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.     

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.     

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 Inhibits Progression of Cells through Mitosis and from G1 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.     

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.     

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.     

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.     

Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65.

A transient expression system was developed which results in efficient synthesis of the regulatory protein Vmw65 of herpes simplex virus type 1 in eucaryotic cells. The gene for Vmw65 was linked to the cytomegalovirus immediate-early (IE) promoter-enhancer region in a plasmid containing the simian virus 40 origin of replication. When transfected into COS cells, Vmw65 was expressed from this vector in 25 to 50% of the cells, with total levels of the protein approaching 20% of those observed in infected cells. Vmw65 expressed in this system is functional for specific DNA-binding complex formation with the host cell octamer-binding protein TRF and for transactivation of IE gene expression. We therefore produced a series of carboxy-terminal truncated forms of Vmw65 to examine the structural requirements of the protein for these activities. Deletion of the acidic carboxy-terminal 56 amino acids had no effect on DNA-binding complex formation but completely abolished the ability to transactivate. Amino acids between residues 434 and 453, a region which exhibits a high negative charge, were critical for IE transactivation. In contrast, the requirements for complex formation are located entirely within the N-terminal 403 amino acids, and our results indicate a requirement for this activity for residues between 316 and 403. Together with our previous work, the results presented here indicate that recruitment of TRF into a specific DNA-binding complex on IE consensus signals is required but not sufficient for functional IE transactivation by Vmw65.     

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.     

The differential requirement for cyclin-dependent kinase activities distinguishes two functions of herpes simplex virus type 1 ICP0

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0(-) mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in 90% of 7134- and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK(cs), a known target of ICP0-directed degradation. The results of the PML and DNA-PK(cs) immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.     

Interaction of Herpes Simplex Virus ICP0 with ND10 Bodies: a Sequential Process of Adhesion,Fusion, and Retention

On entry into the nucleus, herpes simplex virus 1 (HSV-1) DNA localizes to nuclear bodies known as ND10. Gene repression imposed by ND10 is released by a viral protein, ICP0, via degradation of the ND10 constituents promyelocytic leukemia protein (PML) and Sp100 and the subsequent dispersal of ND10 bodies. In order to understand the dynamic interaction between ICP0 and ND10, we carried out deletion mapping to identify the domains of ICP0 responsible for its association with ND10. Here, we report the following. (i) An ND10 entry signal (ND10-ES), located between residues 245 and 474, is required for ICP0 to penetrate and fuse with ND10. ICP0 lacking ND10-ES adheres to the surface of ND10 but fails to enter. (ii) In the absence of ND10-ES, the E3 ubiquitin ligase of ICP0 facilitates the transient adhesion of the truncated ICP0 to the ND10 surface, whereas the presence of ND10-ES in ICP0 renders ND10 fusion regardless of the E3 ligase activity. (iii) The C terminus of ICP0 is required for retention of ICP0 in ND10 but plays no role in the recruitment process. (iv) The adverse effects of an inactive RING domain on viral replication are partially reversed by deleting either ND10-ES or the C-terminal retention domain, suggesting that additional ICP0 functions require the release of ICP0 from ND10. Based on these results, we conclude that association of ICP0 and ND10 is a dynamic process, in which three sequential steps—adhesion, fusion, and retention—are adopted to stabilize the interaction. A faithful execution of these steps defines the ultimate productivity of the virus.