Molecular characterization of UAP56- and pUL69-protein interactions with respect to their impact on nuclear mRNA export during human cytomegalovirus infection [Elektronische Ressource] / vorgelegt von Marco Thomas

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Molecular characterization of UAP56- and pUL69-protein interactions with respect to their impact on nuclear mRNA export during human cytomegalovirus infection Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Marco Thomas aus Halle/Saale Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 27.05.2009 Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch Erstberichterstatter: Prof. Dr. R. Slany Zweitberichterstatter: Prof. Dr. M. Marschall Table of contents I Table of contents I. Summary ..............................................................................................1 I. Zusammenfassung..............................................................................2 II. Introduction...................3 III. Objectives ..........................................................................................14 IV. Materials and Methods .....................................................................15 4.1. Biological Materials............................................................................................ 15 4.1.1. Bacteria ..............................................................................
Publié le : jeudi 1 janvier 2009
Lecture(s) : 21
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2009/1354/PDF/MARCOTHOMASDISSERTATION.PDF
Nombre de pages : 121
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Molecular characterization of UAP56- and pUL69-protein
interactions with respect to their impact on nuclear mRNA export
during human cytomegalovirus infection












Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades








vorgelegt von
Marco Thomas
aus Halle/Saale

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

































Tag der mündlichen Prüfung: 27.05.2009

Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch

Erstberichterstatter: Prof. Dr. R. Slany

Zweitberichterstatter: Prof. Dr. M. Marschall

Table of contents I

Table of contents

I. Summary ..............................................................................................1
I. Zusammenfassung..............................................................................2
II. Introduction...................3
III. Objectives ..........................................................................................14
IV. Materials and Methods .....................................................................15
4.1. Biological Materials............................................................................................ 15
4.1.1. Bacteria ................................................................................................................15
4.1.2. Yeast ............................................................15
4.1.3. Eukaryotic cell cultures .............................15
4.1.4. Virus strains................................................15
4.2. Nucleic acids....................................................................................................... 15
4.2.1. Oligonucleotides .................................................................................................15
4.2.1.1. Primers for PCR cloning 16
4.2.1.2. Sequencing primers 18
4.2.1.3. Probe for FISH 18
4.2.1.4. shRNAs 18
4.2.2. Vectors and plasmids .........................................................................................19
4.2.2.1. Vectors and vector systems 19
4.2.2.2. Ready-to-use DNA constructs 20
4.2.2.3. Newly generated plasmids 22
4.2.3. Additional nucleic acids ............................24
4.3. Antibodies and enzymes ................................................................................... 24
4.3.1. Monoclonal mouse antibodies ...........................................................................24
4.3.2. Polyclonal rabbit antibodies........................................................25
4.3.3. Secondary antibodies .........................................................................................25
4.3.4. Enzymes ...............................26
4.4. Media and chemicals ......................................................................................... 26
4.4.1. Media ....................................................................................................................26
4.4.1.1. Bacterial media 26
4.4.1.2. Yeast media 26
4.4.1.3. Mammalian cell culture media 27
4.4.2. Standard buffers and solutions..........................................................................27
4.4.3. Chemicals................................................28 Table of contents II

4.5. Methods............................................................................................................... 28
4.5.1. Standard molecular biology techniques ...........................................................28
4.5.2. In vitro mutagenesis............................................................................................29
4.5.3. Yeast two-hybrid analyses ..29
4.5.3.1. Small-scale transformation of yeast 29
4.5.3.2. Filter-lift test 30
4.5.4. Eukaryotic cell culture techniques ....................................................................30
4.5.5. Transfection and infection...........................................................30
4.5.5.1. Transfection 30
4.5.5.2. Infection 31
4.5.6. Generation of retrovirally transduced cell lines ...............................................31
4.5.6.1. Generation of infectious retroviruses 31
4.5.6.2. Retroviral transduction and selection of stably transduced cells 32
4.5.7. Indirect immunofluorescence analysis .............................................................32
4.5.8. Fluorescence in situ hybridisation (FISH)..................................33
4.5.9. Heterokaryon assay ............................................................................................33
4.5.10. Nuclear mRNA export assay for pUL69.......................34
4.5.11. Purification and analysis of proteins and protein-protein interactions .........34
4.5.11.1. Purification of FLAG-fusion proteins expressed in mammalian cells 34
4.5.11.2. RIPA cell lysis 35
4.5.11.3. Coimmunoprecipitation (CoIP) 35
4.5.11.4. In vitro kinase assay (IVKA) 35
4.5.11.5. Glutaraldehyde crosslinking 36
4.5.11.6. Gel filtration 36
V. Results ...............................................................................................37
5.1. Molecular interactions of pUL69 and UAP56 .................................................. 37
5.1.1. pUL69 and UAP56/URH49 associate in eukaryotic cells ..................................37
5.1.2. Characterization of the pUL69 – UAP56/URH49 – REF complex......................39
5.1.2.1. pUL69- and REF- interaction of UAP56/URH49 in yeast 39
5.1.2.2. UAP56/URH49´s interaction with pUL69 and REF in human cells 39
5.1.3. UAP56-interaction is crucial for pUL69-mediated nuclear mRNA export .......41
5.2. Molecular characterization of UAP56 and URH49 .......................................... 43
5.2.2. UAP56/URH49 are essential for human cells.....................................................43
5.2.2.1. Coexpression of UAP56 or URH49 enhances protein expression 43
5.2.2.2. Transient knock-down of UAP56 and/or URH49 in eukaryotic cells 44
5.2.2.3. Stable knock-down of UAP56 and/or URH49 in eukaryotic cells 45
5.2.2.4. Impaired mRNA-export in UAP56/URH49 knock-down cells 46
Table of contents III

