The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) as multifunctional oncoprotein [Elektronische Ressource] = Das Tax-Protein des humanen T-Zell-lymphotropen Virus Typ 1 (HTLV-1) als multifunktionelles Onkoprotein / vorgelegt von Karin Andrea Kreß

De
THE TAX PROTEIN OF HUMAN T CELL LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AS A MULTIFUNCTIONAL ONCOPROTEIN DAS TAX PROTEIN DES HUMANEN T-ZELL-LYMPHOTROPEN VIRUS TYP 1 (HTLV-1) ALS MULTIFUNKTIONELLES ONKOPROTEIN Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Karin Andrea Kreß aus Neustadt/Aisch Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 31. Januar 2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Bernhard Fleckenstein Zweitberichterstatter: Prof. Dr. Robert Slany Jeder Augenblick ist von unendlichem Wert. Seneca Im Gedenken an meinen ersten Doktorvater Prof. Dr. Ralph Grassmann † 1957-20084 THE TAX PROTEIN OF HUMAN T CELL LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AS A MULTIFUNCTIONAL ONCOPROTEIN Contents  I. Summary ........................................................................................................................... 6 I. Zusammenfassung ........................................................................................................... 7 II.
Publié le : samedi 1 janvier 2011
Lecture(s) : 25
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Source : D-NB.INFO/1010536974/34
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THE TAX PROTEIN OF HUMAN T CELL
LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AS
A MULTIFUNCTIONAL ONCOPROTEIN

DAS TAX PROTEIN DES HUMANEN T-ZELL-
LYMPHOTROPEN VIRUS TYP 1 (HTLV-1)
ALS MULTIFUNKTIONELLES ONKOPROTEIN






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg
zur Erlangung des
Doktorgrades Dr. rer. nat.










vorgelegt von
Karin Andrea Kreß
aus Neustadt/Aisch























Als Dissertation genehmigt
von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

















Tag der mündlichen Prüfung: 31. Januar 2011

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Bernhard Fleckenstein
Zweitberichterstatter: Prof. Dr. Robert Slany





















Jeder Augenblick ist
von unendlichem Wert.
Seneca

















Im Gedenken an meinen ersten Doktorvater
Prof. Dr. Ralph Grassmann †
1957-20084
THE TAX PROTEIN OF HUMAN T CELL LYMPHOTROPIC VIRUS TYPE 1
(HTLV-1) AS A MULTIFUNCTIONAL ONCOPROTEIN

Contents 

I. Summary ........................................................................................................................... 6

I. Zusammenfassung ........................................................................................................... 7

II. Introduction ...................................................................................................................... 8
1. Human T cell lymphotropic virus type 1 (HTLV-1) ............................................................. 8
2. Differentiation of human T lymphocytes and HTLV-1 persistence................................... 11
3. Cyclic AMP metabolism ........................................................................................................ 13
4. The tumour marker and actin-bundling protein Fascin..................................................... 14

III. Project Rationale............................................................................................................ 16

IV. Material and Methods.................................................................................................... 17
1. Cell culture.............................................................................................................................. 17
2. Nucleic acid detection............................................................................................................. 20
3. manipulation ..................................................................................................... 22
4. Transfection and transduction techniques........................................................................... 25
5. Protein detection..................................................................................................................... 26
6. Cell based assays and functional assays ............................................................................... 29
7. Bioinformatics and statistics.................................................................................................. 33

