SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML.

The E1B-55K product from human adenovirus is a substrate of the small ubiquitin-related modifier (SUMO)-conjugation system. SUMOylation of E1B-55K is required to transform primary mammalian cells in cooperation with adenovirus E1A and to repress p53 tumour suppressor functions. The biochemical consequences of SUMO1 conjugation of 55K have so far remained elusive. Here, we report that E1B-55K physically interacts with different isoforms of the tumour suppressor protein promyelocytic leukaemia (PML). We show that E1B-55K binds to PML isoforms IV and V in a SUMO1-dependent and -independent manner. Interaction with PML-IV promotes the localization of 55K to PML-containing subnuclear structures (PML-NBs). In virus-infected cells, this process is negatively regulated by other viral proteins, indicating that binding to PML is controlled through reversible SUMOylation in a timely coordinated manner. These results together with earlier work are consistent with the idea that SUMOylation regulates targeting of E1B-55K to PML-NBs, known to control transcriptional regulation, tumour suppression, DNA repair and apoptosis. Furthermore, they suggest that SUMO1-dependent modulation of p53-dependent growth suppression through E1B-55K PML-IV interaction has a key role in adenovirus-mediated cell transformation.

DNA viruses and viral proteins that interact with PML nuclear bodies.

Connections between PML nuclear bodies (PML NBs) and DNA virus replication have been investigated from the earliest days of the molecular characterization of PML and associated proteins. It appears to be a general feature of nuclear-replicating DNA viruses that their parental genomes preferentially become associated with PML NBs, and that their initial sites of transcription and development of DNA replication centres are frequently juxtaposed to these domains or their remnants. In addition, regulatory proteins encoded by several DNA viruses associate with and sometimes cause catastrophic changes to PML NBs by a variety of mechanisms. These events can be correlated with the efficiency of viral infection and the functions of viral regulatory proteins, but the underlying molecular connections between PML NB function and viral infection remain poorly understood. This article reviews the latest developments in the interactions between PML NBs and herpesviruses, adenoviruses and papovaviruses.

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 induce the formation of colocalizing, conjugated ubiquitin.

Herpes simplex virus type 1 immediate early protein ICP0 influences virus infection by inducing the degradation of specific cellular proteins via a mechanism requiring its RING finger and the ubiquitin-proteasome pathway. Many RING finger proteins, by virtue of their RING finger domain, interact with E2 ubiquitin-conjugating enzymes and act as a component of an E3 ubiquitin ligase. We have recently shown that ICP0 induces the accumulation of colocalizing, conjugated ubiquitin, suggesting that ICP0 can act as or contribute to an E3 ubiquitin ligase. In this report we demonstrate that the ICP0-related RING finger proteins encoded by other alphaherpesviruses also induce colocalizing, conjugated ubiquitin, thereby suggesting that they act by similar biochemical mechanisms.

Human neuron-committed teratocarcinoma NT2 cell line has abnormal ND10 structures and is poorly infected by herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.

Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0.

Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. Here, we demonstrate that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments we show that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation.

ICP0 induces the accumulation of colocalizing conjugated ubiquitin.

Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 is a general activator of viral gene expression which stimulates the initiation of lytic infection and reactivation from quiescence and latency. The importance of ICP0 to the biology of HSV-1 infection has stimulated interest in its mode of action. Previous studies have reported its interactions with other viral regulatory molecules, with the translation apparatus, with cyclin D3, and with a ubiquitin-specific protease. It has been demonstrated that ICP0 is able to induce the proteasome-dependent degradation of a number of cellular proteins, including components of centromeres and small nuclear substructures known as ND10 or PML nuclear bodies. ICP0 has a RING finger zinc-binding domain which is essential for its functions. In view of several recent examples of other RING finger proteins which modulate the stability of specific target proteins by acting as components of E3 ubiquitin ligase complexes, this study has explored whether ICP0 might operate via a similar mechanism. Evidence that the foci of accumulated ICP0 in transfected and infected cells contain enhanced levels of conjugated ubiquitin is presented. This effect was dependent on the RING finger region of ICP0, and comparison of the properties of a number of ICP0 mutants revealed an excellent correlation between previously established functions of ICP0 and its ability to induce concentrations of colocalizing conjugated ubiquitin. These results strongly support the hypothesis that a major factor in the mechanism by which ICP0 influences virus infection is its ability to induce the degradation of specific cellular targets by interaction with the ubiquitin-proteasome pathway.

