Control of Kaposi's Sarcoma-Associated Herpesvirus Reactivation Induced by Multiple Signals

The ability to control cellular functions can bring about many developments in basic biological research and its applications. The presence of multiple signals, internal as well as externally imposed, introduces several challenges for controlling cellular functions. Additionally the lack of clear understanding of the cellular signaling network limits our ability to infer the responses to a number of signals. This work investigates the control of Kaposi's sarcoma-associated herpesvirus reactivation upon treatment with a combination of multiple signals. We utilize mathematical model-based as well as experiment-based approaches to achieve the desired goals of maximizing virus reactivation. The results show that appropriately selected control signals can induce virus lytic gene expression about ten folds higher than a single drug; these results were validated by comparing the results of the two approaches, and experimentally using multiple assays. Additionally, we have quantitatively analyzed potential interactions between the used combinations of drugs. Some of these interactions were consistent with existing literature, and new interactions emerged and warrant further studies. The work presents a general method that can be used to quantitatively and systematically study multi-signal induced responses. It enables optimization of combinations to achieve desired responses. It also allows identifying critical nodes mediating the multi-signal induced responses. The concept and the approach used in this work will be directly applicable to other diseases such as AIDS and cancer

Virus-host cell interplay in the pathogenesis of Kaposi’s sarcoma herpesvirus

Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of three types of malignancies: Kaposi s sarcoma (KS), multicentric Castleman disease (MCD), and primary effusion lymphoma (PEL). Infection by KSHV displays two different phases: latent and lytic replication phase. By using an unbiased gain-of-function human kinome cDNA screen, the work in this thesis identified two kinases, Pim-1 and -3, to be involved in KSHV reactivation. Ectopic expression of Pim-1 and Pim-3 induced viral lytic replication leading to production of progeny viruses, whereas depletion of Pim-1 and Pim-3 by RNA interference inhibited the induction of lytic reactivation. Pim-1 and -3 was shown to regulate viral reactivation by phosphorylation of LANA, which abolished the LANA-mediated repression of lytic transcription. In this thesis project we developed a novel three-dimensional (3D) cell model to identify novel oncogenic processes involved in the KSHV-induced endothelial cells (EC) transformation. The results demonstrate that KSHV induces transcriptional reprogramming of primary lymphatic endothelial cells (LECs) to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT), a process implicated in promoting tumor growth and cell invasiveness. Two viral gene products, vFLIP and vGPCR, were found to trigger Notch signaling and lead to the KSHV-induced EndMT. Our data further identifies a membrane associated matrix metalloproteinase MT1-MMP as a previously unrecognized regulator downstream of Notch to induce EndMT. 3D KSHV-infected LECs (K-LECs) transcriptome showed significant up-regulation of invasion related genes that were found co-regulated in 3D K-LECs and KS biopsies. The results further demonstrate that 3D culture provides a permissive microenvironment for continuous viral replication and persistence. To summarize, this PhD thesis greatly expands the understanding of host signaling pathways involved in KSHV reactivation and oncogenesis. Furthermore, this thesis provides novel information about cellular targets for pharmacological control in KS and other virus-associated cancers.Ei saatavill

