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

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170 we have, for the first time, demonstrated an interplay between HCV particle interaction with co-receptors, neutralising antibodies and host lipoproteins. We hope to continue this line of investigation, focussing on how HCV LVP composition and structure defines envelope protein exposure, receptor engagement and ultimately infectivity. In continuing our work it is important to continuously evaluate the techniques we use. Cell culture proficient HCV is a very powerful tool, however we must make efforts to understand how our findings relate to the situation within a HCV infected individual. Much of the observations documented in this thesis are virtue of a lab adapted virus and, although fascinating, JFH-1 G451R is emblematic of the problems faced by those using a cell culture proficient HCV. Additionally, some of the inherent limitations in the HCVcc system lie in the cells used to support infection; Huh-7.5 cells are a very good target for infection but doubts remain over how well they represent human hepatocytes. Therefore, the ongoing attempts to propagate patient derived HCV in primary human hepatocytes (51, 216, 217) are an important aspect of HCV research. Consolidating observations from cell culture, animal models and HCV infected individuals will pave the way towards more effective treatments in the future.
7 Bibliography 171 1. Acton, S., D. Osgood, M. Donoghue, D. Corella, M. Pocovi, A. Cenarro, P. Mozas, J. Keilty, S. Squazzo, E. A. Woolf, and J. M. Ordovas. 1999. Association of polymorphisms at the SR-BI gene locus with plasma lipid levels and body mass index in a white population. Arterioscler Thromb Vasc Biol 19:1734-43. 2. Acton, S., A. Rigotti, K. T. Landschulz, S. Xu, H. H. Hobbs, and M. Krieger. 1996. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518-20. 3. Agnello, V., G. Abel, G. B. Knight, and E. Muchmore. 1998. Detection of widespread hepatocyte infection in chronic hepatitis C. Hepatology 28:573-84. 4. Ahras, M., T. Naing, and R. McPherson. 2008. Scavenger receptor class B type I localizes to a late endosomal compartment. J Lipid Res 49:1569-76. 5. Alberti, A., L. Chemello, and L. Benvegnu. 1999. Natural history of hepatitis C. J Hepatol 31 Suppl 1:17-24. 6. Ali, N., K. D. Tardif, and A. Siddiqui. 2002. Cell-free replication of the hepatitis C virus subgenomic replicon. J Virol 76:12001-7. 7. Alter, M. J. 2006. Epidemiology of viral hepatitis and HIV co-infection. J Hepatol 44:S6-9. 8. Andre, P., F. Komurian-Pradel, S. Deforges, M. Perret, J. L. Berland, M. Sodoyer, S. Pol, C. Brechot, G. Paranhos-Baccala, and V. Lotteau. 2002. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76:6919-28. 9. Andreo, U., P. Maillard, O. Kalinina, M. Walic, E. Meurs, M. Martinot, P. Marcellin, and A. Budkowska. 2007. Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection. Cell Microbiol 9:2445-56. 10. Ariumi, Y., M. Kuroki, K. Abe, H. Dansako, M. Ikeda, T. Wakita, and N. Kato. 2007. DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication. J Virol 81:13922-6. 11. Azhar, S., L. Tsai, S. Medicherla, Y. Chandrasekher, L. Giudice, and E. Reaven. 1998. Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis. J Clin Endocrinol Metab 83:983-91. 12. Bacon, B. R., and J. G. McHutchison. 2007. Into the light: strategies for battling hepatitis C. Am J Manag Care 13 Suppl 12:S319-26. 13. Bannert, N., D. Schenten, S. Craig, and J. Sodroski. 2000. The level of CD4 expression limits infection of primary rhesus monkey macrophages by a T-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses. J Virol 74:10984- 93. 14. Baranowski, E., C. M. Ruiz-Jarabo, N. Sevilla, D. Andreu, E. Beck, and E. Domingo. 2000. Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage. J Virol 74:1641-7.
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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
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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
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 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: 169 The G451R mutation disrupts the
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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