5.2.3. Nucleocytoplasmic shuttle activity of UAP56/URH49.......................................48
5.2.3.1. UAP56 and URH49 exert nucleocytoplasmic shuttling activity 49
5.2.3.2. CRM1-independent shuttling of UAP56/URH49 50
5.2.3.3. Mapping of the UAP56 nuclear localization signal 51
5.2.3.4. Mapping of the UAP56 nuclear export signal 52
5.2.3.5. UAP56- and pUL69-interaction with Rae1 53
5.2.3.6. Shuttle activity of mutant UAP56-Q289R+F312A 56
655.2.3.7. UAP56 and URH49 interact with U2AF 57
5.2.4. UAP56 and URH49 form homo- and heterodimers............................................58
5.2.4.1. UAP56/URH49 self-association in yeast 58
5.2.4.2. UAssociation in higher eukaryotes 60
5.2.4.3. Point-mutagenesis within a putative interaction interface of UAP56 61
5.3. Characterization of pUL69-interactions........................................................... 65
5.3.1. Multimerization of pUL69.....................................................................................65
5.3.1.1. Self-association of pUL69 in transfected cells 66
5.3.1.2. Purified pUL69 from eukaryotic cells multimerizes in vitro 67
5.3.1.3. Mapping of the pUL69 self-interaction motif 68
5.3.1.4. In vivo verification of pUL69 self-association 70
5.3.2. Phosphorylation of pUL69 by pUL97-kinase .....................................................72
5.3.2.1. pUL69 is serine-phosphorylated but UAP56 and URH49 not 72
5.3.2.2. Intranuclear accumulation of pUL69 after addition of kinase inhibitors 73
5.3.2.3. pUL69 interacts with pUL97 in infected and transiently transfected cells 74
5.3.2.4. Mapping of the pUL97-interaction motif within pUL69 75
5.3.2.5. Mapping of the pUL69-interaction motif wiUL97 75
5.3.2.6. In vitro phosphorylation of pUL69 by the kinase pUL97 77
5.3.2.7. pUL69-mediated mRNA export is modulated by kinase activity 79
5.4. Interactions of the tegument proteins pUL97, pp65 and pUL69 ................... 80
5.4.1. Interaction of UL97 and pp65 ..............................................................................81
5.4.2. Interaction of pp65 and pUL69 ............................................................................82

VI. Discussion........................................................................................84
VII. Abbreviations ...................................................................................97
VIII. References.................................................98
IX. Appendix.........................................................................................114
X. Acknowledgment ...........................................................................116
Summary 1