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.

ICP0, a regulator of herpes simplex virus during lytic and latent infection.

Cold sores produced by HSV-1 infection are an annoying but trivial recurrent problem for most of us, but the virus can also cause more serious disease. Episodes of active HSV-1 infection, in response to stress or sunlight, are possible because the virus establishes a latent infection in neurones which can not be eliminated. Since vigorous transcription from the whole viral genome during lytic infection contrasts with almost complete quiescence during latency, the mechanisms controlling HSV-1 gene expression have come under close scrutiny. These studies have demonstrated that the viral immediate-early protein ICP0, a promiscuous activator of gene expression, is required for efficient initiation of lytic infection and reactivation from latency. It is proposed that in the absence of functional ICP0, a cellular repression mechanism silences viral transcription. ICP0 appears to counteract this process by stimulating the degradation of a number of cellular proteins via the ubiquitin-proteasome pathway.

Cell cycle regulation of PML modification and ND10 composition.

The nuclear sub-structures known as ND10, PODs or PML nuclear bodies can be rapidly modified by diverse stimuli, and the resultant structural changes correlate with events such as cellular transformation and successful virus infection. We show that the ND10 components PML and Sp100 undergo profound biochemical changes during the cell cycle. Both proteins are conjugated to the ubiquitin-like protein SUMO-1 during interphase, but they become de-conjugated during mitosis and an isoform of PML of distinct electrophoretic mobility appears. This mitosis-specific form of PML is highly labile in vitro, but is partially stabilised by phosphatase inhibitors. Treatment of interphase cells with phosphatase inhibitors induces the production of a PML isoform of similar gel mobility to the mitosis-specific species, and taken together these results suggest that phosphorylation is an important factor in the differential modification of PML during the cell cycle. PML and Sp100 normally tightly co-localise in ND10 in interphase cells, but they become separated during mitosis. Interphase cells treated with phosphatase inhibitors or subjected to heat shock also show structural changes in ND10, accompanied by alterations to the normal pattern of PML modification. Taken with previous findings on the effects of infection by herpes simplex virus and adenovirus on ND10 structure and PML modification, these results suggest that the many factors which have been shown to modify ND10 structure may do so by interaction with the biochemical mechanisms that act on ND10 components during the cell cycle.

The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2.

Herpes simplex virus type 1 (HSV-1), the prototype alpha-herpesvirus, causes several prominent diseases. The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far. IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm. To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait. Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the beta subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped. Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays. hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by anti-hnRNP K serum. Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2. These data provide important new insights into how this key viral regulatory protein exerts its functions.

A dynamic connection between centromeres and ND10 proteins.

ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G(2) phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.

Herpes simplex virus type 1 immediate-early protein Vmw110 inhibits progression of cells through mitosis and from G(1) into S phase of the cell cycle.

