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

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• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2010
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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Ulm University Hospital Institute of Virology Director: Prof. Dr. med. Thomas Mertens

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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 

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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 

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Giada

my parents

and Bernd

You are my towers of strength

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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................................................................................................................... 9

1.5 Aim of the work ....................................................................................................... 14

2.Materials....................................................................................................15 

2.1Instruments..............................................................................................................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

3.8Immunoblot(westerblot)........................................................................................30

3.9 Flow cytometry analyses (FACS) ........................................................................... 31

3.10 Chemotaxis assay................................................................................................... 32

3.11 Sample preparation for Electron microscopy..................................................... 32 3.12 Intracellular Ca 2+ measurements ......................................................................... 33

4.Results........................................................................................................34 

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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

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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

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4.7 HCMV and rpUL128 does not induce release of intracellular Ca 2+ ................... 54

4. Discussion ................................................................................................... 56 

5. Summary .................................................................................................... 62 

6.References..................................................................................................64 

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V

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ECL

EBV

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DTT

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ds

d pi

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dNTP

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FACS

DNase

EtBr

EtOH

EGTA

ELISA

EDTA

EGFR

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VI

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Da

DMEM

DC

double-stranded

dithiothreitol

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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

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fluorescence-activated cell sorter

cDNA

CMV

CpG

CSF

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CCR

CD

CKR

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APS

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bp

BSA

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base pair

cluster of differentiation

chemokine receptor

DMSO

acquired immunodeficiency syndrome

Ammonium persulfate

CC chemokine receptor

bovine serum albumin

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Abbreviation

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Antibody

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AIDS

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I.  Ab

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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

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VII

hour(s) post infection

horseradish peroxidase

herpes simplex virus

heperan sulphate proteoglycans

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immediate early

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indirect immunofluorescence assay

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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

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HCMV

HEPES

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HFF

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FCS

FITC

GM-CSF

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IE

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IIF

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IFN

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Ig

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HLA

HIV

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h pi

HSPGs

HRP

HSV

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LPS

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kDa

MACS

MEM

2-ME

mAbs

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kbp

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IL

MHC

MeOH

MO

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kilo Dalton

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monocyte

monoclonal antibodies

2-mercaptoethanol

lipopolysaccharide

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MAPK

M-CSF

MDM

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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

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PMSF

PI3K

PFU

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RNA

rec

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Tris-(hydroxymethyl)-aminomethan

T helper cell

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Western Blot

monocytic leukemia cell line

Tris-Acetate-EDTA

Toll-like receptor

tumor necrosis factor

PAGE

OD

NK cell

NF-κB

μg μl mw

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MOPS

moi

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rpm

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rt

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RT

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SDS

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SDS-PAGE

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SSC

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TAE

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TEMED

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THP-1

Th cell

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TLR

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Tris

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TNF

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WB

VIII

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1. Introduction

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1.1 The history of Cytomegalovirus

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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.

SincenownoantiviralagenthavebeenapprovedforuseintreatingcongenitalCMV

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.

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1.2 The family of Herpesviruses

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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-

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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:

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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

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Natural Host

Reproductive cycle

Spread in cell culture

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α-Herpesviruses

wide range

relatively short

fast

1.3 The human cytomegalovirus

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1.3.1 Genome orientation and virion structure

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β-Herpesviruses

restricted

prolonged

slowly

γ-Herpesviruses

limited to family

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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 (U S ) and one

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2

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Introduction

long (U L ), 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 TR L /TR S while the internal repeats linking the U S /U L  are referred to as IR S /IR L . The overall genome configuration is TR L -U L -IR L -IR S -U S -TR S  as it is shown in figure 1. Intramolecular homologous recombination of these repeated sequences can result in four different isomeric genome forms, where the U s and the U L regions 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), I L  (L inverted), I S  (S inverted) and I SL  (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 (U S and U L )  are flanked by inverted terminal and internal repeats (TR L /TR S and IR L /IR S ). 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

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