V. Results ............................................................................................................................. 34
1. The costimulatory receptors GITR, 4-1BB and 4-1BB ligand (4-1BBL) are overexpressed
on HTLV-1-transformed cells ............................................................................................... 34
2. HTLV-1-transformed cells exhibit a long-lived phenotype ................................................ 39
3. The transactivating capacity of Tax varies during T cell activation ................................. 43
4. cAMP levels are elevated in HTLV-1-transformed cells .................................................... 46
5. Tax is necessary for the maintenance of intracellular cAMP............................................. 48
6. Phosphodiesterase PDE3B is downregulated in HTLV-1-transformed cells.................... 51
7. PDE3B and cAMP concentrations inversely correlate in HTLV-1-transformed cells..... 55
8. Inhibitory histone modifications are increased at the PDE3B promoter.......................... 57
9. cAMP and PDE3B are deregulated in other lymphomas ................................................... 58
10. HTLV-1-transformed cells communicate with other lymphocytes via gap junctions, but
transfer of ectopic cAMP is not mediated by gap junctions............................................... 60 5
11. The tumour marker Fascin is specifically overexpressed in virus-transformed cells.. 63
12. Tax is necessary and sufficient to induce Fascin............................................................. 65
13. Tax-mediated induction of Fascin requires NF- κB signalling ....................................... 67
14. Fascin is induced by other viral oncogenes...................................................................... 69
15. Knockdown of Fascin reduces the invasive capacity of ATLL-derived cells................ 70

VI. Discussion ....................................................................................................................... 73
1. HTLV-1-transformed cells have a long-lived phenotype with potential relevance to
proliferation and survival.................................................................................................... 73
2. Cyclic AMP and PDE3B are deregulated in HTLV-1-transformed cells.................... 75
3. The tumour marker Fascin is strongly induced by HTLV-1/Tax through NF- κB
signals in T cells .................................................................................................................... 79

VII. References....................................................................................................................... 83

VIII. Publications....... 93

IX. Appendix ........................................................................................................................... 97

6 I. SUMMARY
I. SUMMARY
+Oncogenic transformation of CD4 T cells by human T cell lymphotropic virus type 1
(HTLV-1) is understood as the initial step to adult T cell leukaemia/lymphoma (ATLL), a
+severe neoplasm of CD4 T cells. This process is mainly initiated by perturbation of
cellular signalling by the viral Tax oncoprotein, a potent transcriptional regulator. In this
study, novel Tax functions (1) on the phenotype of HTLV-1-transformed cells, (2) on
cyclic AMP (cAMP) regulation, and (3) on expression of the tumour marker Fascin, were
identified. First, characterisation of the phenotype of HTLV-1-transformed cells revealed
that these cells share phenotypic properties with long-lived T cell populations such as
memory and regulatory T cells (T reg) with regard to the expression of characteristic
surface markers. Given the fact that murine T reg-mediated suppression employs elevated
cAMP levels as a key regulator, cAMP levels were investigated in HTLV-1-transformed
cells next. Elevated cAMP concentrations were a consistent feature of all HTLV-1-
transformed cell lines, including HTLV-1 in vitro-transformed, Tax-transformed, and
patient-derived cells. In transformed cells with conditional Tax expression, high cAMP
coincided with the presence of Tax, but was lost without it. This work shows that the
cAMP-degrading phosphodiesterase 3B (PDE3B) was specifically downregulated in
HTLV-1-transformed cells, which was independent of Tax. PDE3B transcripts and cAMP
levels were inversely correlated in HTLV-1-transformed cells and overexpression of
PDE3B led to a decrease of cAMP. Decreased expression of PDE3B was associated with
inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In search
of novel biomarkers with relevance to oncogenesis, the tumour marker and actin-
bundling protein Fascin (FSCN1) was identified and found to be specifically and strongly
+upregulated in both HTLV-1-transformed and ATLL-patient-derived CD4 T cells.
Fascin is important for migration and metastasis in various types of cancer. In this study,
a direct link between a viral oncoprotein and Fascin expression was identified, as Tax
was sufficient to induce high levels of Fascin. NF- κB signals were important for Tax-
mediated induction of Fascin in T cells providing a new mode of transcriptional
regulation of Fascin. Functionally, knockdown of Fascin reduced the invasive capacity of
ATLL-derived cells into extracellular matrix. In summary, novel functions of Tax were
identified in this work. The data show that HTLV-1-transformed cells assume biological
features of long-lived T cell populations potentially supportive to viral persistence.
Additionally, Tax-transformation and its continuous expression contribute to elevated
cAMP levels, which may be regulated through PDE3B suppression. Identification and
characterisation of the tumour marker Fascin in HTLV-1-transformed cells imply that
Fascin upregulation by Tax contributes to the development of HTLV-1-associated
pathogenesis.


