Herpes simplex virus (HSV) is a clinically important virus, whose life cycle alternates between productive replication and latency. Infected cell protein 0 (ICP0) is generally believed to play a key role in determining the outcome of HSV infections. Synthesis of ICP0 promotes the productive replication of HSV, whereas absence of ICP0 renders HSV prone to establish latent infections. In the first part of the dissertation, I attempt to address the question how is ICP0 gene regulated. To tackle this question, we constructed recombinant HSV that encodes GFP-tagged ICP0 so that the regulation of ICP0 gene can be visualized in real time. Using this reagent, we found that ICP0 gene was subject to potent repression immediately following infection. Surprisingly, HSV's major transcriptional regulator, ICP4, was necessary and sufficient to repress ICP0 gene, and did so in an ICP4-binding-site dependent manner. Synthesis of ICP0 alleviated the ICP4-dependent repression of ICP0 gene. ICP4 co-immunoprecipitated with FLAG-tagged ICP0, thus, a physical interaction between ICP0 and ICP4 likely explains how ICP0 antagonizes ICP4's capacity to silence the ICP0 gene. Therefore, our findings suggest that ICP0 gene is differentially regulated by virus-encoded repressor ICP4 and virus-encoded antirepressor ICP0. In the second part of the dissertation, I attempt to address the question what function does ICP0 assume. Since the discovery of ICP0, the nuclear function of ICP0 has been the focal point of studies, whereas the cytoplasmic function of ICP0 is unknown. While pursuing our first study, we unexpectedly found that GFP-tagged ICP0 was predominantly localized to the cytoplasm during infections. Taking advantage of live-cell imaging, we found that ICP0 translocated from nucleus to cytoplasm during early phase of HSV infections, where it bundled and dispersed microtubules. Synthesis of ICP0 was proved to be necessary and sufficient to dismantle microtubules in HSV-infected and transfected cells. Therefore, our findings suggest ICP0 might play a previously unrecognized role in the cytoplasm through dismantling microtubule networks of the host cells. Furthermore, our study represents the first report showing a virus-encoded E3 ligase disrupts host cell microtubule networks, thus suggests a general function of many other viral E3 ligases.

Herpes simplex virus entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in herpes simplex virus entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on herpes simplex virus capsid transport. Herpes simplex virus can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, herpes simplex virus successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. Herpes simplex virus immediate-early protein ICP0 is a multifunctional regulator of herpes simplex virus infection. Late in infection ICP0 interacts dynamically with cellular proteasomes. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for its IE functions. The fundamental and functional properties of ICP0 that is present in the virion tegument layer have not been well characterized. For these reasons, I sought to characterize tegument ICP0 and determine the role of tegument ICP0 during proteasome-dependent entry of herpes simplex virus. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. Virions with mutations in the RING finger domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins. ICP0 mutations that resulted in the absence of ICP0 in the tegument layer, allow herpes simplex virus to enter cells independently of the proteasome activity. I propose that proteasomal degradation of virion and/or host proteins is regulated by ICP0 to allow for efficient delivery of incoming herpes simplex virus capsids to the nucleus.

No description available.

Infection of most cell types with herpes simplex virus (HSV) mutants lacking the activation functions of VP16 and/or ICP0 results in repression of viral gene expression. However, the human osteosarcoma cell line U2OS supports the replication of VP16 and ICP0 mutants to nearly wild type levels. Prior to the studies presented in this thesis, the basis for the permissivity of U2OS cells to VP16 and ICP0 mutants had not been explored. Here, somatic cell fusion assays were used to determine that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate gene silencing mechanism. The artificial induction of interferon stimulated genes that occurs during the somatic cell fusion assays is not the basis for the observed repression of viral gene expression. As one means of identifying components of the antiviral pathway defective in U2OS cells, restrictive cell types were treated with kinase inhibitors and infected with VP16 and/or ICP0 mutants. Although several compounds were identified which compensate for the defect in gene expression of VP16 mutants, these drugs also stimulate mutant virus gene expression in U2OS. Thus, U2OS are most likely not defective in the cellular signalling pathway(s) targeted by these compound(s). Finally, the importance of VP16 and ICP0 in modulating chromatin structure on the viral genome in both restrictive and permissive cells was examined, uncovering an essential role for both proteins in altering histone occupancy and acetylation levels. Importantly, U2OS cells have a defect in the chromatin-based pathway targeted by ICP0. However, evidence suggests that the ability of VP16 and ICP0 to affect histone occupancy and acetylation levels is not required for viral gene expression. Taken together, the results of this thesis demonstrate that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate antiviral mechanism which does not involve the targets of several well characterized kinase inhibitors. The significance of the defect in a chromatin-based pathway targeted by ICP0 in U2OS cells remains to be elucidated. / Virology

No description available.

