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

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156 interactions and we are preparing to carry out a series of experiments to address this. It has been previously shown that antibodies specific to SR-BI and CD81 block HCV infection in a synergistic manner, suggesting that they function co- operatively during HCV entry (139, 346). Our studies using JFH-1 G451R have for the first time shed light on an inter-dependence between HCV interaction with SR-BI and CD81 (Figure 5-1 & Figure 5-2). Taken together, these data point towards sequential receptor engagement by HCV, much like that seen with other viruses (184, 293, 315, 335). It is believed that HCV interaction with CD81 occurs after a particle has bound to the cell surface (31, 91, 346). Indeed Flint et. al. demonstrated that particle neutralisation by hCD81 LEL can only occur post binding (101), suggesting that HCV attachment primes particle envelope proteins for CD81 engagement. Evans et. al. reported that HCVcc particles bind to CHO-SR-BI cells whilst not to cells expressing CD81 or CLDN1 (91). We hypothesise that HCV engagement of SR-BI precedes and facilitates further interactions with co-receptors such as CD81, indeed, many of the epitopes exposed on sE2 bound to SR-BI are critical for CD81 binding ( Figure 3-3). Building on this model, the G451R mutation may cause a conformational change that primes E2 to interaction with CD81 and therefore negates the requirement for prior attachment to SR-BI. In support of this we demonstrate that JFH-1 G451R infectivity is less dependent on SR-BI (Figure
157 5-1), better able to engage CD81 (Figure 5-2Figure 5-3Figure 5-4) and is highly susceptible to polyclonal neutralising IgG (Figure 5-7) suggesting an alteration in the conformation of E2. Such a hypothesis is not unprecedented, primary attachment of HIV to CD4 + lymphoid cells promotes a conformational change in the viral envelope protein, gp120, which exposes epitopes that are critical for later stages of entry (273, 315, 335). As a result, CD4 expression levels limit HIV entry and cell culture adaptation leads to viruses with reduced dependence on CD4 and a heightened sensitivity to nAbs (13, 38, 86, 129, 249). Similar observations have been made using mutant respiratory syncytial virus (196). Over expression of SR-BI enhanced JFH-1 infection to greater extent than J6/JFH, suggesting strain specific differences in SR-BI dependency. Given our findings using JFH-1 G451R it will be interesting to investigate how this phenotype correlates with particle CD81 engagement and sensitivity to neutralising antibodies. HDL (high density lipoprotein) enhances HCVcc infection, this process is thought to involve SR-BI mediated cholesterol exchange, followed by the liberation of apoC-I from HDL (25, 79, 324, 325). Recent reports suggest that apoC-I integrates into HCV particles and enhances attachment and fusion events (79, 210, 211). Notably, JFH-1 G451R did not respond to supplemental HDL (Figure 5-1), this is most likely linked to its lower dependence on SR-BI. Based on our current understanding, it is difficult to speculate on the mechanisms by which apoC-I promotes infection, however our data suggests
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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
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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
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 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: 155 to over express SR-BI we were a
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
Page 189 and 190: 179 KewalRamani, D. R. Littman, C.
Page 191 and 192: 181 King. 1996. Efficient infection
Page 193 and 194: 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,
Page 199 and 200: 189 243. Perez-Berna, A. J., J. Gui
Page 201 and 202: 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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