Roger D. Everett

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.

Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro

ABSTRACT Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.

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.

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.

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.

The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0

ND10 are nuclear domains of unknown function that become abundant in response to stress. Infection by herpes simplex virus type 1 (HSV-1) causes the apparent disappearance of these domains, an effect that requires the expression of the immediate early protein ICP0. Previously, we have shown that there are a number of cellular antigens in the ND10. In this report, we show that one of these proteins is PML, a member of the C3HC4 zinc-binding domain family which also includes ICP0. The C3HC4 domain of ICP0 is essential for the apparent release of PML from the ND10, although the interaction of ICP0 with ND10 is determined by a small region near its carboxy terminus. PML and other ND10 proteins are not lost after removal from ND10 but deposited at the nuclear envelope or nuclear envelope modifications during later parts of the replication cycle. ICP0 is required for the onset of low multiplicity infections, and has been implicated in the process of reactivation from HSV latency. Therefore, the interaction between ICP0 and the ND10 domains, specifically PML, may be important for the outcome of virus-cell interactions.

Trans activation of transcription by herpes virus products: requirement for two HSV-1 immediate-early polypeptides for maximum activity.

The transcriptional programme of the herpes viruses is organised into three principal phases. The immediate‐early (IE) genes are the first to be transcribed, by the pre‐existing host RNA polymerase II, and their promoters are strongly stimulated by a polypeptide component of the virus particle. The E and L gene promoters become active only after the appearance of IE gene products. Genetic and biochemical evidence has shown that the HSV‐1 IE polypeptide Vmw175 (ICP 4) is essential for the trans activation of HSV early promoters, but the role of none of the other four IE gene products was known. This paper describes functional tests that show, by co‐transfection of recombinant plasmids into HeLa cells, that (i) Vmw175 alone can activate an HSV‐1 E gene promoter, (ii) the four other HSV‐1 IE gene products by themselves are unable to activate transcription, (iii) the combination of Vmw175 plus the product of IE gene 1, Vmw110 (ICP 0), is a much better activator than Vmw175 alone, (iv) cloned IE gene products of human cytomegalovirus (CMV), varicella‐zoseter virus (VZV) and pseudorabies virus (PRV) can also activate transcription from an HSV‐1 early promoter, and (v) this activation also occurs with cellular promoters.

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.

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 Herpes Simplex Virus ICP0 RING Finger Domain Inhibits IRF3- and IRF7-Mediated Activation of Interferon-Stimulated Genes

ABSTRACT Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.