Studies of the association of hepatitis C virus and host lipoproteins in vivo and in cell culture

PhD ThesisThe purpose of this project has been to investigate the mechanism of hepatitis C virus/lipoprotein complexes of lipo-viro-particles (LVP) derived from transplanted liver macerates of an HCV infected immunodeficient patient (S6b) and density-fractionated from an iodixanol gradient. Previous studies have indicated that serum derived HCV may bind to hepatocytes via the LDL-receptor (LDLr) or the scavenger receptor SR-B1. The role of these receptors in the uptake of LLVP in HepG2 cells was investigated by comparing LLVP uptake with that of dioctadecylindocarbocyanine-labeled-low density lipoprotein (DiI-LDL) and similarly labeled oxidized LDL (DiI-oxLDL), know to be taken up predominantly via the LDLr and SR-B1 respectively. DiI-LDL and DiI-oxLDL binding resembled that of LLVP in being significantly increased by insulin and LPDS treatment. DiI-LDL but not DiI-oxLDL binding was also decreased by hydroxycholesterol. These results suggest that all three lipoprotein particles may be taken up via the LDLr which binds predominantly via apolipoprotein B100. To confirm this, the inhibition of binding studies were conducted. Whilst binding of DiI-LDL was reduced by 98% by pre-incubation with anti-apoB100 antibodies, binding of both LLVP and DiI-oxLDL was enhanced. Using confocal microscopy and FACS analysis, we compared the role of glycosaminoglycans (GAGs) in the binding of the three particles types by washing cells with suramine, previously shown to remove GAG bound-LDL. Like LLVP, DiI-LDL bound at 0 degrees Celsius was washed off with suramine but became resistant at 37 degrees Celsius. In contrast, washing off DiI-oxLDL with suramine at both 0 degrees Celsius and 37 degrees Celsius had no significant effect. The binding patterns of LLVP therefore differ from those of DiI-LDL and DiI-oxLDL. To investigate whether such differences might be due to viral glycoprotein E2, the effect of polyclonal antibody to E2 and a monoclonal antibody to the E2-hypervariable region (HVR1) on binding of LLVP to HepG2 cells was assessed. Neither anti-E2 nor anti-HVR1 blocked binding.The Malaysian Government, Wellcome Trust, Professor Geoffrey Tom