I. Summary
The UL69 protein of human cytomegalovirus has counterparts in every mammalian or avian
herpesvirus sequenced so far. Because members of this family of homologous proteins have in
part been reported (i) to facilitate viral mRNA export via interaction with the cellular mRNA
export factor REF, (ii) to multimerize in vivo and (iii) to be regulated in their activities via
phosphorylation by cellular protein kinases, this study was conducted to analyze the β-
herpesviral protein UL69 of HCMV with regard to these features. Similar to its counterparts of
the α- or γ-herpesviruses, pUL69 of HCMV possesses properties of a viral mRNA export factor,
such as RNA-binding capacity and nucleocytoplasmic shuttling activity. This study extends
these attributes by the finding that pUL69 recruits the cellular mRNA export machinery via
interaction with UAP56/URH49, an interaction that was subsequently shown to be a
prerequisite for pUL69-mediated mRNA export. The essential role of UAP56 and URH49 for
cellular mRNA export was accordingly demonstrated, since UAP56/URH49 double knock-down
+cells were not viable due to a retention of poly(A) mRNA within the nucleus. After a series of
mapping studies has been performed, it could finally be shown that neither homo-/
65heterodimerization, nor REF- or U2AF -interaction of UAP56/URH49 were required for pUL69-
interaction. In addition, this work revealed that the cellular mRNA export factors UAP56 and
URH49 exhibit CRM1-independent nucleocytoplasmic shuttle activity via their C-termini – a
region that is also required for pUL69-interaction. Furthermore, it could be demonstrated that
pUL69 self-associates in vitro and in vivo in order to form high-molecular-mass complexes. The
self-interaction domain of pUL69 was mapped to a highly structured central domain of the
protein, thereby indicating that UAP56/URH49-interaction and shuttle activity of pUL69 were
independent from pUL69-multimerization. Finally, this work provides evidence that pUL69 is
serine-phosphorylated and serves as a phosphorylation substrate for the viral protein kinase
pUL97. While presence of catalytically active pUL97-kinase augmented pUL69-mediated
mRNA export, this activity was remarkably reduced after pharmacological inhibiton of cellular
and/or viral kinases. In summary, consistent with its homologs in other herpesviruses, pUL69 of
HCMV (i) acts as a posttranscriptional transactivator in order to promote the accumulation of
unspliced mRNAs in the cytoplasm, (ii) multimerizes via its conserved central domain and (iii) is
regulated in its mRNA export activity by phosphorylation. However, in contrast to its
counterparts, pUL69 recruits UAP56/URH49, which act upstream of REF in the cellular mRNA
export pathway and this work identified pUL69 as the first member of this protein family that is
phosphorylated by a viral kinase, i.e. pUL97. Zusammenfassung 2

I. Zusammenfassung
Homologe zu dem UL69 Protein des humanen Cytomegalovirus (HCMV) findet man in jedem
bisher sequenzierten menschlichen oder aviären Herpesvirus. Nachdem für einige Mitglieder
dieser Proteinfamile bereits gezeigt werden konnte, daß diese (i) den viralen mRNA-Export
durch ihre Interaktion mit dem zellulären mRNA Exportfaktor REF vermitteln, (ii) in vivo
Multimere bilden und (iii) in ihren Aktivitäten durch Phosphorylierung mittels zellulärer
Proteinkinasen reguliert werden, sollte pUL69 der β-Herpesviren bezüglich dieser
Eigenschaften untersucht werden. Analog seiner verwandten Proteine der α- oder γ-
Herpesviren, verfügt auch pUL69 über Characteristika viraler mRNA-Exportfaktoren und zwar
insofern, als es RNA bindet und zwischen Kern und Zytoplasma wandert. Diese Arbeit erweitert
diese Eigenschaften um den Nachweis, daß pUL69 mit den zellulären mRNA Exportfaktoren
UAP56 und URH49 interagiert und dies eine wesentliche Vorraussetzung für den pUL69-
vermittelten mRNA-Export darstellt. Die essenzielle Funktion von UAP56/URH49 für den
zellulären mRNA-Export wurde durch UAP56/URH49 Doppel-knock-down Zellen dokumentiert,
welche auf Grund nukleärer mRNA-Retention nicht mehr lebensfähig waren. Mittels
Kartierungsstudien in UAP56/URH49 konnte ausgeschlossen werden, dass Homo-/
65Heterodimerisierung, REF- oder U2AF -interaktion eine notwendige Vorraussetzung für eine
erfolgreiche pUL69-UAP56/URH49-Interaktion darstellt. In dieser Arbeit wurde erstmalig
gezeigt, daß die zellulären mRNA-Exportfaktoren UAP56 und URH49 eine CRM1-unabhängige
shuttle-Aktivität aufweisen und hierfür, ebenso wie für eine pUL69-Interaktion, ihr C-Terminus
benötigt wird. Weiterhin wurde nachgewiesen, dass pUL69 mit sich selbst assoziiert und
Multimere bildet. Die hierfür benötigte Domäne wurde innerhalb des zentralen Teils des
Proteins kartiert, woraus sich erschließt, daß Multimerisierung von pUL69 keine notwendige
Voraussetzung für seine UAP56/URH49-Interaktion oder shuttle-Aktivität darstellt. Zusätzlich
wurde gezeigt, daß pUL69 Serin-phosphoryliert wird und als Substrat der viralen Kinase pUL97
fungiert. Der pUL69-vermittelte mRNA Export konnte in Anwesenheit katalytisch aktiver pUL97-
Kinase gesteigert werden, wohingegen pharmakologische Inhibition von zellulären und/oder
viralen Kinasen seine mRNA-Exportaktivität verminderte. Zusammenfassend konnte bestätigt
werden, daß pUL69 (i) als posttranskriptionaler Transaktivator die zytoplasmatische
Akkumulation ungespleisster mRNAs vermittelt, (ii) mit Hilfe seiner konservierten zentralen
Domäne multimerisiert und (iii) durch Phosphorylierung in seiner mRNA-Exportaktivität
moduliert wird. Im Gegensatz zu seinen Homologen rekrutiert pUL69 UAP56/URH49, welche
übergeordnet von REF im zellulären mRNA-Exportweg wirken und wird als erstes identifiziertes
Mitglied seiner Proteinfamilie von einer viralen Kinase (pUL97) phosphoryliert. Introduction 3

