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

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120 Figure 4-4 Expression of murine SR-BI in Huh-7.5 cells does not enhance JFH-1 infection. Huh-7.5 cells were transiently transfected to express murine GFP-SR-BI chimera 48 hrs prior to challenge with JFH-1, the infection was allowed to proceed for 72 h. Infected cells were realised by staining for intracellular NS5A followed by an alexaflour 633 conjugated secondary. Flow cytometric analysis allowed quantification of infected cells within moSR-BI –ve and +ve populations. A. Gating of GFP-moSR-BI –ve (black) and GFP-moSR-BI +ve (red) populations, the left hand panel displays untransfected Huh-7.5 cells. In transfected Huh-7.5 cells 18% were GFP-moSR-BI +ve (Cell counts: - ve = 45,000 +ve = 10,000) B. NS5A +ve cells within each population; GFP-moSR-BI –ve = 7.1% (~3000 cells), GFP-moSR-BI +ve = 9.1% (~1000 cells) C. percentage infected cells within each population, difference does not reach significance (unpaired t-test). Errors bars indicate standard deviation from the mean (n=3).
121 4.6 SR-BI/II expression levels modulate plasma-derived J6/JFH infectivity. The production of HCV virions is believed to be dependent on the assembly and secretion of VLDL, indeed Huh-7.5 are thought to produce HCV lipo-viro- particles (63, 109, 131, 224). However, it is accepted that the HCVcc system is only a surrogate model of in vivo virion production and as such may not support authentic HCV-lipoprotein associations. Numerous studies have shown that HCVcc is infectious in animal models such as the chimpanzee (144, 180), virus recovered from infected subjects have a lower buoyant density than cell culture virus (180), suggesting increased lipoprotein association. We therefore used J6/JFH virus recovered from the plasma of infected uPA-SCID mice with transplanted human livers (208, 209), to investigate the relationship between plasma derived virus and SR-BI/II. J6/JFHplasma infection of Huh-7.5 cells was enhanced by over expression of SR-BI/II in a comparable manner to J6/JFHHCVcc(Figure 4-5 A)suggesting that J6/JFHplasma virus has a similar dependence for SR-BI to virus produced in vitro. However, Maillard et. al. reported that human sera derived HCV may interact with SR-BI via associated apoproteins and not virion envelope proteins (190). To address the importance of E2 glycoproteins in J6/JFHplasma infection we pre-incubated particles with ant-E2 mAb C1 prior to inoculation of Huh-7.5 cells (Figure 4-5 B). Both plasma and cell culture derived J6/JFH are neutralised by mAb C1, this implies that E2 is essential for infection by either virus. mAb C1 is reported to inhibit E2-CD81 interaction (172), however its
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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
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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 and 128: 117 4.4 SR-BI expression levels lim
Page 129: 119 4.5 Murine SR-BI does not enhan
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
Page 179 and 180: 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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