Han Xiao PhD thesis - Research@StAndrews:FullText - University of ...

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Aims of Thesis The overall aim of this study is to compare influenza A viruses (FLUAV) and PIV5 in dealing with cells in a pre-existing interferon-induced antiviral state. Since the mechanisms of PIV5 in dealing with the IFN response is well characterized, the knowledge of PIV5 may shed lights on understanding how FLUAV deals with the IFN response. It may facilitate better understanding of viral pathogenesis and epidemiology. In particular, it is of interest to study which stages of influenza A virus life cycle is inhibited by IFN, and to identify the key antiviral molecules induced by IFN in establishing the antiviral state. To achieve this, it requires us to be able to look into each stage of virus life cycle in details and to be able monitor the fate of incoming genome. Since MxA was reported to be an important antiviral molecule induced by IFN and inhibit a number of viruses, it would also be of interest to characterize the antiviral mechanisms of MxA against influenza virus infection. 52
2.1 Mammalian cells and tissue culture 2.1.1 Cell types used in this study A549 cells - Human lung epithelial cells. Vero cells – fibroblast-like cell line originating from kidney cells of African Green monkey. Vero-MxA – Vero cells constitutively expressing human MxA (kindly provided by Prof. Otto Haller, Freiburg). MDCK cells – canine kidney cells. MDCK/V cells – MDCK cells stably expressing the V protein of PIV5 (Precious et al., 2005b). 293T cells – Human embryonic kidney cells constitutively expressing SV40 large T antigen. Kindly provided by Prof. Richard Iggo, University of St Andrews. A549/BVDV-Npro cells – A549 cells constitutively expressing Bovine Diarrhoea Virus (BVDV) Npro protein, which targets IRF-3 for degradation. A549/shMxA cells – A549 cells transducted with lentivirus overexpressing shRNA against MxA. A549/IFNβ-GFP cells – A549 cells overexpressing GFP under the control of IFNβ promoter, generated by Ms. Shu Chen (University of St Andrews). A549/shUbcH8 cells – A549 cells transducted with lentivirus over-expressing shRNA against ISG15 conjugating enzyme E2 (UbcH8). A549/MxA cells – A549 cells constitutively over-expressing MxA. A549/pk-MxA cells – A549 cells constitutively overexpressing pk-tagged MxA. A549/shMxA/wMxA cells – A549 cells with MxA knockdown constitutively overexpressing wobble MxA. 53
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VIRUS AND INTERFERON: A FIGHT FOR S
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Abstract The Interferon (IFN) famil
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3. Permission for electronic public
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Abbreviations ADAR1 Adenosine deami
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PI3K Phosphatidylinositol 3-kinase.
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1.2.2 Classification of paramyxovir
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2.5.1 RNA extraction……………
Page 15 and 16: 1.1 The Interferon system The inter
Page 17 and 18: structural homologies: they both co
Page 19 and 20: 1.1.2 Mechanisms of IFN signalling
Page 21 and 22: promoters, whilst others have both
Page 23 and 24: actively regulates diverse cellular
Page 25 and 26: 2007)]. Mx1 gene expression is norm
Page 27 and 28: (Hsiang, Zhao, and Krug, 2009; Lai
Page 29 and 30: 1.1.4 Virus encoded IFN antagonists
Page 31 and 32: 1.1.5 Virus shut-off of host protei
Page 33 and 34: sector. The aetiological agent is N
Page 35 and 36: 1.2.3 Virion structure The virions
Page 37 and 38: of P and V proteins are unique. 23
Page 39 and 40: species. The expression of P/V/C pr
Page 41 and 42: al., 1992; Riedl et al., 2002). M p
Page 43 and 44: forming the postfusion hairpin conf
Page 45 and 46: antigenically distinct HA subtypes
Page 47 and 48: asic amino acids sequence R-X-R/K-R
Page 49 and 50: egins with PB1 binding to the 5’
Page 51 and 52: stage of replication, full-length p
Page 53 and 54: into infectious virion. Finally, co
Page 55 and 56: with a number of host proteins invo
Page 57 and 58: infection, NS1 is found not only in
Page 59 and 60: 1.3.4.3 NS1 and host immune respons
Page 61 and 62: The precise mechanism for how NS1 i
Page 63 and 64: CPSF30 and PABPII, some IFN-β pre-
Page 65: 2008; Shin et al., 2007). Recently,
Page 69 and 70: supplemented with Roferon recombina
Page 71 and 72: 10. rUd-103/106B: The NS1 protein c