Herpes simplex virus type 1 (HSV-1) immediate-early protein Vmw110 stimulates the onset of virus infection in a multiplicity-dependent manner and is required for efficient reactivation from latency. Recent work has shown that Vmw110 is able to interact with or modify the stability of several cellular proteins. In this report we analyze the ability of Vmw110 to inhibit the progression of cells through the cell cycle. We show by fluorescence-activated cell sorter and/or confocal microscopy analysis that an enhanced green fluorescent protein-tagged Vmw110 possesses the abilities both to prevent transfected cells moving from G(1) into S phase and to block infected cells at an unusual stage of mitosis defined as pseudo-prometaphase. The latter property correlates with the Vmw110-induced proteasome-dependent degradation of CENP-C, a centromeric protein component of the inner plate of human kinetochores. We also show that whereas Vmw110 is not the only viral product implicated in the block of infected cells at the G(1)/S border, the mitotic block is a specific property of Vmw110 and more particularly of its RING finger domain. These data explain the toxicity of Vmw110 when expressed alone in transfected cells and provide an explanation for the remaining toxicity of replication-defective mutants of HSV-1 expressing Vmw110. In addition to contributing to our understanding of the effects of Vmw110 on the cell, our results demonstrate that Vmw110 expression is incompatible with the proliferation of a dividing cell population. This factor is of obvious importance to the design of gene therapy vectors based on HSV-1.

The adenovirus type 5 E1b 55K and E4 Orf3 proteins associate in infected cells and affect ND10 components.

Three early proteins expressed by adenovirus type 5, E1b 55K, E4 Orf3 and E4 Orf6, are involved in regulating late viral gene expression. It has previously been shown that 55K associates with Orf6. Here we show that 55K also associates with Orf3 and that this interaction is necessary for 55K to localize to the nuclear matrix fraction of the cell. From our data, we infer that the Orf3 and Orf6 interactions with 55K may be mutually exclusive. The Orf3 protein is also known to associate with and cause the reorganization of cell nucleus structures known as ND10 or PODs. Consistent with the observed increase in the biochemical interaction between 55K and Orf3 in the absence of Orf6, the 55K association with Orf3 in ND10 was also found to increase in the absence of Orf6. The most studied cellular component of ND10 is PML, a complex protein present in a range of isoforms, some of which are modified by conjugation to the small ubiquitin-like protein PIC-1. The pattern of PML isoforms was altered in adenovirus-infected cells, in that a number of additional isoform bands appeared in an Orf3-dependent manner, one of which became predominant later in infection. As for ND10 reorganization, neither Orf6 nor 55K was required for this effect. Therefore it is likely that these changes in PML are related to the changes in ND10 structure that occur during infection.

Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110.

Examination of cells at the early stages of herpes simplex virus type 1 infection revealed that the viral immediate-early protein Vmw110 (also known as ICP0) formed discrete punctate accumulations associated with centromeres in both mitotic and interphase cells. The RING finger domain of Vmw110 (but not the C-terminal region) was essential for its localization at centromeres, thus distinguishing the Vmw110 sequences required for centromere association from those required for its localization at other discrete nuclear structures known as ND10, promyelocytic leukaemia (PML) bodies or PODs. We have shown recently that Vmw110 can induce the proteasome-dependent loss of several cellular proteins, including a number of probable SUMO-1-conjugated isoforms of PML, and this results in the disruption of ND10. In this study, we found some striking similarities between the interactions of Vmw110 with ND10 and centromeres. Specifically, centromeric protein CENP-C was lost from centromeres during virus infection in a Vmw110- and proteasome-dependent manner, causing substantial ultrastructural changes in the kinetochore. In consequence, dividing cells either became stalled in mitosis or underwent an unusual cytokinesis resulting in daughter cells with many micronuclei. These results emphasize the importance of CENP-C for mitotic progression and suggest that Vmw110 may be interfering with biochemical mechanisms which are relevant to both centromeres and ND10.

The ability of herpes simplex virus type 1 immediate-early protein Vmw110 to bind to a ubiquitin-specific protease contributes to its roles in the activation of gene expression and stimulation of virus replication.

