Anne Orr

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 ∼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.

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

ABSTRACT 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.

Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1 Is Restricted by both PML and Sp100

ABSTRACT 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.

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.

Phenotype of a Herpes Simplex Virus Type 1 Mutant That Fails To Express Immediate-Early Regulatory Protein ICP0

ABSTRACT Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limited-passage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque.

Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7

ABSTRACT Herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 stimulates lytic infection and the reactivation of quiescent viral genomes. These roles of ICP0 require its RING finger E3 ubiquitin ligase domain, which induces the degradation of several cellular proteins, including components of promyelocytic leukemia nuclear bodies and centromeres. ICP0 also interacts very strongly with the cellular ubiquitin-specific protease USP7 (also known as HAUSP). We have shown previously that ICP0 induces its own ubiquitination and degradation in a RING finger-dependent manner, and that its interaction with USP7 regulates this process. In the course of these studies we found and report here that ICP0 also targets USP7 for ubiquitination and proteasome-dependent degradation. The reciprocal activities of the two proteins reveal an intriguing situation that poses the question of the balance of the two processes during productive HSV-1 infection. Based on a thorough analysis of the properties of an HSV-1 mutant virus that expresses forms of ICP0 that are unable to bind to USP7, we conclude that USP7-mediated stabilization of ICP0 is dominant over ICP0-induced degradation of USP7 during productive HSV-1 infection. We propose that the biological significance of the ICP0-USP7 interaction may be most pronounced in natural infection situations, in which limited amounts of ICP0 are expressed.

A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.

Herpes Simplex Virus Type 1 Genomes Are Associated with ND10 Nuclear Substructures in Quiescently Infected Human Fibroblasts

ABSTRACT 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.

Formation of Nuclear Foci of the Herpes Simplex Virus Type 1 Regulatory Protein ICP4 at Early Times of Infection: Localization, Dynamics, Recruitment of ICP27, and Evidence for the De Novo Induction of ND10-Like Complexes

ABSTRACT Herpes simplex virus type 1 (HSV-1) has an intricate association with cellular nuclear structures known as ND10 or promyelocytic leukemia protein (PML) nuclear bodies. Parental viral genomes initially become juxtaposed to ND10, and then viral replication compartments develop from the ND10-associated genomes. Viral immediate-early (IE) regulatory protein ICP0 colocalizes with ND10 and then induces the degradation of critical ND10 component protein PML and therefore the release and dispersal of other ND10 proteins. The IE transcriptional regulatory protein ICP4 also forms foci at early times of infection, many of which are juxtaposed to ND10 and later develop into replication compartments, indicating that at least some of the initial ICP4 foci contain parental viral genomes. Here we report that the ICP4 foci also contain ICP27 and that their formation occurs extremely rapidly at locations just inside the nuclear envelope. By examining developing plaques or thinly seeded cells infected at high multiplicity, we found evidence to suggest that at least some of the ND10-viral nucleoprotein complex association could be attributed to de novo formation of ND10-like structures in response to incoming viral genomes. The ICP4 complexes associated efficiently with ND10 in cells infected with an ICP0-null mutant virus at high but not at low multiplicity, and the degree of association was reduced by the proteasome inhibitor MG132. Therefore, the interaction between viral nucleoprotein complexes and ND10 is in part due to a dynamic response by the cell. This response is modulated by functional ICP0, and cells that are productively or nonproductively infected in the absence of functional ICP0 can be distinguished by the relative locations of ICP4 foci and ND10 proteins.

PML Residue Lysine 160 Is Required for the Degradation of PML Induced by Herpes Simplex Virus Type 1 Regulatory Protein ICP0

ABSTRACT During the early stages of herpes simplex virus type 1 (HSV-1) infection, viral immediate-early regulatory protein ICP0 localizes to and disrupts cellular nuclear structures known as PML nuclear bodies or ND10. These activities correlate with the functions of ICP0 in stimulating lytic infection and reactivating quiescent HSV-1. The disruption of ND10 occurs because ICP0 induces the loss of the SUMO-1-modified forms of PML and the subsequent proteasome-mediated degradation of the PML protein. The functions of ICP0 are largely dependent on the integrity of its zinc-binding RING finger domain. Many RING finger proteins have been found to act as ubiquitin E3 ligase enzymes, stimulating the production of conjugated polyubiquitin chains in the presence of ubiquitin, the ubiquitin-activating enzyme E1, and the appropriate E2 ubiquitin-conjugating enzyme. Substrate proteins that become polyubiquitinated are then subject to degradation by proteasomes. We have previously shown that purified full-length ICP0 acts as an efficient E3 ligase in vitro, producing high-molecular-weight polyubiquitin chains in a RING finger-dependent but substrate-independent manner. In this paper we report on investigations into the factors governing the degradation of PML induced by ICP0 in a variety of in vivo and in vitro assays. We found that ICP0 expression increases the levels of ubiquitinated PML in transfected cells. However, ICP0 does not interact with or directly ubiquitinate either unmodified PML or SUMO-1-modified PML in vitro, suggesting either that additional factors are required for the ICP0-mediated ubiquitination of PML in vivo or that PML degradation is an indirect consequence of some other activity of ICP0 at ND10. Using a transfection-based approach and a family of deletion and point mutations of PML, we found that efficient ICP0-induced PML degradation requires sequences within the C-terminal part of PML and lysine residue 160, one of the principal targets for SUMO-1 modification of the protein.