The study of oligomerization and nuclear import of influenza virus nucleoprotein.

Chan, Wai Hon.Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.Includes bibliographical references (leaves 134-141).Abstracts in English and Chinese.Acknowledgements --- p.2Abstract --- p.3摘要 --- p.5Content --- p.6List of Abbreviations and symbols --- p.10Chapter Chapter 1 --- Introduction --- p.13Chapter 1.1 --- The Severity of Influenza A Virus --- p.14Chapter 1.2 --- Introduction to Influenza A Virus --- p.15Chapter 1.3 --- What is Nucleoprotein? --- p.17Chapter 1.4 --- Multifunctional role of Nucleoprotein --- p.19Chapter 1.4.1 --- Interaction of Nucleoprotein with Other Viral Components --- p.19Chapter 1.4.1 --- Interaction of Nucleoprotein with Cellular Components --- p.22Chapter 1.5 --- Aims of study --- p.23Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- Materials --- p.25Chapter 2.1.1 --- Chemical reagents --- p.25Chapter 2.1.2 --- Buffers --- p.28Chapter 2.1.2.1 --- Preparation of Buffers --- p.28Chapter 2.1.2.2 --- Buffer for Common Use --- p.28Chapter 2.1.3 --- Plasmids and Strains --- p.31Chapter 2.2 --- Methods --- p.32Chapter 2.2.1 --- Molecular Cloning --- p.32Chapter 2.2.2 --- "Expression of the Recombinant NP-WT/ mutants and importin α1, 3and 5 in E.coli" --- p.46Chapter 2.2.3 --- Purification of the NP WT/ variants --- p.50Chapter 2.2.4 --- "Purification of importin αl, 3 and 5" --- p.53Chapter 2.2.5 --- In vitro interaction study between NP and importin α --- p.56Chapter 2.2.6 --- In vivo interaction study between NP and importin α --- p.59Chapter 2.2.7 --- In vivo analysis to study NP-NP homo-oligomerization --- p.64Chapter 2.2.8 --- In vitro static light scattering analysis to determine NP oligomeric state --- p.68Chapter 2.2.9 --- Control experiments to verify NP-polymerases and NP-RNA interaction --- p.69Chapter Chapter 3 --- Functional analysis of influenza H5N1 nucleoprotein tail loop for oligomerization and ribonucleoprotein activitiesChapter 3.1 --- Introduction --- p.72Chapter 3.2 --- Results --- p.75Chapter 3.2.1 --- Tail loop insertion is maintained by intra- and inter-molecular interactions --- p.75Chapter 3.2.2 --- NP mutants display defective transcription-replication activity --- p.77Chapter 3.2.3 --- Expression and purification of defective NP variants --- p.80Chapter 3.2.4 --- Defective NP variants interact with RNA and the polymerase complex --- p.85Chapter 3.2.5 --- Defective NP variants possess abnormal oligomeric states in vitro --- p.91Chapter 3.2.6 --- NP variants with impaired RNP activity cannot form homo-oligomers in vivo --- p.95Chapter 3.3 --- Discussion --- p.98Chapter Chapter 4 --- Biophysical characterization of the interaction between influenza nucleoprotein and importin αChapter 4.1 --- Introduction --- p.105Chapter 4.2 --- Results --- p.108Chapter 4.2.1 --- Expression and Purification of NP-WT/ NLS variants and Importin a --- p.108Chapter 4.2.2 --- Pull down Assay --- p.113Chapter 4.2.3 --- Light scattering analysis (NP-lmportin α5) --- p.114Chapter 4.2.4 --- Binding assay of NP NLS variants with importin α5 --- p.115Chapter 4.2.5 --- BIAcore 3000 Surface Plasmon Resonance (NP-lmportin α5) --- p.118Chapter 4.2.6 --- QRT-PCR (NP-lmportin a5) --- p.123Chapter 4.3 --- Discussion --- p.125References --- p.13