Role of the accessory Vpr and Vpu proteins and upstream LTR U3 sequences in HIV-1 replication and cytopathicity in human lymphoid tissue ex vivo [Elektronische Ressource] / presented by Devi Rajan

Matthias Brenner

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• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2007
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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





Role of the accessory Vpr and Vpu proteins
and upstream LTR U3 sequences in HIV-1 replication
and cytopathicity in human lymphoid tissue ex vivo



Dissertation
To obtain the Doctoral Degree of Human Biology (Dr. biol. hum.)
at the Faculty of Medicine, University of Ulm

Presented by
Devi Rajan
From Ambasamudram, India
2007
Present Dean: Prof. Dr. Klaus-Michael Debatin

1. Reviewer: Prof. Dr. Frank Kirchhoff
2. Reviewer: Prof. Dr. Klaus-Dieter Spindler

Graduation Day: 15.06.2007












1. INDEX 1
1. Index-------------------------------------------------------------------------------------------- 1
2. List of Abbreviations------------------------------------------------------------------------ 3
3. Introduction----------------------------------------------------------------------------------- 6
3.1 Human Immunodeficiency Virus: Discovery and prevalence--------------------- 6
3.2 Morphology and genomic structure of HIV-1 --------------------------------------- 7
3.3 Pathogenesis of HIV-1 infection ------------------------------------------------------ 7
3.4 Viral Protein R (Vpr) ------------------------------------------------------------------- 8
3.5 Viral Protein U (Vpu)------------------------------------------------------------------11
3.6 Long Terminal Repeat (LTR)---------------------------------------------------------12
3.7 Ex vivo Human Lymphoid Tissue (HLT) – A model for AIDS pathogenesis-- 14
3.8 Scientific aims --------------------------------------------------------------------------15
4. Materials--------------------------------------------------------------------------------------17
5. Methods---------------------------------------------------------------------------------------23
5.1 DNA Methods23
5.2 Bacterial Methods ----------------------------------------------------------------------24
5.3 Cell Culture -----------------------------------------------------------------------------24
5.4 Human Lymphoid Tissue -------------------------------------------------------------25
5.5 Mutant Construction -------------------------------------------------------------------25
5.6 Viral Methods---------------------------------------------------------------------------25
5.7 Protein and Enzyme Methods --------------------------------------------------------26
5.8 FACS Analysis -------------------------------------------------------------------------27
5.9 Computer Program and Data Analysis ----------------------------------------------28
6. Results-----------------------------------------------------------------------------------------29
6.1 Effect of R77Q, R77A and R80A mutations in Vpr on HIV-1 replicative
capacity and cytopathicity in HLT ex vivo------------------------------------------------29
6.1.1 Mutations of R77Q, R77A and R80A did not reduce the replication
of X4-tropic HIV-1 ----------------------------------------------------------------------29
6.1.2 Mutant Vpr proteins enhance R5-tropic HIV-1 replication in
macrophages------------------------------------------------------------------------------30
6.1.3 R5-tropic HIV-1 Vpr mutants (R77Q and R80A) show reduced
cytopathicity32
6.1.4 Mutations of R77Q and R80A but not R77A disrupt the proapoptotic
activity of Vpr ----------------------------------------------------------------------------33 1. INDEX 2
6.2 Vpu-mediated CD4 down-modulation is dispensable for efficient HIV-1
replication and CD4+ T cell depletion in HLT ex vivo----------------------------------35
6.3 HIV-1 variants without nef/U3 overlap replicates efficiently and causes
CD4+ T cell depletion in ex vivo-infected HLT------------------------------------------ 38
7. Discussion ------------------------------------------------------------------------------------43
7.1 A naturally occurring sequence variation in Vpr reduces the cytopathicity of
R5-tropic HIV-1------------------------------------------------------------------------------43
7.2 Role of Vpu-mediated CD4 down-modulation in HIV-1 replication
and cytopathicity in HLT ex vivo ----------------------------------------------------------45
7.3 Role of the modulatory U3 region in HIV-1 replication and cytopathicity ------47
7.4 Conclusions-------------------------------------------------------------------------------48
8. Summary -------------------------------------------------------------------------------------49
9. References ------------------------------------------------------------------------------------51 2. LIST OF ABBREVIATIONS 3
2. List of Abbreviations

