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

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4.9 Discussion. 128 Over expression of SR-BI in Huh-7.5 cells increases their susceptibility to HCVcc (Figure 4-2), indicating that infection is limited by SR-BI expression levels. We made similar, though less pronounced, observations using cells over expressing the splice variant SR-BII, suggesting that it is a functionally active receptor for HCV attachment and/or entry (Figure 4-2). Comparable findings were reported for CD4 and chemokine receptor expression levels influencing human immunodeficiency virus cell entry (13, 249). Over expression of SR-BI enhanced JFH-1 infectivity to a much greater extent than J6/JFH; 18-fold and 3-fold, respectively (Figure 4-2), suggesting strain specific variation within genotypic clades. HCVpp expressing diverse glycoproteins vary in their ability to infect HepG2 cells expressing CD81, supporting a model of genotypic variants with different dependencies for the viral co-receptors (101, 202). HCVpp-JFH-1 infection of Huh-7.5 cells over expressing SR-BI was enhanced 2 fold indicating that the restriction occurs at the point of virus entry (Figure 4-4). However, this increase in HCVpp infection, was much less pronounced than that seen with JFH-1 HCVcc, suggesting a disparity between the two model systems. This discrepancy may be accounted for by the increase in JFH-1 infected cell focus size in cells over expressing SR-BI, as foci occur following multiple rounds of replication and HCVpp do not mimic this process. The fact that a large proportion of JFH-1 infected Huh-7.5 TRIP SR-BI cells reside in large foci (Figure 4-3) suggests that the phenotype seen in these
129 cells is largely manifested in secondary and local transmission. It has been recently shown that HCV is able to transfer infectivity between cells, resulting in the formation of large infected cell foci (314). Our data suggest that SR-BI promotes this route of transmission. J6/JFH is infectious in vivo and virus recovered from infected animal models can be recultured in vitro. These HCVplasma particles are of a lower buoyant density and are thought to represent the particle-lipoprotein associations found in human plasma (180). SR-BI over expression enhanced plasma derived virus in a comparable manner to cell culture virus and anti-SR-BI serum inhibited the infection of both viruses (Figure 4-5 & Figure 4-6). The infectivity of both viruses was neutralised by anti-E2 mAb C1 (Figure 4-5), indicating that HCVplasma particle entry is dependent on viral envelope proteins in an analogous manner to HCVcc. These data suggest that particle associated lipoproteins do not alter the relationship with SR-BI, a view supported by our findings using murine SR-BI (Figure 4-4). This implies that HCVcc particles do not interact non-specifically with murine-SR-BI via associated apoproteins and that a specific interaction between HCV E2 and human SR-BI is necessary for enhanced infection in over expressing cells. Interestingly, infection of Huh-7.5 cells over expressing SR-BI by a cell culture adapted JFH-1 variant was only enhanced ~3 fold (Figure 4-7). This observation suggests that adaptation to Huh-7.5 leads to a lower dependency on SR-BI and consequently its expression levels do not limit infection to the same extent. The adaptation of viruses to cell culture is a well documented
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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
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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
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 and 130: 119 4.5 Murine SR-BI does not enhan
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: 127 Figure 4-7 Infection of Huh-7.5
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
Page 181 and 182: 7 Bibliography 171 1. Acton, S., D.
Page 183 and 184: 173 include the CD81 tetraspanin an
Page 185 and 186: 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
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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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