L’infection par le virus de l’herpès simplex de type 1 (HSV-1), un virus pathogène humain majeur, engendre la déstabilisation des centromères. Cette déstabilisation est induite par la protéine virale ICP0, et entraîne la dégradation par ICP0, via le protéasome, des protéines CENP-A, -B et CENP-C. Des résultats obtenus au laboratoire ont mis en évidence le phénomène iCDR (pour interphase Centromere Damage Response) qui implique la redistribution de la coïline, fibrillarine et SMN dans ces structures centromériques déstabilisées par ICP0 mais également par des drogues ou des siRNAs dirigés contre des constituants protéiques essentiels pour la stabilité des centromères. Il a été étudié leur interdépendance dans la réponse iCDR. Il a été ainsi démontré que la redistribution de SMN aux centromères déstabilisés est dépendante de : 1) la présence de la coïline aux centromères, et 2) de son interaction, via son domaine TUDOR, avec l’histone H3 méthylée sur la lysine K79 par l’enzyme Dot1L. L’équipe suggère donc l’hypothèse que ces protéines ont pour rôle de protéger l’ADN nu se trouvant aux centromères après dégradation des histones pour empêcher les cellules de rentrer en apoptose. Ces résultats ont mené à démontrer l’implication de certaines des protéines de l’iCDR et notamment la coïline, dans une réponse apoptotique générale suite à un stress UV. Ces protéines pourraient donc faire partie d’un mécanisme de contrôle qui serait défini comme un checkpoint centromérique / Infection by Herpes Simplex Virus type 1, a major pathogenic virus in human, has been shown to cause centromere destabilization. The infected cell protein 0 (ICP0) induces centromere destabilization and lead to proteasomal-dependent degradation of the proteins of the centromeres, CENP-A, -B and CENP-C. Recent data, obtained in our laboratory, highlights the interphase Centromere Damage Response (iCDR) phenomena. This phenomena involves centromeric accumulation and redistribution of the Cajal body-associated coilin and fibrillarin as well as the Survival Motor Neuron (SMN) proteins by ICP0 or by other drugs or siRNA targeting several constitutive centromere proteins known to play a major role in centromeres stabilization. Our data shows that SMN reditribution in the destabilized centromere is dependent of : 1) centromeric presence and accumulation of the coilin, 2) its interaction, via the TUDOR domain, with the methylated (Lys K79) histone H3. This methylation occurs in the presence of the Dot-1L enzyme. We hypothesize that these proteins play a critical role in safeguarding centromeric DNA to prevent the cells from apoptosis after Histone degradation. These observations, demonstrate the implication of certain iCDR proteins, more specifically the coilin, in the apoptotic response following a UV stress. In conclusion, these proteins could be part of a safeguard mechanism considered as a centromeric checkpoint

In response to viral infection, host cells activate various antiviral defence mechanisms to inhibit virus replication. Therefore in order for a virus to replicate efficiently, it must counteract cellular antiviral defence. Promyelocytic leukemia protein (PML) is a cellular protein involved in intrinsic immunity. It inherently forms nuclear bodies (PML-NBs) that assemble at the site of viral genomes. Proteins related to epigenetic regulation are recruited to PML-NBs, and silence viral gene transcription. This study focuses on the role of two herpes simplex virus type 2 (HSV-2) proteins, ICP0 and Us3, in disrupting PML-NBs and counteracting cellular antiviral defence. En passant mutagenesis was used to create recombinant HSV-2 viruses lacking ICP0, Us3, or both ICP0 and Us3. Growth analysis of these recombinants indicates no growth defects for the ICP0Δ virus, while the Us3Δ virus grows to one log lower titres than wild type virus (WT). By contrast, the ICP0Δ virus displays a delay in PML-NB disruption, but the Us3Δ virus is as efficient as WT. However, Us3 is still important for PML-NB disruption, since the ICP0Δ/Us3Δ double mutant exhibits a greater delay than the ICP0Δ single mutant. Although PML is a mediator of the interferon (IFN) response and it was predicted that ICP0 and Us3 interfere with the IFN response through disruption of PML-NBs, my results show that only some HSV-2 Us3Δ clones are hypersensitive to the effects IFN, and others are resistant. Us3 affects more than one cellular pathway, and those cellular pathways are affected by more than one viral protein. I conclude that the activities of multiple viral proteins create a fine balance between activating cellular pathways to promote virus replication, and inhibiting cellular antiviral defence. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-01-23 10:55:16.715

