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

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A549/NS1B cells – A549 cells constitutively overexpressing pk-tagged influenza B virus NS1 protein. A549/FLAG-E3L cells – A549 cells constitutively overexpressing FLAG-tagged vaccinia virus E3L protein. 2.1.2 Cell maintenance Cell monolayers were maintained in 25cm 2 or 75cm 2 tissue culture flasks (Greiner) in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Biowest) and incubated at 37°C/5% CO 2 incubator. Cells were routinely passaged using trypsin/EDTA (Becton Dickinson Ltd.), at a frequency dictated by the rate of cell growth. 2.1.3 Cell stock preparation and resuscitation Adherent cells were trypsinised, resuspended in DMEM/10% FBS, and centrifuged at ~500×g for 5mins. Pelleted cells were resuspended in DMEM supplemented with 20% FBS and 10% DMSO and aliquoted into cryovials. Cell stocks were frozen at 70 o C before long-term storage in liquid nitrogen. For resuscitation of cells, cryovials were thawed at 37°C before centrifugation at ~500×g. Pelleted cells were then resuspended and grown in DMEM/10% FBS at 37°C/5% CO 2 . Medium was replaced after 24 hours in order to remove traces of DMSO. 2.1.4 Treatment of cells 2.1.4.1 IFN treatment Cells were treated overnight (at least 16 hours, unless where otherwise stated) with media 54
supplemented with Roferon recombinant human IFNα-2a (Roche Diagnostics) or IFN-γ (Invitrogen) at a concentration of 10 3 units/ml. 2.1.4.2 Transfections with plasmid DNA Mutant viruses were generated by substituting the pHH-NS “rescue” plasmid for mutated versions in the rescue transfection (plasmids were kindly provided by Robert A. Lamb, Northwestern University). These plasmids (pHH-NS1-Y89F, pHH-NS1-P164/7A, pHH- NS1-P162/4/7A, pHH-NS1-R38A, pHH-NS1-123/4, pHH-NS1-184-8, pHH-NS1- 103/106, pHH-NS1-Δ99) were generated by site-directed mutagenesis of pHH-NS using the QuikChange mutagenesis kit (Stratagene) and specific primers. The sequence of the mutated plasmids was confirmed by DNA sequencing. Transfection of cells with plasmid DNA was carried out using FuGENE 6 transfection reagent (Roche) according to the manufacturer’s instructions. 55
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VIRUS AND INTERFERON: A FIGHT FOR S
Page 3 and 4:
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 and 66: 2008; Shin et al., 2007). Recently,
Page 67: 2.1 Mammalian cells and tissue cult
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
Page 117 and 118: 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
Page 155 and 156:
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
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
(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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