression.

Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot.

ND10, PML bodies or PODs have become the defining nuclear structure for a highly complex protein complement involved in cell activities such as aging, apoptosis, the cell cycle, stress response, hormone signaling, transcriptional regulation and development. ND10 are present in many but not all cell types and are not essential for cell survival. Here, we review the cellular proteins found in ND10, their few known interactions and their contribution to the ND10 structure per se and to functions elsewhere in the nucleus. The discrepancy between the functions of the ND10 proteins and the nonessential nature of the structure in which they are aggregated at their highest concentrations leads to the conclusion that the proteins function elsewhere. The regulated recruitment of specific proteins into ND10 as well as their controlled release upon external induced stress points to a regulated nuclear depot function for ND10. These nuclear depot functions seem important as nuclear defense against viral attack and other external insults.

Accumulation and intranuclear distribution of unintegrated human immunodeficiency virus type 1 DNA.

The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4(CEM)) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.

Evidence for separate ND10-binding and homo-oligomerization domains of Sp100.

Nuclear domains called ND10 or PML nuclear bodies consist of an aggregation of several proteins, most notably PML and Sp100. PML is essential in the nucleation and formation of ND10 as well as in the recruitment of other ND10-associated proteins such as Daxx, pRb, BLM and Sp100. In cells induced to overexpress Sp100, ND10 binding of Sp100 was saturable and excess Sp100 formed new aggregation sites devoid of other ND10-associated proteins, suggesting that homo-oligomerization is the basis for aggregation. To determine whether Sp100 binds to ND10 through hetero- or oligomerization, Sp100 deletion variants fused with GFP were transfected into cells with and without endogenous Sp100, and the localization of the GFP-labeled fragments was determined relative to ND10. Amino acids 29-152 were sufficient for deposition of the GFP-labeled fragments at ND10 in the absence of endogenous Sp100 (heterologous binding) and for self-aggregation (formation of new Sp100 deposits). None of the shorter fragments was deposited at ND10 or self-aggregated. The 29-152 amino acid fragment and some larger fragments, but not the full-size Sp100, induced elongation of ND10, which at their ends contain only Sp100, probably due to self-aggregation. By fusing a peptide consisting of the p53-binding domain from hMDM2 to the Sp100(29-152) fragment, this self-aggregation could be blocked while retaining the limited ND10 binding capacity, indicating that the Sp100 self-aggregation domain and the ND10 binding domain are separate entities. This fusion peptide was used to demonstrate the potential of ND10 to recruit p53 as a protein not usually present at this site. Such deposited p53 was protected from turnover. The capacity of ND10 to recruit Sp100 may serve primarily to reduce its availability.

[Monoclonal antibodies against protein Daxx and its localization in nuclear domains 10]. / Monoklonal'nye antitela k belku Daxx i ego lokalizatsiia v iadernykh domenakh 10.

Nuclear domains 10 (ND10) were first detected occasionally using antibodies to an antigen of unknown nature (Ascoli, Maul, 1991). Further on it was shown that ND10 were sites of locality of the number of proteins (PML, Sp 100, pRB) (Sterndorf et al., 1992; Kamitani et al., 1998), the majority of which are modified with ubiquitin-like small proteins-modifiers (SUMO) (Ishov, Maul, 1996). In addition, it was shown that ND10 were sites of primary localization, transcription and replication of some DNA-viruses (SV40, virus of simple herpes 1, adenovirus 5) (Ishov, Maul, 1996; Ishov et al., 1997). Except for SV40, these viruses produce proteins able to modify ND10, or leading to degradation of ND10-associated proteins (Maul et al., 1993; Maul, Everett, 1994). This degradation is accompanied with protein desumofication and, later, with hydrolysis on the ubiquitin-proteosomal way (Everett et al., 1998, 1999). Cell incubation with interferon leads to augmentation of the number and dimension of ND10 owing to increased expression of Sp100 and PML (Lavau et al., 1995; Grotzinger et al., 1996). In all, these data make it possible to put forward a hypothesis that ND10 may represent a peculiar cell storage ("dépôt") of proteins regulated according to the "accumulation-drop" principle (Ishov et al., 1997; Maul, 1998). However, this hypothesis requires further factual grounds.

