The role of scavenger receptor BI in hepatitis - eTheses Repository ...

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118 Figure 4-4 Over expression of SR-BI and SR-BII in Huh-7.5 cells enhances HCVpp-JFH infection. Parental and Huh-7.5 cells over expressing human CD9, SR-BI, or SR- BII were incubated with murine leukaemia virus pseudo-particles (MLVpp) (white bars) or HCVpp - JFH-1 (grey bars), for 8 hrs, followed by a 72 hr infection. Infection was realised by flow cytometric detection of a pseudo-particle encoded GFP reporter gene. Over-expression of SR-BI/II enhanced HCVpp - JFH-1 infection by ~2 fold, MLVpp infection remained unaltered. * =
119 4.5 Murine SR-BI does not enhance HCVcc infection. Murine SR-BI is believed to be indistinguishable from human in regards to ligand binding and metabolism, indeed a number of studies have used murine SR-BI expressed in human hepatoma cell lines to study cholesterol uptake and trafficking (279, 300). HCV sE2 does not bind to murine SR-BI (274), however if HCV interaction with SR-BI occurs via apoproteins associated with the particle (190) both human and murine forms of SR-BI may act as co- receptors. To investigate this we challenged Huh-7.5 cells expressing murine SR-BI with JFH-1. The anti-SR-BI serum used in previous experiments is reactive against both species, therefore an N-terminal GFP tagged form of murine SR-BI (GFP-moSR-BI) was used, allowing flow cytometric detection of positive cells. Importantly, the GFP-SR-BI construct has previously been shown to be functional in regards to lipid uptake and metabolism (279). Figure 4-4A displays un-transfected Huh-7.5 cells and those expressing GFP-moSR- BI, ~18 % are positive for exogenous murine SR-BI. Gating of GFP-moSR-BI positive and negative Huh-7.5 cells allowed quantification of JFH-1 infection within each population (Figure 4-4B). Expression of murine SR-BI did not enhance JFH-1 infection of Huh-7.5 cells (Figure 4-4B & C), suggesting that HCV dependence on SR-BI is driven by a specific HCV E2-receptor interaction and not by apoprotein association.
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The role of scavenger receptor B-I
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i Abstract. With 170 million infect
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iii Acknowledgements. I would like
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v 5 Results: Identification of a re
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vii Figure 5-5 Analysis of JFH-1 wt
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1 Introduction 1.1 The disease. 1 D
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3 predominantly immuno-pathogenic i
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5 Aside from considerations of the
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7 Current attempts to design a HCV
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1.2 An elusive pathogen. 9 The emer
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11 PCR). This technique is highly s
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13 Within six years of the descript
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1.3 Basic virology. 15 Like other m
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17 Figure 1-2 HCV genome and polypr
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19 241). E1 and E2 are anchored to
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21 The characterisation of HCV geno
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23 in the E1 and E2 glycoprotein ge
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25 The solution reached by a great
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27 binding to mouse cells. sE2 inte
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29 density lipoprotein (HDL), allow
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31 blood brain barrier is permeable
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33 act as receptors for viral entry
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1.4.2 Attachment factors 35 The tru
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1.4.3 Endocytosis and fusion 37 Aft
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39 1.4.4 Co-receptor interplay and
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41 inhibition of protein kinase A (
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1.5 Lipoproteins 43 Since 1992 ther
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45 Lipoproteins exist in a dynamic
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47 and delivers its cargo via the w
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49 uptake of cholesterol from LDL (
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Figure 1-4 SR-BI-lipoprotein intera
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53 In summary the class B scavenger
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55 In 1992 Thomssen et. al. demonst
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57 lipoprotein association promotes
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59 Figure 1-5 Lipo-viro-particle as
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61 In vitro culture medium, into wh
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2 Materials and methods. 2.1 Cell l
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Preparation of rat anti-E2 mAbs. 65
Page 77 and 78: 2.4 Basic techniques. Flow cytometr
Page 79 and 80: 69 6. Finally, the cells were washe
Page 81 and 82: 71 (Promega, WI, USA) kits accordin
Page 83 and 84: 73 proteins and are incapable of fu
Page 85 and 86: 75 spectrophotometer (Amersham), we
Page 87 and 88: 77 Figure 2-2 Electroporated Huh-7.
