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The gene region UL128-UL131A of human cytomegalovirus (HCMV) is essential for monocyte infection and block of migration [Elektronische Ressource] : characterisation of the infection of primary human monocytes / presented by Sarah Straschewski

85 pages
Ulm University Hospital Institute of Virology Director: Prof. Dr. med. Thomas Mertens The gene region UL128-UL131A of human cytomegalovirus (HCMV) is essential for monocyte infection and block of migration Characterisation of the infection of primary human monocytes Dissertation to obtain the Doctoral Degree of Human Biology (Dr. biol. hum.) at the Faculty of Medicine, University of Ulm presented by Sarah Straschewski from Ulm 2010 Dean of the Faculty: Prof. Dr. rer. nat. Thomas Wirth 1. Reviewer: Prof. Dr. med. Thomas Mertens 2. Reviewer: Prof. Dr. rer. nat. Klaus-Dieter Spindler Day doctorate awarded: 18.2.2011 II To Giada my parents and Bernd You are my towers of strength III Index 1. Introduction ................................................................................................. 1 1.1 The History of cytomegalovirus ............................................................................... 1 1.2 The family of Herpesviruses ..................................................................................... 1 1.3 The human cytomegalovirus .................................................................................... 2 1.4 Monocytes...................................................................................................................
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Ulm University Hospital Institute of Virology Director: Prof. Dr. med. Thomas Mertens
The gene region UL128-UL131A of human cytomegalovirus (HCMV) is essential for monocyte infection and block of migration Characterisationoftheinfectionofprimaryhumanmonocytes
Dissertation to obtain the Doctoral Degree of Human Biology (Dr. biol. hum.) at the Faculty of Medicine, University of Ulm
presented by Sarah Straschewski from Ulm 2010
Dean of the Faculty: Prof. Dr. rer. nat. Thomas Wirth 1. Reviewer: Prof. Dr. med. Thomas Mertens 2. Reviewer: Prof. Dr. rer. nat. Klaus-Dieter Spindler Day doctorate awarded: 18.2.2011
II
To
Giada
my parents
and Bernd
You are my towers of strength
I
I
I
Index
1. Introduction ................................................................................................. 1
1.1TheHistoryofcytomegalovirus...............................................................................1
1.2 The family of Herpesviruses ..................................................................................... 1
1.3 The human cytomegalovirus .................................................................................... 2
1.4 Monocytes................................................................................................................... 9
1.5 Aim of the work ....................................................................................................... 14
2.Materials....................................................................................................15
2.1Instruments..............................................................................................................15
2.2 Other utensils ........................................................................................................... 16
2.3 Chemicals ................................................................................................................. 17
2.4 Antibodies................................................................................................................. 19
2.5 Oligonucleotides....................................................................................................... 20
2.6 Recombinant Proteins ............................................................................................. 20
2.7 Viruses ...................................................................................................................... 21
2.8 Cell types and lines .................................................................................................. 22
2.9 Cell buffer and media.............................................................................................. 22
2.10 Media and standard solutions .............................................................................. 23
3. Methods ...................................................................................................... 27
3.1 Monocytes isolation ................................................................................................. 27
3.2 HFF ........................................................................................................................... 27
3.4 Production of cell-free viral stocks and titration.................................................. 28
3.5 Sequencing of the UL128-UL131A region in viral stocks .................................... 29
3.6 HCMV infection of monocytes and HFF............................................................... 29
3.7 Indirect Immunofluorescence assay (IIF) ............................................................. 29
3.8Immunoblot(westerblot)........................................................................................30
3.9 Flow cytometry analyses (FACS) ........................................................................... 31
3.10 Chemotaxis assay................................................................................................... 32