Lytic but not latent replication of epstein-barr virus is associated with PML and induces sequential release of nuclear domain 10 proteins.

Nuclear domains called ND10 (nuclear domain 10) are discrete nuclear protein aggregations characterized by a set of interferon-upregulated proteins including Sp100 and PML, where papova-, adeno-, and herpesviruses begin their transcription and DNA replication. Both the alpha- and betaherpesvirus subfamilies disrupt ND10 upon infection by dispersing and/or destroying ND10-associated proteins. We studied the effect of the gammaherpesvirus Epstein-Barr virus (EBV) on ND10 and its spatial distribution in the nucleus of cells during latency and lytic reactivation. In latently infected Burkitt's lymphoma, lymphoblastoid, and D98/HR1 cells, ND10 were intact, as judged by immunofluorescence localization of PML, Sp100, NDP55, and Daxx. Fluorescent in situ hybridization revealed no association between viral episomes and ND10 during latency, implying that the maintenance replication of EBV, which depends on host cell proliferation, occurs independent of ND10. As in mitosis, the EBV genomes were attached to interphase chromosomes, suggesting that they are unable to move freely within the interchromosomal space and thus unable to associate with the interchromosomally located ND10 or other nuclear domains. Upon lytic activation, ND10 became dispersed in cells expressing lytic proteins. Redistribution of ND10 proteins occurred sequentially at different stages of the lytic cycle, with Sp100, Daxx, and NDP55 dispersed before and PML dispersed after the onset of lytic replication. ND10 remnants were retained until the early stages of lytic replication, and replicating EBV genomes were frequently found beside this nuclear domain; the number of replication domains was usually lower than the average latent virus frequency. Thus, latency does not require or induce interaction of EBV with ND10 for transcription and replication, whereas lytic replication triggers dispersion of ND10 proteins and occurs in close association with PML aggregates. The required movement of chromosome-attached latent EBV episomes to ND10 after reactivation from latency might include physical release of the chromosome-bound episomes. Only episomes contacting ND10 after such a release might be able to begin lytic replication.

Replication but not transcription of simian virus 40 DNA is dependent on nuclear domain 10.

DNA viruses from several families including herpes simplex virus type 1, adenovirus type 5, and simian virus 40 (SV40), start their transcription and replication adjacent to a specific nuclear domain, ND10. We asked whether a specific viral DNA sequence determines the location of these synthetic activities at such restricted nuclear sites. Partial and overlapping SV40 sequences were introduced into a beta-galactosidase expression vector, and the beta-galactosidase transcripts were localized by in situ hybridization. Transcripts derived from control plasmids were found throughout the nucleus and at highly concentrated sites but not at ND10. SV40 genomic segments supported ND10-associated transcription only when the origin and the coding sequence for the large T antigen were present. When the large T-antigen coding sequence was eliminated but the T antigen was constitutively expressed in COS-7 cells, the viral origin was sufficient to localize transcription and replication to ND10. Deletion analysis showed that only the large T-antigen binding site II (the core origin) was required but the T antigen was needed for detectable transcription at ND10. Large T antigen expressed from plasmids without the viral core origin did not bind or localize to ND10. Blocking of DNA replication prevented the accumulation of transcripts at ND10, indicating that only sites with replicating templates accumulated transcripts. Transcription at ND10 did not enhance total protein synthesis of plasmid transcripts. These findings suggest that viral transcription at ND10 may only be a consequence of viral genomes directed to ND10 for replication. Although plasmid transcription can take place anywhere in the nucleus, T-antigen-directed replication is apparently restricted to ND10.

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces.

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates.

Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10.

Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.

Review: properties and assembly mechanisms of ND10, PML bodies, or PODs.

