A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein.

Herpes simplex virus type 1 immediate-early protein Vmw110 is a non-specific activator of gene expression and is required for efficient initiation of the viral lytic cycle. Since Vmw110-deficient viruses reactivate inefficiently in mouse latency models it has been suggested that Vmw110 plays a role in the balance between the latent and lytic states of the virus. The mechanisms by which Vmw110 achieves these functions are poorly understood. Vmw110 migrates to discrete nuclear structures (ND10) which contain the cellular PML protein, and in consequence PML and other constituent proteins are dispersed. In addition, Vmw110 binds to a cellular protein of approximately 135 kDa, and its interactions with the 135 kDa protein and ND10 contribute to its ability to stimulate gene expression and viral lytic growth. In this report we identify the 135 kDa protein as a novel member of the ubiquitin-specific protease family. The protease is distributed in the nucleus in a micropunctate pattern with a limited number of larger discrete foci, some of which co-localize with PML in ND10. At early times of virus infection, the presence of Vmw110 increases the proportion of ND10 which contain the ubiquitin-specific protease. These results identify a novel, transitory component of ND10 and implicate a previously uncharacterized ubiquitin-dependent pathway in the control of viral gene expression.     

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.     

HSV-1 IE protein Vmw110 causes redistribution of PML.

Herpes simplex virus immediate-early protein Vmw110 is required for fully efficient viral gene expression and reactivation from latency. At early times of viral infection, Vmw110 localizes to discrete nuclear structures (known as ND10, PODs or Kr bodies) which contain several cellular proteins, including PML. Interestingly, the unregulated growth of promyelocytic leukaemia cells is correlated with disruption of the normal state of ND10. In this paper we show that: (i) Vmw110 affects the distribution of PML in the cell; (ii) Vmw110 proteins lacking a functional RING finger zinc-binding domain cause the production of striking abnormal cytoplasmic and nuclear structures, some of which contain PML and other ND10 antigens; (iii) a mutant form of Vmw110 which is confined to the cytoplasm appears to result in cytoplasmic PML in some cells; (iv) normal interaction with the nuclear structures requires the C-terminal portion of Vmw110; (v) the C-terminal portion of Vmw110, when linked to a heterologous protein, disrupts the normal distribution of PML. The results suggest that, in normal cells, the PML protein migrates between nucleus and cytoplasm. These observations present an unexpected link between processes involved in the control of cell growth and viral infection and latency.     

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.     

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.     

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.     

Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures.

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein.     

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.     

Herpes simplex virus type 1 genomes are associated with ND10 nuclear substructures in quiescently infected human fibroblasts

Herpes simplex virus type 1 (HSV-1) genomes become associated with structures related to cellular nuclear substructures known as ND10 or promyelocytic leukemia nuclear bodies during the early stages of lytic infection. This paper describes the relationship between HSV-1 genomes and ND10 in human fibroblasts that maintain the viral genomes in a quiescent state. We report that quiescent HSV-1 genomes detected by fluorescence in situ hybridization (FISH) are associated with enlarged ND10-like structures, frequently such that the FISH-defined viral foci are apparently enveloped within a sphere of PML and other ND10 proteins. The number of FISH viral foci in each quiescently infected cell is concordant with the input multiplicity of infection, with each structure containing no more than a small number of viral genomes. A proportion of the enlarged ND10-like foci in quiescently infected cells contain accumulations of the heterochromatin protein HP1 but not other common markers of heterochromatin such as histone H3 di- or trimethylated on lysine residue 9. Many of the virally induced enlarged ND10-like structures also contain concentrations of conjugated ubiquitin. Quiescent infections can be established in cells that are highly depleted for PML. However, during the initial stages of establishment of a quiescent infection in such cells, other ND10 proteins (Sp100, hDaxx, and ATRX) are recruited into virally induced foci that are likely to be associated with HSV-1 genomes. These observations illustrate that the intimate connections between HSV-1 genomes and ND10 that occur during lytic infection also extend to quiescent infections.     

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0

Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.     

ND10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection

Infections with DNA viruses commonly result in the association of viral genomes and replication compartments with cellular nuclear substructures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. While there is evidence that viral genomes can associate with preexisting ND10, we demonstrate in this study by live-cell microscopy that structures resembling ND10 form de novo and in association with viral genome complexes during the initial stages of herpes simplex virus type 1 (HSV-1) infection. Consistent with previous studies, we found that the major ND10 proteins PML, Sp100, and hDaxx are exchanged very rapidly between ND10 foci and the surrounding nucleoplasm in live cells. The dynamic nature of the individual protein molecule components of ND10 provides a mechanism by which ND10 proteins can be recruited to novel sites during virus infection. These observations explain why the genomes and replication compartments of DNA viruses that replicate in the cell nucleus are so commonly found in association with ND10. These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

Overexpression of promyelocytic leukemia protein precludes the dispersal of ND10 structures and has no effect on accumulation of infectious herpes simplex virus 1 or its proteins

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the M(r) 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of alpha, beta, or gamma groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the alpha 0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.     

Interactions between DNA viruses, ND10 and the DNA damage response

ND10 are small nuclear substructures that are defined by the presence the promyelocytic leukaemia protein PML. Many other proteins have been detected within ND10, a complexity that is reflected in reports of their involvement in multiple cellular pathways that include the regulation of gene expression, chromatin dynamics, protein modification, apoptosis, p53 function, senescence, DNA repair, the interferon response and viral infection. This review summarizes recent evidence of similarities between the behaviour of ND10 components and DNA repair pathway proteins in response to viral infection and DNA damage. ND10 structures become associated with the parental genomes and early replication compartments of many DNA viruses, and DNA repair pathway proteins are also recruited to these sites. Similarly, PML and DNA repair proteins are recruited to sites of DNA damage. The mechanisms by which these events might occur, and the implications for ND10 function in DNA virus infection and chromatin metabolism, are discussed.     

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.