PML Oncogenic Domains Research Articles

Autophagic Degradation of Misfolded Nuclear Receptor Co-repressor (NCoR) Is Linked to the Growth of Tumor Cells in HBX Positive Hepatocellular Carcinoma (HCC).

Hepatitis B virus (HBV) is causally linked to hepatocellular injury and cell death, which are followed by hepatocellular carcinoma (HCC) after a long latent period. The HBV derived X protein (HBX) is the most potent carcinogenic factor for HCC, however, the molecular mechanism of HBX-induced transformation of hepatic cells in HCC is poorly understood. We have shown that nuclear receptor co-repressor (NCoR) is essential for the spatial repression of global transcription by the promyelocytic leukemia oncogenic domains (PODs), a frequent target of viral oncoproteins like HBX and that disintegration of PODs due to misfolded conformation dependent loss (MCDL) of NCoR is linked to promyelocytic and monocytic acute myeloid leukemia (AML). Given the key role of NCoR in cellular homeostasis across various tissue subtypes, we hypothesized that HBX-induced MCDL of NCoR might be linked to HCC through similar mechanism. Based on this hypothesis, the conformation of NCoR in HCC derived tumor cells and primary human tissue sections were analyzed and a selective MCDL of NCoR in HBX positive HCC cells was identified. HBX triggered the misfolding of NCoR through ubiquitination, followed by its degradation by autophagy, thus suggesting a cross talk between ubiquitin proteasome system (UPS) and autophagy lysosomal pathway (ALP) in MCDL of NCoR in HBX positive HCC cells. SiRNA-induced NCoR ablation selectively impaired the growth and survival of HBX positive HCC cells, suggesting a role of MCDL in the growth and survival of HBX positive HCC cells. These finding identify a possible crosstalk between UPS and ALP in the misfolding and loss of NCoR in HBX positive HCC cells and suggest a role of autophagic recycling of misfolded NCoR in the activation of oncogenic metabolic signaling in HCC. The misfolded NCoR reported in this study represents a novel conformation based molecular target which could be valuable in the design and development of tumor cell specific diagnostic and therapeutic approach for HBX positive HCC.

PML isoforms IV and V contribute to adenovirus-mediated oncogenic transformation by functionally inhibiting the tumor-suppressor p53.

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.

Nuclear domain 10 of the viral aspect

Nuclear domain 10 (ND10) are spherical bodies distributed throughout the nucleoplasm and measuring around 0.2-1.0 μm. First observed under an electron microscope, they were originally described as dense bodies found in the nucleus. They are known by a number of other names, including Promyelocytic Leukemia bodies (PML bodies), Kremer bodies, and PML oncogenic domains. ND10 are frequently associated with Cajal bodies and cleavage bodies. It has been suggested that they play a role in regulating gene transcription. ND10 were originally characterized using human autoantisera, which recognizes Speckled Protein of 100 kDa, from patients with primary biliary cirrhosis. At the immunohistochemical level, ND10 appear as nuclear punctate structures, with 10 indicating the approximate number of dots per nucleus observed. ND10 do not colocalize with kinetochores, centromeres, sites of mRNA processing, or chromosomes. Resistance of ND10 antigens to nuclease digestion and salt extraction suggest that ND10 are associated with the nuclear matrix. They are often identified by immunofluorescent assay using specific antibodies against PML, Death domain-associated protein, nuclear dot protein (NDP55), and so on. The role of ND10 has long been the subject of investigation, with the specific connection of ND10 and viral infection having been a particular focus for almost 20 years. This review summarizes the relationship of ND10 and viral infection. Some future study directions are also discussed.

The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes

The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 k in viral late mRNA accumulation.

Daxx-β and Daxx-γ, Two Novel Splice Variants of the Transcriptional Co-repressor Daxx

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-β and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-β and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-β and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-β and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-β/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-β and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

A High-Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains (PODs)

PML is a multi-functional protein with roles in tumor suppression and host defense against viruses. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML. We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may restore the tumor suppressor activity of PML in cancers or possibly promote anti-viral states.

Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2

The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) latent protein latency-associated nuclear antigen 2 (LANA2) has a critical role in KSHV-induced B-cell malignancies. LANA2 increases the level of small ubiquitin-like modifier (SUMO)2-ubiquitin-modified PML and induces the disruption of PML oncogenic domains (PODs) by a process that requires a non-covalent SUMO interaction domain (SIM) in LANA2. We now demonstrate that LANA2 is covalently conjugated to SUMO1 and SUMO2 both in vitro and in latently KSHV-infected B-cells. We show that a LANA2 SIM mutant exhibits a slightly altered sumoylation pattern, which suggests that non-covalent SUMO interactions represent a mechanism for determining SUMO substrate recognition and modification. In addition, several lysine residues were mapped as SUMO conjugation sites. A sumoylation-deficient mutant shows impaired ability to induce disruption of PODs, which suggests that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interaction between LANA2 and other SUMO-modified or SUMO-interacting proteins required for disruption of PODs.

Daxx Is a Transcriptional Repressor of CCAAT/Enhancer-binding Protein β

CCAAT/enhancer-binding Protein beta (C/EBPbeta) is a member of the bZIP transcription factor family that is expressed in various tissues, including cells of the hematopoietic system. C/EBPbeta is involved in tissue-specific gene expression and thereby takes part in fundamental cellular processes such as proliferation and differentiation. Here, we show that the activity of C/EBPbeta is negatively regulated by the transcriptional co-repressor Daxx. C/EBPbeta was found to directly interact with Daxx after overexpression as well as on the endogenous level. Glutathione S-transferase pulldown assays showed that Daxx binds via amino acids 190-400 to the C-terminal part of C/EBPbeta. Co-expression of C/EBPbeta changed the sub-nuclear Daxx distribution pattern from predominantly POD-localized to nucleoplasmic. Daxx suppressed basal and p300-enhanced transcriptional activity of C/EBPbeta. Furthermore, Daxx decreased the C/EBPbeta-dependent phosphorylation of p300, which in turn was associated with a diminished level of p300-mediated C/EBPbeta acetylation. Co-expression of promyelocytic leukemia protein abrogated the repressive effect of Daxx on C/EBPbeta as well as the direct interaction of Daxx and C/EBPbeta, presumably by re-recruiting Daxx to PML-oncogenic domains. In acute promyelocytic leukemia (APL) cells, C/EBPbeta activity is known to be required for all-trans-retinoic acid-induced cell differentiation and disease remission. We show that all-trans-retinoic acid as well as arsenic trioxide treatment leads to a reduced C/EBPbeta fraction associated with Daxx suggesting a relief of Daxx-dependent C/EBPbeta repression as an important molecular event leading to APL cell differentiation. Overall, our data identify Daxx as a new negative regulator of C/EBPbeta and provide first clues for a link between abrogation of Daxx-C/EBPbeta complex formation and APL remission.

Kaposi's Sarcoma-Associated Herpesvirus Protein LANA2 Disrupts PML Oncogenic Domains and Inhibits PML-Mediated Transcriptional Repression of the Survivin Gene

Infection by herpesviruses causes a dramatic disturbance of PML oncogenic domains (PODs) that has been suggested to be essential for viral lytic replication. Several proteins from Kaposi's sarcoma-associated herpesvirus (KSHV) have been tested as putative POD-disrupting factors with negative results. Here, we show that LANA2, a viral protein that is absolutely required for the viability and proliferation of KSHV-infected primary effusion lymphoma (PEL) cells, increases the levels of SUMO2-ubiquitin-modified PML and induces the disruption of PODs by a proteasome-mediated mechanism. In addition, we demonstrate that this disruption is largely dependent on both the integrity of a SUMO interaction motif in LANA2 and the lysine 160 from PML. Moreover, silencing of LANA2 expression in PEL cells by RNA interference led to an increase in the PML levels. Finally, we demonstrate that LANA2 relieves PML-mediated transcriptional repression of survivin, a protein that directly contributes to malignant progression of PEL. This represents the first example of inactivation of these important antiviral structures by KSHV.

Expression Pattern and Subcellular Localization of Human Papillomavirus Minor Capsid Protein L2

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18(+) cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18(+) high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18(+) cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.