AIDS Acquired Immune Deficiency Syndrome
bp Base Pairs
BSA Bovine Serum Albumin
BWK Bundeswehrkrankenhaus
CD Cluster Designation
°C Degree Celsius
CTL Cytotoxic T Lymphocyte
dATP Deoxyadenosine Triphosphate
dCTP Deoxycytidine
dGTP Deoxyguanosine Triphosphate
dTTP Deoxythymidine
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethyl Sulfoxide
DTT Dithiothreitol
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide Triphosphate
ds Double strand
E.coli Escherichia coli
EDTA Ethylenediaminetetraacetic Acid
ELISA Enzyme-Linked Immunosorbent Assay
Env Envelope
FCS Fetal Calf Serum
Fig Figure
g Gram
Gag Group Specific Antigen
Gp Glycoprotein
HBS HEPES Buffered Saline
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
HIV Human Immunodeficiency Virus
HLT Human Lymphoid Tissue
HNO Hals Nasen Ohren 2. LIST OF ABBREVIATIONS 4
HRP Horse Radish Peroxidase
kb Kilobase
kg Kilogram
kd Kilodalton
L Liter
LB medium Luria Bertani medium
L-Glu L-Glutamine
LTR Long Terminal Repeat
-3m milli (10 )
-6µ micro (10 )
M Molarity (mol/l)
MA Matrix
MCSF Macrophage Colony Stimulating Factor
Min Minute
MDM Monocyte-derived macrophage
MHC Major histocompatibility complex
-9n nano (10 )
nm nanometer
nM nanomolar
N Normality
Nef Negative Factor
ORF Open Reading Frame
PAA Polyacrylamide
PAGE Polyacrylamide Gel Electrophoresis
PBMC Peripheral Blood Mononuclear Cell
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PHA Phytohaemagglutinin
Pol Polymerase
PPT Polypurinetract
PR Protease
Rev Regulator of expression of virion proteins
RIPA buffer Radioimmunoprecipitation buffer 2. LIST OF ABBREVIATIONS 5
RNA Ribonucleic acid
RPM Rotation per Minute
RPMI Roswell Park Memorial Institute medium
RT Reverse Transcriptase
SD Standard deviation
SDS Sodium dodecyl sulfate
SEM Standard Error of Mean
SHIV Simian-Human Immunodeficiency Virus
SIV Simian Immunodeficiency Virus
SOE-PCR Splice Overlap Extension-PCR
ss Single strand
SV 40 Simian Virus 40
TAT Transactivator of transcription
Tm Melting temperature
Tab Table
TEMED Tetramethylethylenediamine
Tris Trishydroxymethylaminomethane
TM Transmembrane Protein
Vif Viral Infectivity Factor
Vol Volume
VPR Viral Protein R
VPU Viral Protein U
WT Wildtype
W/V Weight per Volume
Amino acids:

Alanine ala A Glycine gly G Proline pro P

Arginine arg R Histidine his H Serine ser S

Asparagine asn N Isoleucine ile I Threonine thr T

Aspartic acid asp D Leucine leu L Tryptophan trp W

Cysteine cys C Lysine lys K Tyrosine tyr Y

Glutamine gln Q Methionine met M Valine val V

Glutamic acid glu E Phenylalanine phe F
3. INTRODUCTION 6
3. Introduction

3.1 Human Immunodeficiency Virus: Discovery and prevalence
In 1981, Gottlieb et al. published a report about 5 cases of Pneumocystis carinii
pneumonia (PCP), a rare form of fungal pneumonia, associated with severe defects in the
immune system of homosexual men in Los Angeles. This disease was originally referred to
as GRID, or Gay-Related Immune Deficiency. A year later it was renamed as Acquired
Immunodeficiency Syndrome (AIDS), a condition in which the immune system fails,
leading to life-threatening opportunistic infections. The causative agent of AIDS was
isolated in 1983 from a patient blood and named lymphadenopathy-associated virus
(Barre-Sinoussi et al., 1983), which was later designated as Human Immunodeficiency
Virus type-1 (HIV-1) (Coffin et al., 1986). The virus was originally transmitted from
chimpanzees infected with a Simian Immunodeficiency Virus (SIVcpz) to humans on at
least three independent occasions resulting in the HIV-1 groups M, N and O (Sharp et al.,
2005). Group M has spread worldwide and caused the global AIDS pandemic (Kandathil et
al., 2005). It is divided into 11 subtypes or clades named A through K (Requejo et al.,
2006). Group N isolates have been detected only in a few individuals in West Africa
(Kandathil et al., 2005). Group O accounts for less than 10% of HIV-1 infections
world