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

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vi List of Figures. Figure 1-1 Phylogentic analysis of HCV........................................................... 4 Figure 1-2 A cartoon of a Hepatitis C virion. .................................................. 15 Figure 1-3 HCV genome and polyprotein....................................................... 17 Figure 1-4 Cellular receptors for HCV............................................................ 34 Figure 1-5 The basic structure of a lipoprotein............................................... 44 Figure 1-6 SR-BI-lipoprotein interactions within a hepatocyte. ...................... 51 Figure 1-7 Lipo-viro-particle assembly........................................................... 59 Figure 2-1 In vitro transcribed HCV RNA....................................................... 75 Figure 2-2 Electroporated Huh-7.5 cells. ....................................................... 77 Figure 2-3 Basic plasmid map of pJZ189....................................................... 82 Figure 2-4 Restriction Map............................................................................. 83 Figure 2-5 SR-BII restriction digest................................................................ 84 Figure 2-6 Basic plasmid map of PCDNA3.1 H E2....................................... 86 Figure 2-7 PCDNA3.1 H E2 digest................................................................. 86 Figure 2-8 sE2 coding region PCR. ............................................................... 88 Figure 2-9 sE2 sequencing. ........................................................................... 89 Figure 2-10 Expression of sE2....................................................................... 90 Figure 2-11 Titration of sE2............................................................................ 92 Figure 3-1 Expression of SR-BI on CHO cells. .............................................. 94 Figure 3-2 Activity of rat anti-E2 monoclonal antibodies................................ 96 Figure 3-3 Ability of anti-E2 mAb to recognise SR-BI bound E2.................... 97 Figure 3-4 Soluble HCV E2 binding to cell expressed SR-BI......................... 98 Figure 3-5 Position of SR-BI mutations........................................................ 101 Figure 3-6 Expression of SR-BI mutants in CHO cells................................. 102 Figure 3-7 Antigenic characterisation of SR-BI mutants. ............................. 104 Figure 3-8 Expression of SR-BII in CHO cells. ............................................ 106 Figure 4-1 Over expression of human SR-BI/II in Huh-7.5 cells. ................. 112 Figure 4-2 Over expression of SR-BI and SR-BII in Huh-7.5 cells enhances HCVcc infection............................................................................................ 114 Figure 4-3 Over expression of SR-BI and SR-BII in Huh-7.5 cells increases infected cell focus size. ................................................................................ 116 Figure 4-4 Over expression of SR-BI and SR-BII in Huh-7.5 cells enhances HCVpp-JFH infection. .................................................................................. 118 Figure 4-5 Expression of murine SR-BI in Huh-7.5 cells does not enhance JFH-1 infection............................................................................................. 120 Figure 4-6 Over expression of SR-BI and SR-BII in Huh-7.5 cells enhances the infectivity of cell culture- and plasma derived J6/JFH. ........................... 123 Figure 4-7 Anti-SR-BI serum inhibits cell culture- and plasma-derived J6/JFH infectivity. ..................................................................................................... 125 Figure 4-8 Infection of Huh-7.5 over expression SR-BI by cell culture adapted JFH-1 HCVcc. .............................................................................................. 127 Figure 5-1 JFH-1 G451R has an altered dependence on SR-BI. ................ 133 Figure 5-2 CD81 dependence of JFH-1 wt and G451R infection................. 135 Figure 5-3 JFH-1 wt and G451R soluble E2 interaction with CHO cells expressing SR-BI and CD81........................................................................ 138 Figure 5-4 JFH-1 wt and G451R soluble E2 interaction with recombinant CD81............................................................................................................ 139
vii Figure 5-5 Analysis of JFH-1 wt and G451R buoyant density. .................... 142 Figure 5-6 Relationship between particle density and co-receptor dependency. ..................................................................................................................... 143 Figure 5-7 JFH-1 G451R demonstrates an increased sensitivity to neutralisation by gp-specific antibodies. ...................................................... 146 Figure 5-8 Association between JFH-1 particle density and sensitivity to neutralising antibodies. ................................................................................ 148 Figure 6-1 An overview of antibody dependent neutralisation of virus infectivity. ..................................................................................................... 159 Figure 6-2 Model lipo-viro-particle................................................................ 168
Page 1 and 2: The role of scavenger receptor B-I
Page 3 and 4: i Abstract. With 170 million infect
Page 5 and 6: iii Acknowledgements. I would like
Page 7: v 5 Results: Identification of a re
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
Page 57 and 58: 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
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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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