Immunofluorescence imaging of the influenza virus M1 protein is dependent on the fixation method

The distribution of the matrix (M1) protein of influenza virus in infected cells was examined using immunostaining. The fixation method influenced strongly the immunofluorescence pattern of the M1 protein. The M1 protein was distributed uniformly in both the cytoplasm and in nuclei when cells that had been infected with virus were fixed with paraformaldehyde. In cells that had been fixed with methanol, however, nuclear dots of the M1 protein were clearly visible. The dots were evident at 8h post-inoculation. Up to 6h post-inoculation, only a diffuse distribution of the M1 protein was observed. The dots were co-localized with promyelocytic leukemia (PML) protein, a major component of nuclear domain 10 (ND10), also called PML oncogenic domains (PODs) or PML-nuclear bodies (NBs). These results indicate that the nuclear dots of the M1 protein in cells that had been fixed with methanol are not artifacts of the fixation method. Furthermore, methanol fixation is preferred for localization of the influenza M1 protein in nuclei using immunostaining.

Therapeutic targeting of nuclear receptor corepressor misfolding in acute promyelocytic leukemia cells with genistein

We have recently reported that PML-RAR-induced misfolding of the N-CoR protein could be reversed by retinoic acid (RA), a therapeutic agent that promotes differentiation of acute promyelocytic leukemia (APL) cells. This finding suggests a role of misfolded N-CoR in the differentiation arrest of APL cells and highlights its significance as a potential molecular target in protein conformation-based therapy for APL. Based on this hypothesis, we investigated the therapeutic potential of several protein conformation modifiers on APL-derived cell lines NB4 and NB4-R1. Through a small-scale screening of these selected compounds, we identified genistein as a potent inhibitor of growth of both RA-sensitive and RA-resistant APL cells. Genistein inhibited the growth of NB4 cells through its collective regulatory effects on cell cycle progression, differentiation, and apoptosis. Genistein-induced apoptosis of NB4 cells was mediated by activation of caspase-9 and caspase-3 and was associated with a decrease in mitochondrial transmembrane potential and cytosolic release of cytochrome c. Genistein promoted differentiation of both RA-sensitive and RA-resistant NB4 cells and induced cell cycle arrest by blocking the G(2)-M transition. Genistein up-regulated the expression of PML and N-CoR proteins, promoted degradation of PML-RAR, and reorganized the microspeckled distribution of PML oncogenic domains to a normal dot-like pattern in NB4 cells. Moreover, genistein significantly reversed the PML-RAR-induced misfolding of N-CoR protein by possibly inhibiting the selective phosphorylation-dependent binding of N-CoR to PML-RAR. These findings identify genistein as a potent modifier of N-CoR protein conformation and highlights its therapeutic potential in both RA-sensitive and RA-resistant APL cells.

Induction of promyelocytic leukemia (PML) oncogenic domains (PODs) by papillomavirus

Promyelocytic leukemia oncogenic domains (PODs), also called nuclear domain 10 (ND10), are subnuclear structures that have been implicated in a variety of cellular processes as well as the life cycle of DNA viruses including papillomaviruses. In order to investigate the interplay between papillomaviruses and PODs, we analyzed the status of PODs in organotypic raft cultures of human keratinocytes harboring HPV genome that support the differentiation-dependent HPV life cycle. The number of PODs per nucleus was increased in the presence of HPV genomes selectively within the poorly differentiated layers but was absent in the terminally differentiated layers of the stratified epithelium. This increase in PODs was correlated with an increase in abundance of post-translationally modified PML protein. Neither the E2-dependent transcription nor viral DNA replication was reliant upon the presence of PML. Implications of these findings in terms of HPV's interaction with its host are discussed.

Human T-Cell Leukemia Virus Oncoprotein Tax Represses Nuclear Receptor–Dependent Transcription by Targeting Coactivator TAX1BP1

Human T-cell leukemia virus type 1 oncoprotein Tax is a transcriptional regulator that interacts with a large number of host cell factors. Here, we report the novel characterization of the interaction of Tax with a human cell protein named Tax1-binding protein 1 (TAX1BP1). We show that TAX1BP1 is a nuclear receptor coactivator that forms a complex with the glucocorticoid receptor. TAX1BP1 and Tax colocalize into intranuclear speckles that partially overlap with but are not identical to the PML oncogenic domains. Tax binds TAX1BP1 directly, induces the dissociation of TAX1BP1 from the glucocorticoid receptor-containing protein complex, and represses the coactivator function of TAX1BP1. Genetic knockout of Tax1bp1 in mice abrogates the influence of Tax on the activation of nuclear receptors. We propose that Tax-TAX1BP1 interaction mechanistically explains the previously reported repression of nuclear receptor activity by Tax.