Cell and Receptor Tropism of γ2-Herpesviruses

Das Kaposi-Sarkom-Herpesvirus (KSHV), das einzige Rhadino- oder γ2-Herpesvirus im Menschen, ist mit dem Kaposi Sarkom (KS) und zwei B-Zell-Lymphomen, dem primären Effusionslymphom und der multizentrischen Castleman-Erkrankung, assoziiert. Da der Mechanismus der Primärinfektion, die Ausbreitung im Wirt und die Entstehung Virus-assoziierter Erkrankungen zumindest teilweise durch den viralen Zell- und Gewebetropismus bestimmt werden, ist es entscheidend den Beitrag spezifischer Interaktionen zwischen viralen Glykoproteinen und zellulären Rezeptoren zur Infektion unterschiedlicher Zelltypen zu verstehen. Der Fokus dieser Arbeit liegt hierbei auf der Familie der Eph Rezeptortyrosinkinasen, die an der Infektion diverser adhärenter Zelllinien beteiligt sind. Indes der KSHV gH/gL Glykoproteinkomplex die höchste Affinität für EphA2 aufweist, wurden weitere Eph Rezeptoren des A-Typs als Bindepartner von KSHV beschrieben. Obwohl die gH/gL-Eph Interaktion Gegenstand verschiedener Studien war, gibt es weiterhin unbeantwortete Fragen bezüglich der Rolle von Eph Rezeptoren für den KSHV Tropismus und KSHV-assoziierte Pathologien. Unsere Zielsetzung lag demnach in der Identifikation von Aminosäuren in KSHV gH/gL, welche essentiell für die Eph-Interaktion sind, in der Konstruktion von rekombinanten Viren mit Mutationen in den identifizierten Aminosäuren, sowie in der Charakterisierung der Eph Rezeptor Nutzung auf BJAB Zellen, einem Modell für die Zell-assoziierte KSHV Infektion von B-Zellen. Analog zu KSHV interagiert das verwandte Rhesusaffen Rhadinovirus (RRV) mit Eph Rezeptoren, zeigt hierbei jedoch abweichende Affinitäten für einzelne Mitglieder der Eph Familie. Durch Vergleiche zwischen beiden Viren konnten wir konservierte Aminosäuren in der N-terminalen Region von gH identifizieren, welche essentiell für die gH/gL-Eph Interaktion sind. Mutation dieser Aminosäuren in KSHV und RRV verhinderte die Interaktion mit Eph Rezeptoren und ermöglichte uns die Analyse des Zelltypspezifischen Beitrags von Eph Rezeptoren zur KSHV und RRV Infektion. Dieses System kam außerdem in unserer Studie zum Einsatz, die zwei weitere Eph Rezeptoren des A-Typs als funktionelle KSHV und RRV Rezeptoren auf BJAB Zellen charakterisierte. Die Funktion von EphA5 und EphA7 in der KSHV Zell-Zell Übertragung, sowie in der zellfreien RRV Infektion wurde durch Knockout mit Hilfe der CRISPR/Cas9 Methode nachgewiesen. Ferner beschäftigten wir uns mit der Frage, ob weitere, Eph-unabhängige Interaktionspartner des gH/gL Komplexes die Infektion verschiedener Zelltypen durch Rhadinoviren beeinflussen. Wir identifizierten die Plexin domain containing Proteine 1 und 2 (Plxdc1/2) als spezifische Interaktionspartner von RRV im Gegensatz zu KSHV und beschrieben ein essentielles Plxdc-Interaktionsmotiv nahe dem Eph-Interaktionsmotiv in RRV gH. Die Plxdc-Rezeptorfunktion wurde mittels lentiviraler Überexpression sowie mit Hilfe von zur Plxdc-Bindung unfähigen RRV Deletionsmutanten nachgewiesen. Zusammengenommen beschreiben die vorliegenden Studien weitere Eph Rezeptoren vom A-Typ als funktionelle Rezeptoren für KSHV und RRV, charakterisieren die Funktion einer neuen Rezeptorfamilie für die RRV Infektion und verdeutlichen die Bedeutung der N-terminalen Region des rhadinoviralen gHs als konservierte Rezeptorbindedomäne, welche die Interaktion von KSHV und RRV mit Eph Rezeptoren und die Interaktion von RRV mit Plxdc Rezeptoren vermittelt.Kaposi’s sarcoma-associated herpesvirus (KSHV), the only rhadino- or γ2-herpesvirus of humans, is associated with Kaposi’s sarcoma (KS) and two B cell proliferative malignancies, primary effusion lymphoma (PEL) and a variant of multicentric Castleman’s disease (MCD). As routes of primary infection, dissemination through the host, and development of virus-associated pathologies are at least partially shaped by viral cell and tissue tropism, it is crucial to understand the contribution of distinct viral glycoproteins and cellular receptor interactions to cell type-specific infection. In this context, the present thesis focuses on members of the Eph family of receptor tyrosine kinases, which were shown to play a role in KSHV infection of various adherent cell lines. While the KSHV gH/gL glycoprotein complex exhibits the highest affinity for EphA2, additional A-type Ephs have been described as interaction partners of KSHV. Even though the gH/gL-Eph interaction was subject of various studies, key questions regarding the role of Ephs in KSHV tropism and pathology remained unanswered. We therefore aimed to identify amino acid residues on the KSHV gH/gL complex that critically mediate the Eph interaction, create Eph detargeted virus recombinants mutated in the identified amino acid residues, and characterize the Eph usage on BJAB cells, as model for cell-to-cell transmission of KSHV into B cells. Similar to KSHV, the related rhesus monkey rhadinovirus (RRV) interacts with Eph receptors while exhibiting differing affinities for individual Eph family members. Comparison of the two viruses allowed us to identify conserved amino acid residues in the N-terminal domain of gH which are critical for the gH/gL-Eph interaction. Mutation of these amino acids in KSHV and RRV recombinants abrogated the viral interaction with Eph receptors and allowed us to analyze the cell type-specific contribution of the Eph family to KSHV and RRV infection. This system was also employed in our second study which identified two additional A-type Ephs as functional KSHV and RRV receptors on BJAB cells. The role of EphA5 and EphA7 in KSHV cell-to-cell transmission and RRV cell-free infection was demonstrated using CRISPR/Cas9-mediated knockout. We furthermore addressed the question whether additional cellular, Eph-independent interaction partners of the gH/gL complex shape the rhadinoviral infection of different cell types. We identified the Plexin domain containing proteins 1 and 2 (Plxdc1/2) as specific interactors for RRV, but not KSHV, and characterized a crucial Plxdc-interaction motif in close proximity to the identified Eph-interacting residues on RRV gH. Receptor function of Plxdcs was demonstrated by lentiviral overexpression of Plxdc1 and 2 in target cells and a Plxdc-detargeted RRV deletion mutant. Collectively, the present studies identify additional A-type Eph members as functional receptors for KSHV and RRV, characterize the role of a novel family of gH/gL-interacting proteins for RRV infection, and underline the importance of the N-terminal domain of the rhadinoviral gH as conserved receptor-binding domain, which mediates the interaction of KSHV and RRV with Eph receptors and the independent interaction of RRV with Plxdc family members