Nuclear domain 10 (ND10), also referred to as PML bodies or PODs, are discrete interchromosomal accumulations of several proteins including PML and Sp100. We describe here developments in the visualization of ND10 and the mechanism of ND10 assembly made possible by the identification of proteins that are essential for this process using cell lines that lack individual ND10-associated proteins. PML is critical for the proper localization of all other ND10-associated proteins under physiological conditions. Introducing PML into a PML -/- cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10, attesting to its essential nature in ND10 formation. This recruitment includes Daxx, a protein that can bind PML and is highly enriched in condensed chromatin in the absence of PML. The segregation of Daxx from condensed chromatin to ND10 by increased accumulation of Sentrin/SUMO-1 modified PML suggests the presence of a variable equilibrium between these two nuclear sites. These findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML. Additional adapter proteins are suggested to exist by the behavior of Sp100, and Sp100 will provide the basis for their identification. Further information about the dynamic balance of proteins between ND10 and the actual site of functional activity of these proteins will establish whether ND10 function as homeostatic regulators or only in storage of excess proteins destined for turnover.

Differential regulation of sentrinized proteins by a novel sentrin-specific protease.

Sentrin-1, also called SUMO-1, is a protein of 101 residues that is distantly related to ubiquitin and another ubiquitin-like protein, NEDD8. Here we report the cloning of a novel sentrin-specific protease, SENP1, which has no homology to the known de-ubiquitinating enzymes or ubiquitin C-terminal hydrolases. However, SENP1 is distantly related to the yeast Smt3-specific protease, Ulp1. A COS cell expression system was used to demonstrate the activity of SENP1 in vivo. When HA-tagged sentrin-1 was co-expressed with SENP1, the higher molecular weight sentrin-1 conjugates were completely removed. Surprisingly, the major sentrinized band at 90 kDa remained intact. The disappearance of the high molecular weight sentrin-1 conjugates also coincided with an increase in free sentrin-1 monomers. SENP1 is also active against proteins modified by sentrin-2, but not those modified by ubiquitin or NEDD8. In addition, sentrinized PML, a tumor suppressor protein that resides in the nucleus, was selectively affected by SENP1, whereas sentrinized RanGAP1, which is associated with the cytoplasmic fibrils of the nuclear pore complex, remained intact. The inability of SENP1 to process sentrinized RanGAP1 in vivo is most likely due to its nuclear localization because SENP1 is active against sentrinized RanGAP1 in vitro. The identification of a nuclear-localized, sentrin-specific protease will provide a unique tool to study the role of sentrinization in the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

A novel nuclear substructure, ND10: distribution in normal and neoplastic human tissues.

ND10 are recently characterized nuclear domains that are composed of 0.5 microm sized, precisely circumscribed dots in cultured human cell lines. To investigate the distribution and number of ND10 on various types of normal and neoplastic human tissues, we carried out immunostaining and immunoprecipitation analyses with monoclonal antibodies 138 and 1150. The number of ND10 varied from 1 to 10 or more in various tissues as did their size. ND10 were diffusely located in early embryonic and normal tissues, except for the exocrine and endocrine cells of the pancreas and for hepatocytes. In normal squamous mucosa, basal cells had more ND10 than did differentiated superficial squamous cells. The number and size of ND10 were markedly increased in malignant neoplasms but were similar in benign tumors and corresponding normal tissues. Sex hormone-related normal tissues, such as the endometrium or myometrium, and neoplasms strongly stained for ND10. The distribution pattern of ND10 in human tissues indicates that they are conserved nuclear substructures that are closely associated with cellular differentiation, hormonal stimulation, and oncogenesis.

Nuclear domain 10, the site of DNA virus transcription and replication.

Within the highly organized nuclear structure, specific nuclear domains (ND10) are defined by accumulations of proteins that can be interferon-upregulated, implicating ND10 as sites of a nuclear defense mechanism. Compatible with such a mechanism is the deposition of herpesvirus, adenovirus, and papovavirus genomes at the periphery of ND10. However, these DNA viruses begin their transcription at ND10 and consequently initiate replication at these sites, suggesting that viruses have evolved ways to circumvent this potential cellular defense and exploit it. Other ND10-associated proteins belong to ubiquitin-related pathways. These findings, together with the accumulation of various overexpressed cellular and viral proteins, suggest that ND10 function as nuclear dumps or as nuclear depots. Consistent with the recruitment or deposition of various proteins and viral genomes adjacent to ND10, ND10 themselves may only be protein accumulations at specific but as yet undefined nuclear deposition sites. The concept of specific nuclear deposition sites may explain the juxtaposition of various nuclear bodies and allows testable predictions about a potential supramolecular regulatory mechanism whereby proteins are selectively segregated or released by global changes induced in nuclear functions such as viral infections, stress, or hormonal induction.