I. ZUSAMMENFASSUNG 7
I. ZUSAMMENFASSUNG
+Onkogene Transformation von CD4 T-Zellen durch das Humane T-Zell-lymphotrope Virus
Typ 1 (HTLV-1) wird als initialer Schritt bei der Entwicklung der adulten T-Zell-
Leukämie/Lymphom (ATLL) verstanden. Die Transformation wird hauptsächlich durch
Störung zellulärer Signaltransduktion durch das virale Onkoprotein Tax, einem starken
transkriptionellen Regulator, initiiert. In dieser Arbeit wurden neue Funktionen von Tax in
Hinblick auf (1) den Phänotyp der HTLV-1-transformierten Zellen, (2) die Regulation von
zyklischem AMP (cAMP) und (3) die Expression des Tumormarkers Fascin identifiziert.
Zunächst zeigte die Charakterisierung des Phänotyps der HTLV-1-transformierten Zellen,
dass sich diese Zellen hinsichtlich der Expression charakteristischer Oberflächenmarker
phänotypische Eigenschaften mit langlebigen T-Zell-Populationen wie Gedächtniszellen oder
regulatorischen T-Zellen (T reg) teilen. Davon ausgehend, dass murine T reg-Zellen ihr
suppressives Potential über cAMP vermitteln, wurden cAMP-Konzentrationen in HTLV-1-
transformierten Zellen analysiert. Es konnte gezeigt werden, dass erhöhte cAMP-
Konzentrationen durchgängige Eigenschaft aller HTLV-1-transformierter Zellen,
einschließlich in vitro-transformierter, Tax-transformierter und Patienten-abgeleiteter Zellen,
waren. In konditionell Tax-exprimierenden Zellen koinzidierten hohe cAMP-Spiegel mit der
Expression von Tax, wohingegen sie ohne Tax sanken. In HTLV-1-transformierten Zellen
war die Expression der cAMP-spaltenden Phosphodiesterase PDE3B, unabhängig von Tax,
spezifisch herunter reguliert. PDE3B Transkripte und cAMP-Konzentrationen korrelierten
invers in HTLV-1-transformierten Zellen und Überexpression von PDE3B führte zu einer
Abnahme von cAMP. Die verminderte PDE3B Expression in HTLV-1-transformierten
Zellen war mit inhibitorischen Histon-Modifikationen am PDE3B Promoter und dem
PDE3B-Lokus assoziiert. Auf der Suche nach einem neuen Biomarker mit
onkogenetischer Relevanz, konnte spezifische und starke Expression des Tumormarkers
und Aktin-bündelnden Proteins Fascin in HTLV-1-transformierten und ATLL-
+abgeleiteten CD4 T-Zellen identifiziert werden. Bei zahlreichen Krebsarten ist Fascin
für Migration und Metastasierung wichtig. In dieser Arbeit konnte erstmals gezeigt
werden, dass ein direkter Zusammenhang zwischen der Expression eines viralen
Onkoproteins und der Expression von Fascin hergestellt werden kann, da Tax
ausreichend war, die Expression von Fascin stark zu induzieren. NF- κB Signale waren
für die Tax-vermittelte Fascin-Induktion in T-Zellen nötig, womit ein bislang
unbekannter Mechanismus der Fascin-Regulation aufgedeckt wurde. Funktionelle
Analysen zeigten, dass Herunterregulation von Fascin zu verminderter Invasion ATLL-
abgeleiteter Zellen in extrazelluläre Matrix führte. Zusammenfassend konnten in dieser
Arbeit neue Tax-Funktionen identifiziert werden. Die Daten zeigen, dass HTLV-1-
transformierte Zellen biologische Eigenschaften langlebiger T-Zell-Populationen
aufweisen, was die virale Persistenz begünstigen könnte. Zudem tragen Transformation
durch Tax sowie kontinuierliche Tax-Expression zu erhöhten cAMP-Spiegeln bei, die
zudem über Suppression von PDE3B reguliert sein können. Die Identifizierung und
Charakterisierung des Tumormarkers Fascin in HTLV-1-transformierten Zellen lassen
vermuten, dass die Tax-vermittelte Induktion von Fascin zur Entwicklung HTLV-1-
assoziierter Pathogenität beiträgt. 8 II. INTRODUCTION
II. INTRODUCTION