Oncogenic KSHV induces ALT to facilitate break-induced viral genome replication

Infection with Kaposi’s Sarcoma herpesvirus (KSHV) has been linked to multiple cancers including Kaposi’s sarcoma (KS), Primary effusion lymphoma (PEL), and Multicentric Castleman’s disease (MCD). One of the hallmarks of transformed cancer cells is the activity of telomere maintenance mechanisms. The present study uncovers the onset of Alternative lengthening of telomeres (ALT) in response to KSHV infection from a proteomic screen of telomere-associated DNA damage response proteins by Proteomics of isolated chromatin fragments (PICh). In several initially telomerase+ cell lines, features of ALT activation are present in response to KSHV infection including increased telomere sister-chromatid exchanges, C-circles, telomere clustering in interphase, ALT-associated proteins at telomere clusters, and telomere fragility. Binding of shelterin to telomeric DNA was increased upon infection with KSHV. Interestingly, cells which are latently infected with KSHV are dependent on ALT factors for their efficient proliferation in clonogenic assays and such factors may be essential for the maintenance of viral episomes in infected cells as shown by episome qPCR. Moreover, preliminary experiments by ChIP indicate an increase in heterochromatin marks at telomeres upon infection, similar to the marks documented on the KSHV episome. Analysis of primary tumour material from 8 KS and 7 PEL patients suggests that this not limited to the in vitro systems considered in this study. Taken together, this work demonstrates for the first time the capability of latent KSHV to trigger telomere maintenance via ALT and suggests a model in which KSHV episomes are replicated in tandem with telomeres by BIR in such cells. This provides a unique susceptibility of KSHV infected cancer cells to inhibition of ALT, which may be utilized for the trial of future treatment for KSHV-associated cancers.Open Acces