3.11 Sample preparation for Electron microscopy..................................................... 323.12 Intracellular Ca2+measurements ......................................................................... 33
4.Results........................................................................................................34
4.1 The complex III is essential for infection of primary human monocytes ........... 34
4.2 The chemokine-driven migration of monocytes is blocked by the viral protein pUL128...........................................................................................................................39
V I
4.3 The block of chemokine-driven migration in HCMV-infected monocytes is not associated to modification of the cell cytoskeleton ..................................................... 44
4.4 Infection with gCIII competent HCMV strains specifically reduces the surface expression of chemokine receptors on monocytes ...................................................... 48
4.5 The chemokine receptors are internalised but not degraded by HCMV infection as well as by treatment with the recombinant rpUL128 ............................................ 52
4.6 rpUL128 has chemoattractant potency
................................................................. 53
4.7 HCMV and rpUL128 does not induce release of intracellular Ca2+................... 54
4. Discussion ................................................................................................... 56
5. Summary .................................................................................................... 62
6.References..................................................................................................64
V
ECL
EBV
DTT
ds
d pi
dNTP
FACS
DNase
EtBr
EtOH
EGTA
ELISA
EDTA
EGFR
VI
Da
DMEM
DC
double-stranded
dithiothreitol
day(s) post infection
ethylene diamine tetraacetic acid
epidermal growth factor receptor
Epstein-Barr virus
enhanced chemiluminescence
ethidium bromide
ethanol
ethylene glycol tetraacetic acid
enzyme-linked immunosorbent assay
CXCR
DNA
fluorescence-activated cell sorter
cDNA
CMV
CpG
CSF
CCR
CD
CKR
APS
bp
BSA
base pair
cluster of differentiation
chemokine receptor
DMSO
acquired immunodeficiency syndrome
Ammonium persulfate
CC chemokine receptor
bovine serum albumin
Abbreviation
Antibody
AIDS
I. Ab
cytomegalovirus
complementary DNA
Dalton
dentritic cells
CXC chemokine receptor
colony-stimulating factor
Dulbecco's modified Eagle medium
cytosine-phosphate guanine dinucleotide
deoxyribonucleic acid
Dimethyl sulfoxide
deoxinucleosid-triphosphate
deoxyribonuclease
magnetic-activated cell sorting
mitogen-activated protein kinase
monocytes-derived macrophages
minimum essential medium
macrophage CSF
major histocompatibility complex
methanol
interleukin
kilo base pair
interferon
immunoglobulin
VII
hour(s) post infection
horseradish peroxidase
herpes simplex virus
heperan sulphate proteoglycans
immediate early
indirect immunofluorescence assay
fetal calf serum
fluorescein isothiocyanate
granulocyte-macrophage CSF
glycoprotein
hour(s)
human cytomegalovirus
N-2-hydroxyethylpiperazine-N-2 ethanesulfonic acid
human immunodeficiency virus
human foreskin fibroblast
human histocompatibility leukocyte Ag
g
h
HCMV
HEPES
HFF
FCS
FITC
GM-CSF
IE
IIF
IFN
Ig
HLA
HIV
h pi
HSPGs
HRP
HSV
LPS
kDa
MACS
MEM
2-ME
mAbs
kbp
IL
MHC
MeOH
MO
kilo Dalton
monocyte
monoclonal antibodies
2-mercaptoethanol
lipopolysaccharide
MAPK
M-CSF
MDM
standard saline citrate
N,N,N,N  Tetramethylethylendiamin
sodium deodecyl sulfate
SDS-Poly-acrylamid gel electrophoreses
room temperature
reverse transcriptase
microliter
revolutions per minute
ribonucleic acid
molecular weight
phenylmethylsulfonyl fluoride
recombinant
phosphonoformic acid
natural killer cell
Phosphatidylinositol 3-kinases
plaque-forming unit
optical density
polymerase chain reaction
phosphate buffered saline
peripheral blood mononuclear cells
polyacrylamide gel electrophoresis
nuclear factorκB
microgram
multiplicity of infection 4-morpholinepropanesulfonic acid
PFA
PCR
PBS
PBMC
PMSF
PI3K
PFU
RNA
rec
Tris-(hydroxymethyl)-aminomethan
T helper cell
Western Blot
monocytic leukemia cell line
Tris-Acetate-EDTA
Toll-like receptor
tumor necrosis factor
PAGE
OD
NK cell
NF-κB
μg μl mw
MOPS
moi
rpm
rt
RT
SDS
SDS-PAGE
SSC
TAE
TEMED
THP-1
Th cell
TLR
Tris
TNF
WB
VIII
1. Introduction
1.1 The history of Cytomegalovirus
Introduction
The intranuclear inclusions that now are recognized as typical features of cytomegalovirus
infections were first noticed in 1881 but wrongly attributed to protozoa. The first portray of
CMV infected cells was done by Jesionek and Kiolemenoglou in 1904 who described
protozoan-like owls eyes cells from the kidney of an alleged luetic foetus. The interest
to identify the etiological agent was manly driven by the role of CMV in congenital
infection. The first tissue isolation of CMV was achieved in the early 1950s by three
groups at the same time. Weller, Smith and Rowe independently isolated and grew CMV
from man and mice in cell-culture. Finally, the common name cytomegalovirus was
proposed by Thomas Huckle Weller to reflect both virus-induced cytopathic effects and
the role of the virus in congenitally acquired cytomegalic inclusion disease. The
importance of CMV has risen over the last decades with the increase in solid organ
transplantations and the increase in acquired immunodeficiency syndrome (AIDS). But
even if HCMV can be detected and often treated in AIDS- and transplant patients, the
relevance of CMV during pregnancy and the severe damages for the unborn is still high.