Page 89 and 90: 79 5. Data is expressed as percenta
Page 91 and 92: 81 (Invitrogen). The samples to be
Page 93 and 94: 83 1. The SR-BII coding region was
Page 95 and 96: 2.7 Cloning and synthesis of JFH-1
Page 97 and 98: The sequence encoding amino acids 3
Page 99 and 100: Figure 2-6 sE2 sequencing. 89 JFH-1
Page 101 and 102: 91 7. Large scale transfections wer
Page 103 and 104: 93 3 Results: Investigations using
Page 105 and 106: 95 3.2 CHO cells expressing human S
Page 107 and 108: Figure 3-3 Ability of anti-E2 mAb t
Page 109 and 110: 99 3.3 The interaction of sE2 with
Page 111 and 112: 101 Figure 3-5 Position of SR-BI mu
Page 113 and 114: 103 Table 3-2 Investigation of cell
Page 115 and 116: 105 3.4 Expression of SR-BII in CHO
Page 117 and 118: 3.5 Discussion. 107 We have demonst
Page 119 and 120: 109 may offer a tool to study the i
Page 121 and 122: 111 Figure 4-1A displays endogenous
Page 123 and 124: 113 4.2 Exogenous expression of SR-
Page 125 and 126: 115 4.3 Evidence of enhanced JFH-1
Page 127: 117 4.4 SR-BI expression levels lim
Page 131 and 132: 121 4.6 SR-BI/II expression levels
Page 133 and 134: 123 Figure 4-5 Over expression of S
Page 135 and 136: 125 Figure 4-6 Anti-SR-BI serum inh
Page 137 and 138: 127 Figure 4-7 Infection of Huh-7.5
Page 139 and 140: 129 cells is largely manifested in
Page 141 and 142: 131 5 Results: Identification of a
Page 143 and 144: 133 Figure 5-1 JFH-1 G451R has an a
Page 145 and 146: 135 Figure 5-2 CD81 dependence of J
Page 147 and 148: 137 G451R sE2 with hCD81 LEL we com
Page 149 and 150: 139 Figure 5-4 JFH-1 wt and G451R s
Page 151 and 152: 141 demonstrated a lower dependence
Page 153 and 154: 143 Figure 5-6 Relationship between
Page 155 and 156: 145 flow cytometry (data not shown)
Page 157 and 158: 147 5.6 Low density JFH-1 particles
Page 159 and 160: 5.7 Discussion. 149 We have demonst
Page 161 and 162: 151 Numerous reports have used dens
Page 163 and 164: 153 demonstrate that low density JF
Page 165 and 166: 155 to over express SR-BI we were a
Page 167 and 168: 157 5-1), better able to engage CD8
Page 169 and 170: 159 Figure 6-1 An overview of antib
Page 171 and 172: 161 it is believed that ligation of
Page 173 and 174: 163 Given our and other’s finding
Page 175 and 176: 6.3 HCV lipo-viro-particles. 165 As
Page 177 and 178: 167 represent the hyper-variable re
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169 The G451R mutation disrupts the
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7 Bibliography 171 1. Acton, S., D.
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173 include the CD81 tetraspanin an
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175 receptor class B type I and inh
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177 83. Drummer, H. E., K. A. Wilso
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179 KewalRamani, D. R. Littman, C.
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181 King. 1996. Efficient infection
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183 hepatitis C virus envelope glyc
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185 recovered chimpanzees exhibit r
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187 218. Monazahian, M., I. Bohme,
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189 243. Perez-Berna, A. J., J. Gui
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191 single-cycle production assay t
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193 298. Strickland, G. T., S. S. E
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195 Remaley, G. Csako, T. L. Eggerm
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197 2007. Scavenger receptor class
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