Cell plasticity in cancer : Cues from virus-host interactions

Human tumorigenesis is a process in which a normal cell needs to acquire multiple characteristics to become malignant and metastatic. In short, these so called cancer hallmarks include increased proliferation and cell survival, as well as the ability to invade into the surroundings, induce angiogenesis, and finally metastasize to distant sites. These traits are regulated in a variety of different ways. However, some embryonic signaling pathways, including the Notch pathway, are able to regulate many of these processes. Furthermore, it has been shown that these signaling pathways can be deregulated in cancer, and that their untimely activation can lead to malignancies. In this study, Kaposi's sarcoma herpesvirus (KSHV) associated malignancies, namely Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), as well as melanoma have been used as model cancers. In all these malignancies, the tumor cells show alterations in cell identity and lineage marker expression, i.e. signs of cellular de- or transdifferentiation. In addition, the Notch pathway has been shown to be overly active in all of them. Thus, this thesis has focused on how the pro-tumorigenic traits are affected by cell plasticity and reprogramming in these cancers, and how the signaling pathways leading to these phenotypes, most notably Notch, are in turn regulated. Firstly, the results show that in vivo expression of a KSHV oncogene, viral (v-)cyclin, leads to activation of Notch signaling through Notch3 upregulation as well as fine-tuning of the NF-κB pathway through Cdk6 mediated phosphorylation. These changes in turn lead to defects in T-lymphocyte differentiation and immune functions, as well as to the development of T-cell lymphomas. Secondly, this work demonstrates that KSHV infection in primary lymphatic endothelial cells (LECs) in three dimensional (3D) cell culture model leads to activation of a morphogenic process, endothelial to mesenchymal transition (EndMT), and increased invasiveness through activation of the Notch pathway and matrix metalloproteinase MT1-MMP. Lastly, the data show that the changes in cell plasticity contributing to tumorigenic traits are not confined to virally induced cancers. Melanoma cell interaction with LECs leads to activation of the Notch pathway and increased adhesive, invasive, and metastatic properties of the tumor cells. In conclusion, the results show that regulation of cell plasticity through the Notch pathway takes place in different types of cancers, and it can affect several steps of tumorigenesis. A thorough and comprehensive understanding of the processes discovered herein may help develop better and more efficient treatments for these largely fatal malignancies.Monien normaalin solun ominaisuuksien tulee muuttua, ennen kuin solu muuntuu syöpäsoluksi ja pystyy leviämään elimistössä. Solun täytyy muun muassa pystyä jakautumaan hallitsemattomasti, tunkeutumaan ympäristöönsä, erittämään veri- ja imusuonien kasvuun vaikuttavia tekijöitä sekä lopulta pystyä hyödyntämään veri- ja imusuonistoa levitäkseen ympäriä kehoa. Syöpäsolujen eri ominaisuuksia säätelevät tyypillisesti eri signalointireitit, mutta eräät sikiönkehityksen aikana aktiivisesti toimivat reitit, kuten Notch-signalointi, voivat vaikuttaa moniin syöpäsolun ominaisuuksiin. Tällaisten signalointireittien yliaktiivisuus onkin liitetty syövän syntyyn ja leviämiseen. Tässä tutkimuksessa on käytetty malleina Kaposin sarkoomaan liittyvän herpesviruksen (KSHV) aiheuttamia syöpiä, Kaposin sarkoomaa (KS) ja primaaria efuusiolymfoomaa (PEL), sekä melanoomaa. Näissä kaikissa syövissä on havaittavissa, että syöpäsolujen soluidentiteetti on heterogeeninen, ja että syöpäsolut pystyvät ohjelmoitumaan uudelleen kasvuolosuhteidensa mukaan. Näille syöville on yhteistä myös, että Notch-signalointireitti on aktivoitunut. Tutkimukseni aiheena oli solujen muovautuvuus- ja ohjelmoitumiskyvyn vaikutukset syövän syntyyn ja leviämiseen ja näiden prosessien säätely. Työni ensimmäisessä osassa osoitin, kuinka KSHV:n onkogeenin v-sykliinin ilmentyminen hiiressä johtaa Notch- ja NF-κB- signalointireittien aktivoitumiseen ja sitä kautta T-solujen erilaistumisen ja toiminnan häiriöön sekä T-solulymfooman kehittymiseen. Seuraavaksi näytin, kuinka KSHV-infektio imusuonten seinämän soluissa johtaa Notch-signaloinnin aktivoitumiseen ja solujen uudelleenohjelmoitumiseen mesenkyymisolujen kaltaisiksi, jolloin ne pystyvät tehokkaammin tunkeutumaan ympäristöönsä. Lopuksi osoitin, että edellä kuvatut mekanismit eivät ole aktiivisia ainoastaan syöpävirusten aiheuttamissa kasvaimissa, vaan että myös melanoomasolujen interaktio imusuonten solujen kanssa aktivoi Notch-signalointia ja johtaa syöpäsolujen lisääntyneeseen adheesio-, invaasio- ja metastasoimiskykyyn. Notch-signalointi ja syöpäsolujen muovautumis- ja uudelleenohjelmoitumiskyky säätelevät siis monentyyppisten syöpien kehittymistä ja leviämistä. Näiden prosessien perusteellinen tuntemus mahdollistaa parempien ja tehokkaampien hoitojen kehittämisen näitä huonoennusteisia syöpiä vastaan

