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

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22 HCV assembly and release is intrinsically linked to the production of very low density lipoproteins (VLDL) by hepatocytes (63, 109, 131). Lipoproteins are spherical particles that deliver dietary lipids to tissues throughout the body (321). The mechanisms by which this occurs are pertinent to the work documented in this thesis and will be addressed in greater detail in sections 1.5 and 1.7. As discussed earlier, HCV is genetically diverse, however variability is not confined to the global level; an infected individual will harbour a population of viruses known as a quasispecies swarm (44, 197). A HCV patient will produce around 10 12 RNA copies per day (229), such a large population will contain genetic variants covering each base position of the entire genome (289). Although the swarm will display a sequence consensus at any given time, it is highly able to respond to new evolutionary pressures as they arise (44). This phenomena will become increasingly important as new anti-HCV treatments become available. As seen in HIV, quasispecies swarms are quick to develop drug resistance if treatment is not managed appropriately (69, 176). Rapid quasispecies evolution also contributes to the ability of HCV to continually escape host immune responses (85, 327). Analysis of genetic variation within genotypic or quasispecies populations has identified highly conserved and diverse regions of the genome. There is pressure to conserve sequences that encode indispensable functions such as the IRES or the NS5B polymerase (289). Whereas other regions, particularly
23 in the E1 and E2 glycoprotein genes, display high variation (292, 332). Two particular stretches of E2, known as the hypervariable regions I and II (HVR-I and HVR-II), are thought to represent neutralisation epitopes under persistent immunological pressure (reviewed in (292)). Our understanding of HCV has expanded greatly since the turn of the century, largely due to the technological breakthroughs of HCV replicons and HCVcc in vitro assays. However, many processes remain poorly understood, none more so than the events leading to HCV internalisation.
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 and 8: v 5 Results: Identification of a re
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: 21 The characterisation of HCV geno
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
Page 59 and 60: 49 uptake of cholesterol from LDL (
Page 61 and 62: Figure 1-4 SR-BI-lipoprotein intera
Page 63 and 64: 53 In summary the class B scavenger
Page 65 and 66: 55 In 1992 Thomssen et. al. demonst
Page 67 and 68: 57 lipoprotein association promotes
Page 69 and 70: 59 Figure 1-5 Lipo-viro-particle as
Page 71 and 72: 61 In vitro culture medium, into wh
Page 73 and 74: 2 Materials and methods. 2.1 Cell l
Page 75 and 76: 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
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73 proteins and are incapable of fu
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75 spectrophotometer (Amersham), we
Page 87 and 88:
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
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
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109 may offer a tool to study the i
Page 121 and 122:
111 Figure 4-1A displays endogenous
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113 4.2 Exogenous expression of SR-
Page 125 and 126:
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)
Page 157 and 158:
147 5.6 Low density JFH-1 particles
Page 159 and 160:
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
Page 187 and 188:
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
Page 195 and 196:
185 recovered chimpanzees exhibit r
Page 197 and 198:
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
Page 203 and 204:
193 298. Strickland, G. T., S. S. E
Page 205 and 206:
195 Remaley, G. Csako, T. L. Eggerm
Page 207:
197 2007. Scavenger receptor class
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