Role of SUMO-Interacting Motif in Daxx SUMO Modification, Subnuclear Localization, and Repression of Sumoylated Transcription Factors

Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

Daxx Represses Expression of a Subset of Antiapoptotic Genes Regulated by Nuclear Factor-κB

Daxx is a nuclear protein that localizes to PML oncogenic domains, sensitizes cells to apoptosis, and functions as a transcriptional repressor. We found that Daxx represses the expression of several antiapoptotic genes regulated by nuclear factor-kappaB, including cIAP2, in human tumor cell lines. Daxx interacts with RelB and inhibits RelB-mediated transcriptional activation of the human cIAP2 gene promoter. Daxx also forms complexes with RelB while bound to its target sites in the cIAP2 promoter, as shown by electrophoretic mobility shift assays and chromatin immunoprecipitation experiments. Using cells from daxx-/- mouse embryos, we observed that levels of the corresponding murine c-IAP mRNA and protein are increased in cells lacking Daxx. Conversely, c-IAP mRNA and protein levels were reduced in relB-/- cells. Taken together, these observations provide a mechanism that links two previously ascribed functions of Daxx: transcriptional repression and sensitization to apoptosis.

Inhibition of SUMO-independent PML oligomerization by the human cytomegalovirus IE1 protein

In human cytomegalovirus-infected cells, the immediate-early IE1 protein disrupts the subnuclear structures known as the PML oncogenic domains or PODs, via the induction of PML desumoylation. This activity correlates with the functions of IE1 in transcriptional regulation and in the stimulation of lytic infection. Here, the effects of IE1 in induction of desumoylation of PML were characterized. IE1 did not interfere with the formation of sumoylated forms of PML in vitro. In in vitro assays using the sumoylated proteins, a SUMO-specific protease SENP1 desumoylated both PML and IE1. However, the IE1 proteins generated from bacteria or insect cells were unable to desumoylate PML in the same conditions. Although both IE1 and SUMO proteases such as SENP1, Axam and SuPr-1 efficiently desumoylated PML in co-transfection assays, they exerted different effects on the localization of PML. In cells transfected with either SENP1 or SuPr-1, the number of PML foci was reduced significantly and these remnant PML foci were devoid of SUMO-1 signals. However, in cells co-transfected with both SUMO proteases and IE1, these SUMO-independent PML foci were also completely disrupted. Furthermore, IE1, but not SENP1, was shown to disrupt the PML foci generated via transfection of a sumoylation-deficient mutant of PML. These data suggest that IE1 exhibits neither an inhibitory effect on sumoylation of PML nor intrinsic SUMO protease activity against PML in vitro. The finding that IE1 is capable of disrupting SUMO-independent PML aggregates suggests that inhibition of PML oligomerization by IE1 may play an important role in inducing PML desumoylation in vivo.

Intracellular localization of human ZBP1: Differential regulation by the Z-DNA binding domain, Zα, in splice variants

We investigated the subcellular distribution of human ZBP1, which harbors the N-terminal Z-DNA binding domains, Zα and Zβ. ZBP1 was distributed primarily in the cytoplasm and occasionally as nuclear foci in interferon (IFN)-treated primary hepatocellular carcinoma cells, and in several other transfected cell types. In leptomycin B (LMB)-treated cells, endogenous ZBP1 efficiently accumulated in nuclear foci, which overlapped PML oncogenic domains (PODs) or nuclear bodies (NBs). In transfection assays, the unique C-terminal region of ZBP1 was necessary for its typical cytoplasmic localization. Interestingly, the Zα-deleted form displayed an increased association with PODs compared to wild-type and, unlike wild-type, perfectly accumulated in PODs in LMB-treated cells, implying that the presence of Zα domain also facilitates the cytoplasmic localization. Our results demonstrate that ZBP1 is localized primarily in the cytoplasm but also associated with nuclear PODs in IFN or LMB-treated cells. Given that about half of ZBP1 mRNA lacks exon 2 encoding the Zα domain, our data also suggest that the localization of ZBP1 may be differentially regulated by the Z-DNA binding domain, Zα, in splice variants.

Sumoylation of Daxx Regulates IFN-Induced Growth Suppression of B Lymphocytes and the Hormone Receptor-Mediated Transactivation

Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.

Interactions among Four Proteins Encoded by the Human Cytomegalovirus UL112-113 Region Regulate Their Intranuclear Targeting and the Recruitment of UL44 to Prereplication Foci

Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.