1. Human T cell lymphotropic virus type 1 (HTLV-1)

Human T cell lymphotropic virus type 1 (HTLV-1), a delta-retrovirus, is the causative agent
+of a severe and fatal lymphoproliferative disorder of CD4 T cells, adult T cell
leukaemia/lymphoma (ATLL), and of a neurodegenerative, inflammatory disease, HTLV-1-
associated myelopathy/ tropical spastic paraparesis (HAM/TSP) (Poiesz et al., 1980; Yoshida
et al., 1982; Yoshida et al., 1984; Gessain et al., 1985; Osame et al., 1986). Both diseases can
develop as a consequence of prolonged viral persistence in T cells after a clinical latency of
decades. The risk of developing ATLL among virus carriers is estimated to be 6.6% for males
and 2.1% for females, while 1-4% of the infected individuals may develop HAM/TSP
(Verdonck et al., 2007; Matsuoka and Jeang, 2007). HTLV-1 has developed a unique strategy
for lifelong persistence in the presence of an active immune system. This is achieved by
replication of the virus mainly in its provirus form, stimulation of cell division by the virus,
and, as a consequence, clonal amplification of infected cells. Virus-infected clones are
detectable and can persist over many years even in non-leukaemic individuals (Bangham and
Osame, 2005).

It has been estimated that 15-20 million people are infected with HTLV-1 world-wide
although the exact number of HTLV-1-carriers is unknown (Hlela et al., 2009). Endemic
regions for HTLV-1 infections are Japan, Melanesia, parts of Sub-Saharan Africa, the
Caribbean, South America, central parts of Australia, and the Middle East (Cooper et al.,
2009; Proietti et al., 2005). The prevalence is very high in some regions of southern Japan
where more than 10% of the population are infected (Verdonck et al., 2007), while in other
endemic regions the prevalence lies between 1-5%. In Europe, immigration rates from
endemic regions are associated with the prevalence of virus infection in the respective
countries. While HTLV-1 prevalence is up to 1% in the United Kingdom and France, it is
estimated that 6000 people are infected in Germany (Proietti et al., 2005).

+In the host, CD4 T cells are the preferential target for HTLV-1 infection. In contrast to HIV,
+cell-free infection of CD4 T cells with HTLV-1 is very inefficient and free virions can hardly
be detected in infected individuals. Therefore, transmission of the virus occurs via body fluids
such as blood, semen and breast milk (Matsuoka and Jeang, 2007). Cell-cell contact is a II. INTRODUCTION 9
prerequisite for efficient virus propagation involving polarisation of the cytoskeleton of the
infected cell and establishment of a so called virological synapse with the target cell (Igakura
et al., 2003; Nejmeddine et al., 2009). Recent studies have shown that also viral biofilms on
the surface of infected cells may be responsible for virus transmission (Pais-Correia et al.,
2010). Given the fact that HTLV-1 can infect many different cell types in vitro, the widely
expressed glucose transporter GLUT-1 has initially been proposed to be the HTLV-1-receptor
(Manel et al., 2003). Up to now, neuropilin-1 and heparan sulfate proteoglycans have been
described to be important for binding of virions to the target cell (Lambert et al., 2009). New
studies have shown that cell-free infection of dendritic cells mediates efficient infection and
+transformation of CD4 T cells in vitro (Jones et al., 2008).

Upon infection, HTLV-1 integrates into the host cell genome and is mainly maintained in its
provirus form (9.1 kb) which is flanked by long terminal repeats (LTR) in both the 5’ and 3’
region. In addition to structural proteins common for retroviruses, protease, and reverse
transcriptase, HTLV-1 encodes accessory and regulatory proteins (Matsuoka and Jeang,
2007). While the accessory proteins p12, p30, p13 and HBZ are important for viral infectivity
and replication, they are not required for lymphocyte immortalization (Mesnard et al., 2006;
Nicot et al., 2005). The regulatory proteins Tax and Rex are both essential for viral replication
(Kashanchi and Brady, 2005). While Tax strongly enhances viral mRNA synthesis by
transactivating the HTLV-1-LTR promoter, Rex controls the synthesis of the structural
proteins on a posttranscriptional level (Bogerd et al., 1992; Gröne et al., 1996).

