Molecular studies on dengue virus-host interaction [Elektronische Ressource] / presented by Anil Kumar Victoria Ansalem

Anil Kumar

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Molecular Studies on Dengue Virus-Host
Interaction







Dissertation
Submitted to the
Combined Faculties for the natural sciences and for Mathematics
Of the Ruperto-Carola University of Heidelberg, Germany
For the degree of
Doctor of Natural Sciences


Presented by
Anil Kumar Victoria Ansalem
Born in Trivandrum, India
Oral Examination:


















Referees: Prof. Dr. rer. nat. Ralf Bartenschlager
Prof. Dr. rer. nat. Oliver Fackler



Declaration
The applicant, Anil Kumar Victoria Ansalem declares that I am the
sole author of the submitted dissertation and no other sources or help
from those specifically referred to have been used. Additionally, the
applicant declares that he has not applied for permission to enter ex-
amination procedure at another institution and this dissertation has
not been presented to other faculty and not used in its current or in
any other form in another examination

_____________ __________________
Date Signature



Table of Contents
1. List of Figures ............................................................................ v
2. List of Tables ........... vii
3. List of Abbreviations............................... viii
4. Acknowledgement ..... x
5. Summary .................................................................................. xi
6. Zusammenfassung . xiii
I. Introduction ............. 1
I.1 Dengue ..................................... 1
I.1.1 History and Epidemiology ......................................... 1
I.1.2 Transmission, Symptoms and Pathogenesis ................ 3
I.1.3 Vaccines and Treatment ........... 5
I.2 The Dengue Virus ..................................................................................... 5
I.2.1 Taxonomy and Evolution .......... 5
I.3 Molecular biology of DENV ........................................................................ 7
I.3.1 Genome organization ............... 7
I.3.2 Translation and polyprotein processing ...................................................... 8
I.3.3 Structure and Assembly of DENV particles .................. 9
I.3.4 DENV proteins and functions ................................................................... 10
I.3.5 DENV infection cycle .............. 11
I.4 Dengue NS5 ............................................................ 13
I.4.1 NS5 phosphorylation .............................................. 14
I.4.2 Immune response modulation by NS5 ...................... 14
I.4.3 Nuclear Localization of NS5 .................................... 15
I.5 Cellular proteins involved in DENV replication cycle ............................... 15
I.5.1 RNAi Screens ........................................................ 16
I.5.2 RNAi screens for identification of cellular genes influencing the viral life cycle17
I.6 Objectives of this work ........................................... 19
I.6.1 Studying the role of nuclear NS5 in viral replication and modulation of innate
immune response ......................................................... 19
1.6.2 Identification of cellular kinases involved DENV infection and replication by
using genome-wide RNAi screen. .................................................................... 19
II. Materials and Methods .......................... 20
II.1 Materials ............................................................... 20
II.1.1 Antibodies ........................................................... 20
II.1.2 Bacterial Strains................................................... 21
i

II.1.3 DNA and RNA oligonucleotides ............................................................... 21
II.1.4 Instruments ........................................................ 21
II.1.5 Enzymes, Kits and other reagents used ................... 23
II.1.6 Buffers and Solutions ............................................ 25
II.1.7 Radioactive Reagents ........................................... 29
II.1.8 Cell lines ............................................................. 29
II.1.9 Cloning Vectors and Plasmids ................................ 29
II.1.9 siRNAs ................................ 30
II.1.10 Virus . 31
II.2 Methods ................................ 32
II.2.1 Cell culture and Viruses ........................................................................ 32
II.2.1.1 Cell culture .................... 32
II.2.1.2 Long term storage of Cell lines ......................................................... 33
II.2.1.3 Counting cells using Haemocytometer ............... 33
II.2.1.4 Transfection of Eukaryotic cells with plasmid DNA .............................. 33
II.2.1.5 Infection with DENV2 ...................................................................... 34
II.2.1.6 Passaging DENV2 ........... 34
II.2.1.7 Concentrating virus stock 34
II.2.1.8 Virus Titer estimation: Plaque Assay ................................................. 34
II.2.1.9 Virus Titer Estimation: TCID method .............. 35 50
II.2.1.10 Electroporation of DENV RNAs into mammalian cells ......................... 36
II.2.1.11 Firefly Luciferase based Virus replication assays ............................... 36
II.2.1.12 Visualization of protein localization by immunofluorescence ............... 36
II.2.2 Working with DNA and RNA ................................................................... 37
II.2.2.1 Transformation of competent bacteria ............... 37
II.2.2.2 Plasmid purification ........ 38
II.2.2.3 DNA purification or extraction from agarose gels 38
II.2.2.4 DNA sequencing ............................................................................. 38
II.2.2.5 Polymerase Chain Reaction (PCR) ..................... 38
II.2.2.6 Site-directed Mutagenesis ............................................................... 39
II.2.2.7 In vitro Transcription ...... 39
II.2.2.8 Isolation of total cellular RNA ........................... 40
II.2.2.9 RNA quantification by RT-PCR. ......................... 40
II.2.3 Working with Proteins ........................................................................... 40
II.2.3.1 Subcellular fractionation of infected cells 40
II.2.3.2 Immunoprecipitation ....... 41
II.2.3.3 Standard SDS PAGE ........ 41
II.2.3.4 Western Blot Analysis ..................................................................... 42
ii

II.2.4.1 NS5 expression in Rosetta (DE3) ...................................................... 42
II.2.4.2 NS5 purification ............................................. 43
II.2.4.3 Polymerase assay ........... 43
II.2.4.4 Methyl transferase assay ................................................................ 44
II.2.5 Plasmids and viral constructs . 44
II.2.5.1 Plasmid construction ....... 44
II.2.6 RNAi Screen ........................................................................................ 47
II.2.6.1 Preparation of siRNA spotted chambered slides .................................. 47
II.2.6.2 Cell seeding and Infection ............................... 47
II.2.6.3 Immunostaining ............................................. 47
I.2.6.4 Image acquisition ............................................ 48
II.2.6.5 Image processing and statistical analysis .......... 48
II.2.6.6 Validation siRNA screen in 96-well format 49
III. Results ................................................................................ 51
III.1 NS5 nuclear accumulation and viral replication ................................... 51
III.1.1 Effect of extrinsic factors on NS5 nuclear accumulation ............................ 51
III.1.1.1 Localization of NS5 upon DENV infection in various cell types ............. 51
III.1.1.2 Study on phosphorylation of NS5 .................................................... 52
III.1.1.3 Biochemical fractionation of NS5 ..................... 53
III.1.1.4 Role of DENV proteins in NS5 nuclear transport ................................ 54
III.1.1.5 Effect of Casein kinases on NS5 nuclear accumulation ........................ 55
III.1.1.6 Study of mobility of NS5 between cellular compartments ................... 56
III.1.2 Identification of determinants within NS5 affecting nuclear accumulation ... 58
III.1.2.1 Introduction of restriction sites for cloning NLS mutants ..................... 58
III.1.2.2 Role of -NLS and  -NLS on NS5 nuclear localization........................ 59
III.1.2.3 Construction and immunolocalization of NLS mutants 60
III.1.3 Viral replication and NS5 nuclear accumulation ....................................... 62
III.1.3.1 Effect of NS5 nuclear accumulation on viral replication ....................... 62
III.1.3.2 Transcomplementation of replication deficient NLS mutants ................ 64
III.1.3.3 Effect of addition of NLS and NES to NS5 ......... 66
III.1.4 Effect of NLS mutations on enzymatic activity of NS5 .............................. 67
III.1.4.1 Bacterial expression and purificatio