Le virus Herpes Simplex de type 1 (HSV-1) possède un mode d’infection particulier dit bimodal. Il peut soit se répliquer de manière active lors d’une phase dite lytique soit migrer dans les neurones et rester en latence. Il peut réactiver pour rétablir une infection lytique. Une protéine virale majeure dans la réactivation de HSV-1 est ICP0. C’est une protéine nucléaire à activité E3 ubiquitine ligase, qui possède la particularité d’induire la dégradation par le protéasome de plusieurs protéines centromériques constitutives, ce qui provoque une déstabilisation du centromère interphasique. Mon équipe a découvert une réponse cellulaire à l’instabilité centromérique, induite par la protéine ICP0, et nommée iCDR (pour interphase Centromere Damage Response.). L’objectif général de ma thèse est de déterminer les modifications structurales que subissent les centromères endommagés par ICP0 à l’origine de l’iCDR et probablement de la réactivation. J’ai pu démontrer qu’ICP0 affectait toute la structure protéique étroitement associée aux centromères durant l’interphase. Suite à ces résultats, j’ai pu démontrer, par des analyses de digestion de chromatine à la nucléase microccocale (MNAse), que l’occupation nucléosomique de la chromatine centromérique suite à l’activité d’ICP0 était affectée de façon significative. Une étude in vivo effectuée à partir de tissus nerveux provenant de souris infectées de manière latente, a permis de démontrer une co-localisation entre les génomes HSV-1 latents et les centromères. Cette co-localisation est associée à une répression transcriptionnelle du virus. Les résultats de ma thèse montrent donc que les effets d’ICP0 sur la déstabilisation des centromères sont en relation avec un rôle de ces centromères durant la latence. Ceci suggère fortement une implication de la déstabilisation des centromères dans le processus de réactivation contrôlé par ICP0. / The Herpes Simplex type 1 (HSV-1) virus possesses a bimodal mode of infection. It can either replicates in an active way during the lytic cycle, or it can infect neurons and stay in latency. HSV-1 reactivates from latently infected neurons for re-establishing a lytic infection. A major viral protein implicated in reactivation is ICP0. It is a nuclear E3 ubiquitin-ligase, which has the particularity to induce the proteasome-mediated degradation of several constitutive centromeric proteins. This activity severely destabilizes the interphase centromere. My team has discovered a novel cellular response triggered by the estabilization of centromeres by ICP0, called iCDR (interphase Centromere Damage Response). The general aim of my thesis is to determine the centromere structural modifications induced by ICP0 that can trigger the iCDR and probably the reactivation. I was able to demonstrate that ICP0 affected the entire proteinacious structure of interphase centromeres. Following this, I showed by micrococcal nuclease (MNase) digestion approach that the nucleosomal organization of centromeric chromatin was significantly affected by ICP0. An in vivo study in nervous tissues coming from latently infected mice enabled to show a co-localization between latent HSV-1 genomes and centromeres. This co-localisation is linked to a transcriptional repression of the virus. The results of my thesis show that the destabilization of centromere by ICP0 correlates with a role of the centromeres during latency. This strongly suggests an implication of centromere destabilization in the ICP0-controlled reactivation process.

The detection of immunostimulatory DNA is well documented to occur at several cellular sites, but there is limited evidence of nuclear innate DNA sensing. Prior to this study, the detection of herpesviral DNA was thought to be restricted to the cytosol so as to limit the sensing of host DNA in the nucleus. However, given the nuclear lifecycle of these viruses, we hypothesized that viral DNA could be sensed in the nucleus of infected cells. To test this hypothesis we examined the activation of interferon regulatory factor 3 (IRF-3) in response to herpes simplex virus 1 (HSV-1) infection of primary human foreskin fibroblasts (HFF). Using a mutant defective for expression of all viral genes, we observed that the release of viral DNA into the nucleus is necessary to activate IRF-3 signaling. Furthermore, we determined this response to be dependent on nuclear-localized interferon inducible protein 16 (IFI16) and the cytoplasmic stimulator of interferon genes (STING) adaptor protein.

The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)