Nuclear redistribution of BRCA1 during viral infection.

The functions and the intracellular localization of the breast/ovarian susceptibility gene product, BRCA1, has been controversial. To arrive at a clear understanding of its localization and relative position to other nuclear structures, a new monoclonal antibody was produced and characterized by immunohistochemical techniques with other BRCA1 antibodies. Each of the antibodies specifically detected BRCA1 as localized to specific nuclear domains and did so in a variety of cells and in a cell cycle-dependent manner. However, all antibodies also cross-reacted with the centrosomal domain, suggesting that BRCA1 is also localized to this important mitotic component. We found that the BRCA1-containing nuclear domains are different than any of the well-defined nuclear domains. However, a cell cycle-related partial overlap was found for HP1alpha, a chromo-domain-containing protein involved in heterochromatin maintenance. Cellular stimuli, such as heat shock and herpes virus infection, dispersed BRCA1 from its domains. In contrast, infection with adenovirus 5 recruited BRCA1 to regions of viral transcription and replication. These disparate distributions of BRCA1 may provide clues to its function.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

Characteristic immunolocalization of Ku protein as nuclear matrix.

Two hybridoma clones, NMB1 and NML90, were established using nuclear matrix proteins from normal human thymi or malignant lymphoma as immunogens. They reacted with human Ku70 and Ku80, respectively, by immunoblotting. When HeLa cell nuclear proteins were fractionated and applied to immunoblotting, both Ku70 and Ku80 were detected in the nuclear matrix as well as the soluble nuclear protein fractions. By confocal scanning microscopy, the immunoreactivity of Ku70 and Ku80 was localized to distinct nucleoplasmic fibrillar network and fine granules in the interphase cell nuclei. When HeLa cells were fractionated in situ using DNase I and buffers containing 0.25 M (NH4)2SO4 and 2 M NaCl, the nucleoplasmic reticular structure was largely preserved, but granules disappeared. The nucleoplasmic distribution of Ku in the tissue and in cultured cells was distinct from each other. In the adult tissue, it consisted mostly of either distinct curvilinear lines along the nuclear periphery or of tangled, beaded lines throughout the nuclei. When xenotransplants of HeLa cell in Scid mice were examined, the "tissue type" immunolocalization pattern was reproduced consistently. In most fetal tissues, "tissue type" and "cell type" patterns were admixed. Monoclonal antibodies described here are useful tools for studying the structure and function of the nuclear matrix.

Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

The nuclear domain 10 (ND10) is disrupted by the human cytomegalovirus gene product IE1.

The nuclear domain 10 (ND10) is modified during the life cycle of a number of viruses. In this study we report the effect of infection with human cytomegalovirus (HCMV) on the ND10 proteins PML, Sp100, and NDP52. Immunofluorescence analyses revealed that 1-2 h after infection (p.i.) with HCMV the immediate early gene (IE) products IE1 and IE2 transiently colocalize with ND10 proteins. At 4 h p.i. the IE gene products were distributed throughout the nucleus, which was accompanied by a complete disruption of ND10, affecting all analyzed proteins. Transfection studies using different HCMV-cDNA expression plasmids revealed that the expression of IE1 alone was sufficient to induce this disruption. As reported for other ND10-modifying viral proteins, no direct interaction between IE1 and the analyzed ND10 proteins could be detected. The disruption of ND10 by HCMV IE1 is very similar to that described for HSV-1 ICP0. Although there is no sequence similarity between proteins, this observation might suggest similar functions in virus-host interactions.