Tax confers the transforming properties on HTLV-1 as it can immortalise
T lymphocytes (Grassmann et al., 1989; Grassmann et al., 1992; Grassmann et al., 2005), and
induce leukaemia in transgenic mice (Hasegawa et al., 2006). Several Tax functions may
contribute to its transforming capacity, including interference with cell cycle check points,
tumour suppressors and DNA repair. To promote cell proliferation, Tax can stimulate the
expression of cellular proteins controlling proliferation and survival (Grassmann et al., 2005;
Boxus et al., 2008). In particular, Tax-mediated modulation of cellular gene expression may
explain the resistance of HTLV-1-positive cells to various proapoptotic stimuli (Copeland et
al., 1994). Tax is capable of stimulating cellular transcription by interacting with various
signalling pathways such as both the canonical and non-canonical nuclear factor kappa B
(NF- κB) pathways, the former by binding IKK γ, a component of the I κB kinase (IKK)
complex (Grassmann et al., 2005; Matsuoka and Jeang, 2007). This binding triggers the 10 II. INTRODUCTION
phosphorylation of IKK α and IKK β, which form the IKK complex with IKK γ. Activation of
the IKK complex leads to phosphorylation of the inhibitor of NF- κB (I κB), which is
thereafter ubiquitinated and proteasomally degradated. This process frees NF- κB subunits
such as e.g. p50 and RelA which can then translocate into the nucleus to bind respective NF-
κB-responsive elements in promoters and induce or support transcription of cellular genes
(Figure 1). Activation of the non-canonical NF- κB pathway is mediated by Tax’s capacity to
bind IKK γ and p100 in an IKK α-IKK γ-p100 complex, which leads to an IKK α-dependent
processing of the NF-κB p100 precursor protein to its active form p52 (Peloponese et al.,
2006). Transactivation of another set of cellular promoters is mediated by Tax via direct
contact with the transcriptional activators cAMP response element-binding protein (CREB),
serum response factor (SRF) and with the coactivators p300/CREB-binding protein
(p300/CBP; Fujii et al., 1995; Kashanchi and Brady, 2005).
vviirus replirus repliccaattionion
RelAp50NF- κB
TaxI κB host cell
IKK γIKKγ transcription
IKKα IKK βIKK
prolifprolifproliferation,
p50p50 RelARelANFNF-- κκBB trtrtrananansformasformasformatititiononon
PI κB P
Ub apoptosis control
Ub Ub
nunuclcleueuss +1+1
p50p50 ReRelAlA
tartartargggetetet genegenegene
NF- κB

Figure 1. Model of Tax-functions and Tax-mediated NF- κB activation. On the one hand, Tax stimulates
virus replication, on the other hand, Tax interferes with cellular transcription. Many of the described effects of
Tax on host cell transcription are mediated by activation of the canonical NF- κB pathway. Tax binds IKK γ,
thereby activating the IKK complex which leads to phosphorylation of I κB followed by its ubiquitination (Ub)
and proteasomal degradation. p50/RelA heterodimers can then enter the nucleus and bind to NF- κB responsive
promoter elements leading to target gene transcription.

It is believed that an accumulation of genetic alterations over decades leads to the
establishment of malignant T cell clones and ATLL. Clinically, ATLL is characterized by
increased numbers of lymphocytes with multi-lobulated nuclei (flower cells). ATLL is
accompanied by hypercalcaemia, infiltrations into various organs, including skin, and
opportunistic infections (e.g. Strongyloides stercoralis) (Verdonck et al., 2007). Thus far,
ATLL cannot be cured. Therapy of ATLL involves chemotherapy (CHOP) and combined

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