Analysing nuclear import of parvoviruses- effects of parvovirus H-1 on the nuclear envelope (NE) [Elektronische Ressource] / vorgelegt von Manvi Porwal

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 44
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Analysing nuclear import of Parvoviruses - Effects of
parvovirus H-1 on the nuclear envelope (NE)





















Inauguraldissertation

Zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
des Fachbereiches Biologie und Chemie
der Justus-Liebig-Universität Gießen

vorgelegt von M.Sc. Manvi Porwal
aus Indien

Gießen
2007




































This project was funded by DFG (Deutsche Forschungsgemeinschaft) with in the Graduiertenkolleg,
Biochemie von Nucleoproteinkomplexen and the Universite Victor Segalen Bordeaux 2.
I
Aus dem Institute für Medizinische Virologie
am Fachbereich Humanmedizin
der Justus-Liebig-Universität Gie βen







Analysing nuclear import of Parvoviruses - Effects of
parvovirus H-1 on the nuclear envelope (NE)







Inauguraldissertation

Zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
des Fachbereiches Biologie und Chemie
der Justus-Liebig-Universität Gießen

vorgelegt von M.Sc. Manvi Porwal
aus Indien

Gießen
2007


II
Mit Genehmigung des Fachbereichs der Justus-Liebig-Universität Gie βen



Dean: Prof. Dr. Wolfgang Weidner





Supervisor: Prof. Dr. Michael Kann

Reviewer: Prof. Dr. Alfred M Pingoud



















III DECLARATION

I, Manvi Porwal, certify that the work presented in the thesis is original work conducted by myself,
unless otherwise specified, under the supervision of Prof. Dr. Michael Kann. All sources of
information have been specifically acknowledged. No part of this thesis has been submitted for a
degree at any other university.













Date: (Manvi Porwal)
Place:







IV Abbreviations

°C Degree centigrade
aa Aminoacids
ATP Adenosine triphosphate
bp Base pair
BSA Bovine serum albumin
CPV Canine Parvovirus
C-terminal Carboxy terminal
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic Acid
DTT Dithiothreitol
E.coli Escherichia coli
ECL Enhanced Chemiluminescence
EDTA Ethylene-diamintetraaceticacid
EGTA (Ethylenebis(oxyethylennitrilo))tetraaceticacid
ER Endoplasmic reticulum
FCS Fetal calf serum
FG repeat Phenylalanine glycine repeat
FITC Fluorescein Isothiocynate
kDa Kilo-Dalton
Min Minute
MVM virus of mice
NE Nuclear envelope
NEBD envelope breakdown
NES export signal
NLS localization signal
NP-40 Nonidet P-40
NPC pore complex
NS Proteins Non structural proteins
N-terminal Amino-terminal
Nup Nucleoporin
PAGE Polyacrylamide gel electrophorosis
PBS Phosphate buffer saline
PFA Paraformaldehyde
PKC Protein kinase C
POD Peroxidase
PV Parvovirus
PVDF Polyvinylidene fluoride
RNA Ribonucleic acid
rpm Rotations per minute
RRL Rabbit reticulate lysate
RT Room temperature
SDS Sodiumdodecylsulphate
ss-DNA Single-stranded DNA
SV40 Simian virus 40
TB Transport buffer
Tris Trishydroxymethyl aminomethane
VP Viral proteins
WGA Wheat germ agglutinin
V Index
1. Introduction ............................................................................................................ 1
2. Review of the literature.......................................................................................... 3
2.1 Overview:- How viruses enter into animal cells ................................................................. 3
2.1.1 Attachment ........................................................................................................................ 3
2.1.2 Penetration......................................................................................................................... 3
2.1.3 Viral uncoating.................................................................................................................. 4
2.1.4 Entry.................................................................................................................................. 4
2.1.5 Assembly, maturation and release of progeny virus ......................................................... 4
2.2 Overview:- Parvovirus H-1 ...................................................................................................5
2.2.1 Parvovirus infection and diseases ..................................................................................... 5
2.2.2 Taxonomy.......................................................................................................................... 5
2.2.3 Virus structure......... 5
2.2.3.1 Morphology ............................................................................................................................................ 5
2.2.3.2 The virion................................................................................................................................................ 5
2.2.3.3 Genome.................................. 6
2.2.3.4 Proteins ................................................................................................................................................... 6
2.2.4 Virus life cycle .................................................................................................................. 6
2.2.5 Genome organization and gene expression....................................................................... 7
2.2.6 Use of parvoviruses in medicines...................................................................................... 9
2.2.7 Parvovirus in gene therapy.............................................................................................. 10
2.3 Overview:- Mechanisms of nuclear transport ................................................................... 11
2.3.1 Endocytosis ..................................................................................................................... 11
2.3.2 Acidification.................................................................................................................... 13
2.3.3 Signal- mediated nuclear import ..................................................................................... 15
2.3.4 The Nuclear Pore Complex (NPC).................................................................................. 15
2.3.5 Nuclear localisation signal (NLS) receptors and import factor recycling....................... 16
2.3.6 Nuclear envelope (NE) and nuclear envelope breakdown (NEBD) .............................. 17
2.3.6.1 Mitosis.......................................................................................................................... 17
2.3.6.2 Apoptosis........ 18
3. Aim of the project................................................................................................. 22
4. Material and Methods.......................................................................................... 23
4.1 Materials ............................................................................................................................... 23
4.1.1 Antibiotics, Chemicals and Enzymes.............................................................................. 23
4.1.2 Inhibitors ......................................................................................................................... 24
4.1.3 Antibodies and Beads...................................................................................................... 24
4.1.4 Cell Lines......... 24
4.1.5 Primers.......... 25
4.1.6 Plasmid 25
4.1.7 Equipments....... 25
4.1.8 Kits and Extras ................................................................................................................ 25
4.1.9 Buffers and Solutions...................................................................................................... 26
4.2 Methods................................................................................................................................. 28
4.2.1 Cell Culture ..................................................................................................................... 28
4.2.2 Virus preparation and Virus purification ........................................................................ 28