II. Introduction
Human cytomegalovirus
Human Cytomegalovirus (HCMV) is a member of the family of Herpesviridae.
Herpesviruses are enveloped double-stranded DNA viruses that share their typical morphology
and their characteristic to persist livelong latently within the host after primary lytic infection.
The eight human pathogenic herpesviruses can be subdivided by their host range and
replication strategy into three subfamilies: the α-herpesviruses (Herpes simplex virus type 1
and 2, and Varicella zoster virus) have a broad host range and a short life cycle. In contrast,
HCMV (and human herpesvirus type 6 and 7), belonging to the β-herpesviruses, and the γ-
herpesviruses (Epstein-Barr virus and human herpesvirus type 8) are characterized by a
restricted host range and a long replication cycle of approximately 48-72 hours (Matthews,
1982; Roizman and Knipe, 2001; Roizmann et al., 1992).

Epidemiology and pathogenesis
HCMV is a ubiquitous pathogen with a seroprevalence of 40-60% in Europe and the
USA but it can reach up to 100% in developing countries (Pass, 2001). Horizontal transmission
occurs by saliva, blood, urine, semen, breast milk as well as solid organ and bone marrow
transplantation. A vertical transmission from mother to child can occur during pregnancy or
during birth (Lang and Kummer, 1975; Reynolds et al., 1973; Stagno et al., 1981).
In general, pathogenicity of HCMV is very low. Thus, primary and secondary infections
of healthy individuals, as well as reactivations are in most cases asymptomatic while, rarely,
symptoms resembling mononucleosis with fatigue, fever and mild hepatitis can be observed
(Cohen and Corey, 1985; Jordan et al., 1973). However, in immunocompromised or
immunosuppressed patients like allograft recipients, tumor or AIDS patients, HCMV often leads
to life-threatening diseases comprising pneumonitis, hepatitis, retinitis, encephalitis or
gastroenteritis (Drew, 1992; Vancikova and Dvorak, 2001). Congenital infections occur mainly
during primary infection of the mother during pregnancy and are the leading cause of virus
associated birth defects including neurological dysfunctions like mental retardation or hearing
loss (Revello and Gerna, 2002). Besides this, HCMV is also discussed to be involved in age-
related T-cell immunosenescence (Koch et al., 2007) and atherosclerosis (Adam et al., 1997).