CHARACTERIZATION OF THE ROLE OF CELLULAR SIGNALING PATHWAYS ON THE PATHOGENESIS OF KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multiple malignancies, including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). KSHV belongs to the gammaherpesvirus family, and it contains a large double-stranded DNA genome encoding a plethora of viral genes and non-coding RNAs. Understanding the interplay between the virus and the host is crucial for identifying therapeutical targets. KSHV viral interleukin-6 (vIL-6) is a viral homolog of human IL-6. This viral protein is expressed in KSHV-associated malignancies and is known to deregulate signaling pathways. We found that vIL-6 induces the expression of integrin β3 (ITGB3), resulting in the surface expression of integrin αVβ3. We discovered that JAK/STAT signaling pathway is necessary for the induction of ITGB3. Moreover, we found that vIL-6 can induce the expression of ITGB3 in a paracrine manner which is physiologically relevant as vIL-6 can be detected circulating in patients suffering from KSHV-associated malignancies. Importantly, depletion of ITGB3 hindered the ability of vIL-6 to promote angiogenesis. Therefore, we identified ITGB3 as a potential therapeutic target for KSHV-associated malignancies. The PI3K/Akt/mTOR pathway has been shown to contribute to cell survival and proliferation and is constitutively activated by some KSHV proteins. Unsurprisingly, the pathway is commonly deregulated in malignancies including non-Hodgkin lymphomas (NHL). Given the importance of the pathway for cell survival, we reasoned that inhibiting the network with novel inhibitors should be a viable therapeutical approach. Miransertib and MK-4440 are small molecules that effectively inhibit Akt and have entered clinical development. Using in vitro and in vivo models of NHL, we explored targeting the Akt alone or in combination with the mTORC1 inhibitor, sirolimus. We found that the combination of miransertib and sirolimus synergistically reduced cell proliferation and tumor growth in NHL, including the KSHV-associated PEL. These data suggest that targeting the PI3K/Akt/mTOR pathway with Akt inhibitors such as miransertib in combination with mTOR inhibitors serves as a broadly applicable therapeutic in NHL. In sum, with this dissertation, I undertook two different routes to better understand the importance of signaling pathways in KSHV-associated malignancies, with implications beyond viral cancers.Doctor of Philosoph

The role of host cell factors in the lytic reactivation of Kaposi’s sarcoma-associated herpesvirus from latency