Page 73 and 74: Confluent monolayers of cells were
Page 75 and 76: 2.3 DNA subcloning 2.3.1 cDNA synth
Page 77 and 78: appropriate time and temperature fo
Page 79 and 80: 2.4.3 Immunofluorescence For immuno
Page 81 and 82: 2.4.4 Preparation of radio-labelled
Page 83 and 84: emove unincorporated DIG nucleotide
Page 85 and 86: 2.6 Miscellaneous assays 2.6.1 Lent
Page 87 and 88: Antibodies Antibodies were used in
Page 89 and 90: 2.8 Primers PRIMERS DESCRIPTION SEQ
Page 91 and 92: 2.9 Plasmids PLASMIDS pMD-VSVG pCMV
Page 93 and 94: A549 cells is that, IFN released fr
Page 95 and 96: 3.1.3 Study of the mechanism of hos
Page 97 and 98: 3.1.4 PIV5 can dismantle the IFN-in
Page 99 and 100: the majority of cells remained nega
Page 101 and 102: 3.1.6 Influenza virus and the IFN-i
Page 103 and 104: these events occur only at the begi
Page 105 and 106: were detecting only the incoming vR
Page 107 and 108: naïve A549 cells and MxA-knockdown
Page 109 and 110: 3.2.6 Effects of introducing MxA ba
Page 111 and 112: expression. However, over-expressio
Page 113 and 114: expression of MxA alone is insuffic
Page 115 and 116: numbers both around 100-fold, sugge
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3.3.4 IFNγ does not have a signifi
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4.2 Comparison of the mechanisms of
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4.3 Influenza A virus shut-off of h
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4.5 MxA recognition of NP protein E
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ability of IFN to block the nuclear
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Akira, S., Uematsu, S., and Takeuch
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Bonjardim, C. A. (2005). Interferon
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Cuddihy, A. R., Li, S., Tam, N. W.,
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Falvo, J. V., Thanos, D., and Mania
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Greenspan, D., Palese, P., and Krys
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Hirano, A., Wang, A. H., Gombart, A
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Jiang, D., Guo, H., Xu, C., Chang,
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Landis, H., Simon-Jodicke, A., Klot
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Martin, J., Wharton, S. A., Lin, Y.
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Neumann, G., Hughes, M. T., and Kaw
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(NAT) 2 acetylator status and age o
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Rodriguez, A., Perez-Gonzalez, A.,
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Staeheli, P., Pitossi, F., and Pavl
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Uze, G., and Monneron, D. (2007). I
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Wong, A. H., Tam, N. W., Yang, Y. L
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A.1 Plasmid map of the pLKO.1 with
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A.2 Plasmid map of the pdlNotI’IR
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Virus Mutation Comments rUd-NS1-R38
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Fig. 1.2. Schematic representation
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Page 167 and 168:
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(A) (B) Fig. 1.8. Replication and t
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(A) (B) Hours p.i. Mock PIV5 12 18
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Mock 103/106 rUd-184-8(L) rUd-184-8
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-antibody +antibody (A) (B) 16h (C)
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(A) 24h 48h -IFN +IFN (B) NS1 MxA M
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(A) 50 Percentage of cells positive
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(A) -IFN +IFN vRNA Merge vRNA Merge
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-IFN +IFN NP Merge NP Merge Mock A5
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Mock A549 A549-MxA -IFN +IFN Fig.3.
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(A) NP MxA Merge -IFN Mock +IFN (B)
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(A) A549 A549-MxA shMxA -7 -7 -7 -I
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(A) Hep2 Hep2/Npro Hep2/shISG56 (B)
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Fig. 3.22. IFNγ does not have an o
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