SincenownoantiviralagenthavebeenapprovedforuseintreatingcongenitalCMV
infection and also the treatment of HCMV infection in transplanted patients is one major
problem since a HCMV-induced inflammation of the transplanted organ can initiate
rejection.
1.2 The family of Herpesviruses
The family ofHerpesviridaeincludes human- and animal pathogens. The members of this
family are divided into three subfamilies. The membership into these subfamilies is based
on the architecture of the viral particles, the pathogenesis, the cell tropism and the length of
the replication cycle. The three subfamilies are theAlpha-, Beta-and Gammaherpesvirinae. The members of theAlphaherpesvirinae the genera are Simplexvirus (HSV-1 and HSV-2; HHV-1 and HHV-2) and Varicellavirus (VZV; HHV-
1
Introduction
3). The subfamily of theBetaherpesvirinaecontains the genera Cytomegalovirus (HCMV; HHV-5), Muromegalovirus (murine cytomegalovirus) and Rosealovirus (HHV-6 and HHV-7). The last subfamily is represented by theGammaherpesvirinaeand contains the
Lymphocryptovirus (HHV- 4 or Epstein-Barr-Virus) and the Rhadinovirus (HHV-8). All
of the Herpesviruses share four significant biological properties:
1) A huge viral genome which encodes a large array of enzymes involved in the nucleic metabolism, DNA synthesis and processing of proteins.
2) DNA and the capsid assembly occur in the nucleus ofThe synthesis of viral infected host cells.
3) The production of new viral particles leads always to a disruption of the host cell. 4) in their natural hosts. In cells harbouring latent virusThe ability to remain latent only a small subset of viral genes are expressed and infectious progenies are not
produced.
The variety of the three Herpesvirus subfamilies is summarised in Table 1:
Table 1: Differences betweenα-,β- andγ-Herpesviruses
Natural Host
Reproductive cycle
Spread in cell culture
α-Herpesviruses
wide range
relatively short
fast
1.3 The human cytomegalovirus
1.3.1 Genome orientation and virion structure
β-Herpesviruses
restricted
prolonged
slowly
γ-Herpesviruses
limited to family
The human cytomegalovirus (HCMV) belongs to thehateeprerivseani Band like other
Herpesviruses HCMV has adapted to its host and has evolved multiple strategies to escape
the immune system.
Among all herpesviruses, HCMV has the highest coding capacity. The genome is 230kbp
in size and encodes for more than 200 gene products [47; 17]. Like all herpesviruses, HCMV has a double-stranded linear DNA (dsDNA) genome that consists of two
covalently linked sequences, each comprising two unique regions, one short (US) and one
2
Introduction
long (UL), flanked by short regions of sequences repeated in inverted and directed ways. The terminal repeats, flanking both sides of the genome are referred to as TRL/TRSwhile the internal repeats linking the US/UL are referred to as IRS/IRL. The overall genome configuration is TRL-UL-IRL-IRS-US-TRS as it is shown in figure 1. Intramolecular homologous recombination of these repeated sequences can result in four different isomeric genome forms, where the Usand the ULregions are differently orientated to each other. In HCMV all four isotypes can be found in the same amount and they are termed the P (prototype), IL (L inverted), IS (S inverted) and ISL (L and S inverted) genome arrangements [45; 15, 60; 84; 17].
Figure 1: Genomic structure of HCMV.upper line represents the structure of the wholeThe
genome. Unique regions (USand UL)are flanked by inverted terminal and internal repeats (TRL/TRSand IRL/IRS). The bottom line represents the size of the genome in kilo base pairs (kbp). The origin of replication in the case of a lytic replicative cycle (oriLyt(L)) is also shown. Scheme source: [45]
The virion structure of HCMV is typical for Herpesviruses. Figure 2 schematically pictures
the architecture of the HCMV virion. The virions are 150-200 nm in diameter and consist
of more than 30 different proteins. The centre of the viral particle is formed by a protein
matrix associated with the linear double-stranded (dsDNA) genome, what is called the
virus-core. The core is surrounded by a capsid measuring 100 nm in diameter. It has an
icosahedral symmetry with a triangulation number of T=16 and consists of 163 capsomers
[93].
The capsid is surrounded by a lipid bilayer envelope, where numerous different
glycoproteins are embedded. Lots of them are linked by disulfide bonds and organised in
glycoprotein complexes, however a large number of envelope proteins still remain
uncharacterised. As far as it is known today, some of these glycoproteins are predicted to
play an important role in each step of the viral replication cycle, but also for initiation of a
neutralising antibody response of the immune system. The outer membrane of the viral
3
 
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