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) has two stages to its life cycle; latency and lytic replication. KSHV latent infection is associated with the B-cell tumour, Primary Effusion Lymphoma (PEL). During latency the viral episome is maintained, few viral genes are expressed and no infectious virions are produced. The switch between phases is controlled by the viral transcription factor, RTA encoded by ORF50. Lytic replication, which results in the production of progeny virions, can be triggered by a variety of causes suggesting KSHV has the ability to reactivate in response to a multitude of scenarios. However, many of the stimuli described, to date, have no clear physiological relevance. This thesis investigates how cellular transcription factors can induce the KSHV lytic cycle. Firstly, a model system for monitoring KSHV lytic replication is developed and characterised. This system is then employed to demonstrate how the cellular transcription factor XBP-1s is able to induce KSHV reactivation. XBP-1 is responsible for the terminal differentiation of B-cells into plasma cells (PCs) and is a major regulator of the unfolded protein response (UPR). PEL do not express the transcription factor X-box binding protein-1 (XBP-1). When spliced active XBP-1s is supplied, PEL cells differentiate towards a PC and induce KSHV lytic replication. B-cell terminal differentiation is therefore a physiological trigger of KSHV lytic reactivation. To understand the role of host cell factors in this interaction further, the gene expression changes that occur when XBP-1s is supplied to PEL are also examined. Finally, we investigate the specific roles of XBP-1s and HIF-1α in the KSHV reactivation seen in response to hypoxia. We conclude that both B-cell differentiation and hypoxia are physiologically relevant triggers for KSHV lytic cycle induction, highlighting some of the many complex interactions between the virus and its host

Utilising Omics Approaches to Understand Kaposi's Sarcoma-associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus associated with a number of malignancies, including Kaposi’s sarcoma. Similar to all herpesviruses, KSHV establishes either latent or lytic infections in host cells. The latent stage involves minimal viral gene expression, enabling the virus to remain dormant, maintaining genome integrity and enabling viral persistence. Conversely, lytic replication is characterised by the expression of a highly regulated and coordinated cascade of viral gene expression, ultimately resulting in the production of new mature, infectious virions. Importantly, lytic replication is necessary for the development and spread of Kaposi’s sarcoma. State of the art high throughput, high resolution “omics” approaches, such as mass spectrometry-based quantitative proteomics and next generation sequencing-based transcriptomics, are rapidly becoming the techniques of choice for discovering novel biological phenomena due, in part, to their sensitivity and the ability to repeatedly ask new questions of an existing dataset. Herein, three high throughput, high resolution approaches, namely SILAC-based quantitative proteomics, miRNA sequencing and mRNA sequencing are employed in an attempt to identify and characterise novel interactions between KSHV and the host cell. Utilising quantitative proteomics, the essential host cell splicing factor Prp19 is identified as a novel interacting partner of the lytic KSHV ORF57 protein in subnuclear bodies. This interaction, surprisingly, does not contribute to viral mRNA maturation, but instead has implications for the cellular DNA damage response, appearing to limit the effectiveness of this important pathway, possibly to reduce any negative effects on viral replication. Proteomic analyses also highlighted a link between KSHV lytic replication and host miRNA biogenesis pathways. Through the application of miRNA sequencing, two host miRNAs, namely miR-151a-5p and miR-365a-3p, were found to be dysregulated during KSHV infection. Importantly, neither of these miRNAs appear to represent a host antiviral response. Instead, cellular target mRNAs are identified for miR-365a-3p, through the use of mRNA sequencing. These targets, termed DOCK5 and PRUNE2 are rapidly degraded during KSHV lytic replication via the viral-mediated upregulation of miR-365a-3p expression. Subsequent analysis of DOCK5 function during lytic replication suggests this interaction may promote viral egress. The data presented herein sheds light on previously unidentified mechanisms employed by KSHV to hijack the host cell, and may aid in the development of novel therapeutics against this important pathogen

Rewiring of signaling pathways by HCMV-encoded GPCRs

Smit, M.J. [Promotor]Siderius, M.H. [Copromotor