Herpes simplex virus type 1 immediate-early protein Vmw110 stimulates the onset of virus infection and is required for efficient reactivation from latency. In transfection assays, Vmw110 is a potent activator of gene expression, but its mode of action has yet to be determined. Previous work has shown that Vmw110 localizes to specific intranuclear structures known as ND10, PML bodies, or PODs and causes the disruption of these domains. The ability of Vmw110 to disrupt ND10 correlates with its biological activities in infected and transfected cells. It has also been found that Vmw110 binds strongly and specifically to a ubiquitin-specific protease known as HAUSP, itself a component of a subset of ND10. In this study we have investigated the role of HAUSP in Vmw110 activity; single amino acid residues of Vmw110 required for the interaction were identified, and the effects of mutation of these residues in infected and transfected cells were then assayed. The results indicate that the ability to bind to HAUSP contributes to the functional activities of Vmw110.

Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase.

Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is by itself sufficient for the effect. We found that proteasome inhibitors prevented the degradation of DNA-PKcs, indicating the involvement of a proteasome pathway. Furthermore, the continued activity of DNA-PK during infection in the presence of these inhibitors indicated that Vmw110 does not directly alter the enzyme activity of DNA-PKcs prior to its degradation in a normal infection. Indeed, Vmw110 was found to bind to neither the catalytic nor Ku subunits of DNA-PK. Using mutant Vmw110 viruses we show that the RING finger domain of Vmw110 is essential for the induced degradation of DNA-PKcs but that the ability of Vmw110 to bind to a cellular ubiquitin-specific protease (HAUSP) is not required. When expressed in the absence of other viral proteins, Vmw110 was sufficient to cause the degradation of DNA-PKcs, indicating that the effect on the stability of DNA-PKcs was a direct consequence of Vmw110 activity and not an indirect Vmw110-dependent effect of virus infection. Finally, the Vmw110-induced degradation of DNA-PKcs and loss in DNA-PK activity appears to be beneficial to HSV-1 infection, as virus replication was more efficient in cells lacking DNA-PKcs, especially at low multiplicities of infection.

A viral activator of gene expression functions via the ubiquitin-proteasome pathway.

The ability of herpes simplex virus type 1 (HSV-1) to attain a latent state in sensory neurones and reactivate periodically is crucial for its biological and clinical properties. The active transcription of the entire 152 kb viral genome during lytic replication contrasts with the latent state, which is characterized by the production of a single set of nuclear-retained transcripts. Reactivation of latent genomes to re-initiate the lytic cycle therefore involves a profound change in viral transcriptional activity, but the mechanisms by which this fundamentally important process occurs are yet to be well understood. In this report we show that the stimulation of the onset of viral lytic infection mediated by the viral immediate-early (IE) protein Vmw110 is strikingly inhibited by inactivation of the ubiquitin-proteasome pathway. Similarly, the Vmw110-dependent reactivation of quiescent viral genomes in cultured cells is also dependent on proteasome activity. These results constitute the first demonstration that the transcriptional activity of a viral genome can be regulated by protein stability control pathways.

The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms.

The small nuclear structures known as ND10 or PML nuclear bodies have been implicated in a variety of cellular processes including response to stress and interferons, oncogenesis, and viral infection, but little is known about their biochemical properties. Recently, a ubiquitin-specific protease enzyme (named HAUSP) and a ubiquitin-homology family protein (PIC1) have been found associated with ND10. HAUSP binds strongly to Vmw110, a herpesvirus regulatory protein which has the ability to disrupt ND10, while PIC1 was identified as a protein which interacts with PML, the prototype ND10 protein. We have investigated the role of ubiquitin-related pathways in the mechanism of ND10 disruption by Vmw110 and the effect of virus infection on PML stability. The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes. The PML isoforms that are most sensitive to virus infection correspond closely to those which have recently been identified as being covalently conjugated to PIC1. In addition, a large number of PIC1-protein conjugates can be detected following transfection of a PIC1 expression plasmid, and many of these are also eliminated in a Vmw110-dependent manner during virus infection. These observations provide a biochemical mechanism to explain the observed effects of Vmw110 on ND10 and suggest a simple yet powerful mechanism by which Vmw110 might function during virus infection.