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

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iv Table of contents. 1 Introduction............................................................................................. 1 1.1 The disease. ..................................................................................... 1 1.2 An elusive pathogen. ......................................................................... 9 1.3 Basic virology................................................................................... 15 1.4 Attachment and entry....................................................................... 24 1.4.1 Co-receptors............................................................................. 26 1.4.2 Attachment factors.................................................................... 35 1.4.3 Endocytosis and fusion............................................................. 37 1.4.4 Co-receptor interplay and localisation. ..................................... 39 1.5 Lipoproteins ..................................................................................... 43 1.6 Scavenger receptor B-I.................................................................... 48 1.7 HCV and lipoproteins....................................................................... 54 1.7.1 Assembly and release. ............................................................. 57 1.7.2 Low density lipoprotein receptor and virus entry. ..................... 60 1.8 Project Objectives............................................................................ 62 2 Materials and methods......................................................................... 63 2.1 Cell lines. ......................................................................................... 63 2.2 Antibodies........................................................................................ 63 2.3 Plasmids and proteins. .................................................................... 66 2.4 Basic techniques.............................................................................. 67 2.5 Cell culture proficient hepatitis C virus............................................. 74 2.6 Cloning of TRIP SR-BII.................................................................... 82 2.7 Cloning and synthesis of JFH-1 wt and G451R soluble E2............. 85 3 Results: Investigations using CHO cell expressed SR-BI. ............... 93 3.1 Expression of SR-BI in CHO cells. .................................................. 93 3.2 CHO cells expressing human SR-BI bind soluble HCV E2 glycoprotein. ............................................................................................... 95 3.3 The interaction of sE2 with a panel of SR-BI mutants. .................... 99 3.4 Expression of SR-BII in CHO cells. ............................................... 105 3.5 Discussion. .................................................................................... 107 4 Results: Scavenger Receptor BI and BII Expression Levels Modulate HCV Infectivity............................................................................................ 110 4.1 Over expression of SR-BI/II in Huh-7.5 cells. ................................ 110 4.2 Exogenous expression of SR-BI/II in Huh-7.5 cells enhances HCVcc infection. ................................................................................................... 113 4.3 Evidence of enhanced JFH-1 transmission in cells over expressing SR-BI. ....................................................................................................... 115 4.4 SR-BI expression levels limit the entry of HCV pseudo-particles. . 117 4.5 Murine SR-BI does not enhance HCVcc infection......................... 119 4.6 SR-BI/II expression levels modulate plasma-derived J6/JFH infectivity................................................................................................... 121 4.7 Anti-SR-BI serum inhibits cell culture and plasma derived J6/JFH infection. ................................................................................................... 124 4.8 Cell culture adaptation of JFH-1 HCVcc reduces SR-BI dependency. ....................................................................................................... 126 4.9 Discussion. .................................................................................... 128
v 5 Results: Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and particle sensitivity to neutralising antibodies........... 131 5.1 JFH-1 G451R has a reduced dependence on SR-BI. ................... 131 5.2 Cell culture adapted JFH-1 G451R has an increased sensitivity to neutralisation by soluble CD81................................................................. 134 5.3 sE2 glycoprotein bearing the G451R mutation demonstrates increased binding to CD81. ...................................................................... 136 5.4 Relationship between JFH-1 and G451R particle density, infectivity and co-receptor interactions..................................................................... 140 5.5 Adapted JFH-1 demonstrates an increased sensitivity to nAbs. ... 144 5.6 Low density JFH-1 particles are less sensitive to nAbs................. 147 5.7 Discussion. .................................................................................... 149 6 Discussion and conclusions. ............................................................ 154 6.1 SR-BI and HCV attachment and entry........................................... 154 6.2 Antibody mediated neutralisation of HCV...................................... 158 6.3 HCV lipo-viro-particles................................................................... 165 6.4 Closing remarks............................................................................. 169 7 Bibliography........................................................................................ 171