Introduction 4

Morphology and replication
HCMV has a size of approximately 150-250nm and shows the typical morphology of
herpesviruses. Its linear, double-stranded DNA genome (229-240kb) encodes about 200 gene
products (Chee et al., 1990). The viral DNA is associated with a fibrillar protein matrix that
forms the virus core. The core is surrounded by an icosahedral capsid with a diameter of about
100 nm. The outermost boundary of the virus is a lipid bilayer that originates from intracellular
membranes of the host cell and is modified by incorporation of viral glycoproteins which are
required for the adsorption of the virus to its host cell but are also responsible for the induction
of a neutralizing antibody response. The area between envelope and capsid is composed of a
pleomorphic protein matrix called tegument. The tegument consists of viral regulatory proteins
as for instance the phosphoproteins pUL69, pUL82/pp71, pUL97 and, most abundantly,
UL83/pp65, that are supposed to exert essential functions during the early phase of viral
infection (Baldick, Jr. et al., 1997; Baldick and Shenk, 1996; Roby and Gibson, 1986; van Zeijl
et al., 1997; Winkler et al., 1994). Additionally, a subset of viral and cellular mRNAs has been
shown to be incorporated into the viral particle by a still unknown mechanism (Bresnahan and
Shenk, 2000; Greijer et al., 2000).
In vivo, HCMV infects a broad cell range within its host, including parenchymal and
connective tissue cells, various hematopoietic cells, specialized neuronal cells from brain and
retina, whereas the predominant target for HCMV infection are fibroblasts, endothelial,
epithelial and smooth muscle cells (Sinzger et al., 2008; Sinzger and Jahn, 1996). HCMV
adsorbs by glycoproteins within its lipid bilayer to heparansulfate on the surface of a host cell
(Compton et al., 1992; Compton et al., 1993; Taylor and Cooper, 1990), whereupon the
membranes of virus and host fuse to release capsid and tegument into the cytoplasm. The
capsids associate with microtubules and are thereby transported to the nuclear pores where
the viral genome is released into the nucleoplasm (Ogawa-Goto et al., 2003), followed by
circularization of the genome to an extrachromosomal episome. Viral tegument proteins that
have a nuclear localisation signal are also imported into the nucleus. Expression of the viral
genome occurs in a cascade with three phases (Demarchi, 1981; McDonough and Spector,
1983; Wathen and Stinski, 1982). During the initial immediate early phase (IE), viral regulatory
proteins like IE1 and IE2 are transcribed. Viral transactivator proteins of the tegument like pp71
and pUL69, cooperatively with pUL35 and pUL26, enhance the expression of these regulatory
proteins by activating the major immediate early enhancer promoter (Liu and Stinski, 1992;
Schierling et al., 2004; Stamminger et al., 2002; Winkler et al., 1994; Winkler et al., 1995;
Winkler and Stamminger, 1996). As a prerequisite for efficient HCMV replication IE2 and
pUL69 both induce a G1 phase cell cycle arrest (Lu and Shenk, 1996; Lu and Shenk, 1999; Introduction 5

Murphy et al., 2000; Wiebusch and Hagemeier, 1999). Thereafter, IE1 and IE2 transactivate
the promoters of early genes, thereby initiating the early phase (E). During this phase proteins
are synthesized that are required for viral DNA replication and the induction of the late phase.
Viral DNA replication starts at defined elements (ori ) following the principle of a rolling circle, lyt
thus synthesizing multiple copies of the viral genome in a concatemeric arrangement (Anders
et al., 1992). At the time DNA synthesis starts, the late phase (L) of viral gene expression is
initiated and structural proteins of the capsid and the tegument as well as glycoproteins are
synthesized. The assembly of virus particles takes place in the nucleus (Gibson, 1996). After
162 capsomers have built up a capsid, one genome equivalent is introduced and with the help
of a viral endonuclease cut from the following genome of the concatemeric DNA (Bogner et al.,
1998; Spaete and Mocarski, 1985). Thereafter, capsids leave the nucleus via disruption of the
nuclear lamina by the nuclear egress complex (NEC), comprising cellular p32, lamin B
receptor, PKC and viral pUL50, pUL53 and the kinase pUL97 (Marschall et al., 2005; Milbradt
et al., 2009). By budding into the trans-golgi network, viruses obtain their final envelope
(Gibson, 1996; Tooze et al., 1993), maturate and are then transported to the cell surface where
they finally leave the cell by an exocytotic-like pathway (Mettenleiter, 2002). During latency viral
DNA is localized as an episome within the nucleoplasm, propagated by cellular DNA
polymerase and distributed to daughter cells. It still remains unclear which mechanisms are
required for establishment and maintenance of latency and how reactivation of the lytic
+replication cycle occurs. Also, the site of latency is still not finally proven – currently, CD34
hematopoetic progenitor cells are discussed to be the reservoir of HCMV during latency
(Khaiboullina et al., 2004; Mendelson et al., 1996).

Prophylaxis and therapy
Due to the worldwide distribution of HCMV an exposure prophylaxis seems to be
impossible. The problem of high seropositivity is especially of importance for transplantation
and transfusion medicine: since not all CMV-positive sera can be excluded, risk of infection by
blood donation is about 2.4% per transfusion (Bowden et al., 1995). Today, mainly two anti-
HCMV therapeutics are available: ganciclovir and foscarnet. The nucleoside analog ganciclovir
is first monophosphorylated by the viral protein kinase pUL97, further phosphorylated by
cellular kinases to a triphosphate and then selectively incorporated into newly synthesized viral
DNA, ultimately leading to a polymerisation stop. The non-nucleoside foscarnet, however,
selectively inhibits the viral DNA polymerase pUL54 by non-competitive interaction with its
phosphate binding site. Immunoglobulins are given to counteract perinatal infections or to avoid
virus spread in transplant patients. To date, no vaccine is available and because of numerous

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