Page 1 and 2: The role of scavenger receptor B-I
Page 3 and 4: i Abstract. With 170 million infect
Page 5: iii Acknowledgements. I would like
Page 9 and 10: vii Figure 5-5 Analysis of JFH-1 wt
Page 11 and 12: 1 Introduction 1.1 The disease. 1 D
Page 13 and 14: 3 predominantly immuno-pathogenic i
Page 15 and 16: 5 Aside from considerations of the
Page 17 and 18: 7 Current attempts to design a HCV
Page 19 and 20: 1.2 An elusive pathogen. 9 The emer
Page 21 and 22: 11 PCR). This technique is highly s
Page 23 and 24: 13 Within six years of the descript
Page 25 and 26: 1.3 Basic virology. 15 Like other m
Page 27 and 28: 17 Figure 1-2 HCV genome and polypr
Page 29 and 30: 19 241). E1 and E2 are anchored to
Page 31 and 32: 21 The characterisation of HCV geno
Page 33 and 34: 23 in the E1 and E2 glycoprotein ge
Page 35 and 36: 25 The solution reached by a great
Page 37 and 38: 27 binding to mouse cells. sE2 inte
Page 39 and 40: 29 density lipoprotein (HDL), allow
Page 41 and 42: 31 blood brain barrier is permeable
Page 43 and 44: 33 act as receptors for viral entry
Page 45 and 46: 1.4.2 Attachment factors 35 The tru
Page 47 and 48: 1.4.3 Endocytosis and fusion 37 Aft
Page 49 and 50: 39 1.4.4 Co-receptor interplay and
Page 51 and 52: 41 inhibition of protein kinase A (
Page 53 and 54: 1.5 Lipoproteins 43 Since 1992 ther
Page 55 and 56: 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 (
Page 61 and 62:
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
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69 6. Finally, the cells were washe
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71 (Promega, WI, USA) kits accordin
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73 proteins and are incapable of fu
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75 spectrophotometer (Amersham), we
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77 Figure 2-2 Electroporated Huh-7.
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79 5. Data is expressed as percenta
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81 (Invitrogen). The samples to be
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83 1. The SR-BII coding region was
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2.7 Cloning and synthesis of JFH-1
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The sequence encoding amino acids 3
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Figure 2-6 sE2 sequencing. 89 JFH-1
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91 7. Large scale transfections wer
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93 3 Results: Investigations using
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95 3.2 CHO cells expressing human S
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Figure 3-3 Ability of anti-E2 mAb t
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99 3.3 The interaction of sE2 with
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101 Figure 3-5 Position of SR-BI mu
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103 Table 3-2 Investigation of cell
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105 3.4 Expression of SR-BII in CHO
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3.5 Discussion. 107 We have demonst
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109 may offer a tool to study the i
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111 Figure 4-1A displays endogenous
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113 4.2 Exogenous expression of SR-
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115 4.3 Evidence of enhanced JFH-1
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117 4.4 SR-BI expression levels lim
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119 4.5 Murine SR-BI does not enhan
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121 4.6 SR-BI/II expression levels
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123 Figure 4-5 Over expression of S
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125 Figure 4-6 Anti-SR-BI serum inh
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127 Figure 4-7 Infection of Huh-7.5
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129 cells is largely manifested in
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131 5 Results: Identification of a
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133 Figure 5-1 JFH-1 G451R has an a
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135 Figure 5-2 CD81 dependence of J
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137 G451R sE2 with hCD81 LEL we com
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139 Figure 5-4 JFH-1 wt and G451R s
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141 demonstrated a lower dependence
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143 Figure 5-6 Relationship between
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145 flow cytometry (data not shown)
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147 5.6 Low density JFH-1 particles
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5.7 Discussion. 149 We have demonst
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151 Numerous reports have used dens
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153 demonstrate that low density JF
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155 to over express SR-BI we were a
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157 5-1), better able to engage CD8
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159 Figure 6-1 An overview of antib
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161 it is believed that ligation of
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163 Given our and other’s finding
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6.3 HCV lipo-viro-particles. 165 As
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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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