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Title: The interaction of hepatitis C virus and intracellular lipid metabolism
Author: Hubb, Jonathan Ramsay
ISNI:       0000 0004 2669 6886
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2008
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Chronic hepatitis C virus (HCV) infection causes inflammation of the liver, which can lead to fibrosis and cirrhosis over time. Whether liver damage is a consequence of viral infection or is due to an immune mediated response is not clear. Steatosis is a histopathological feature often found in HCV infected patients. Steatosis is the accumulation of intracytoplasmic lipid droplets within hepatocytes. It has been linked to the progression of fibrosis (Adinolfi et al., 2001). Steatosis was found significantly more frequently in patients infected with HCV genotype 3 than those infected with genotype 1 (Mihm et al., 1997). Currently there is no cell-based method of investigating the life cycle of HCV genotype 3 and transgenic mice studies have been restricted to genotype 1 proteins. Three chimpanzees experimentally infected with HCV showed differential regulation of genes encoding enzymes concerned with lipid metabolism. Treatment of HCV genotype 1b replicon containing cells with cerulenin, which inhibits fatty acid synthase, reduced replication of HCV RNA in a dose dependent manner (Su et al., 2002). Polyunsaturated fatty acids (PUFAs) have recently been shown to inhibit replication of a genotype 1b sub-genomic replicon. PUFAs are essential and are known to down regulate lipogenic gene expression. However, the inhibitory effect of PUFAs on HCV RNA levels was thought to be independent of their inhibitory effect on fatty acid biosynthesis (Kapadia et al., 2005). To assess the effects of cerulenin and fatty acids on HCV genome replication we measured replication by northern blot analysis of total HCV RNA and using a replicon expressing luciferase. HCV protein production was measured by western blot using an antibody to the NS5A protein. To examine the effect on long chain fatty acid synthesis, we measured incorporation of 14C acetate into total cellular lipids. Toxicity was assayed using mitochondrial enzyme activity assays. Treating genotype 1b replicon cells with 30 μM cerulenin led to inhibition of fatty acid biosynthesis and a corresponding inhibition of HCV RNA replication. However, at this level of cerulenin, only 60 % of cells were viable. Inhibition of fatty acid biosynthesis was not observed at the lower non-toxic concentrations of 10 μM and 3 μM, although HCV replication was inhibited. These experiments were repeated using more frequent media changes and different suppliers of cerulenin. However, similar results were obtained. When a genotype 2a replicon expressing cell line (JFH1) was treated with cerulenin it was possible to inhibit both HCV RNA levels and fatty acid biosynthesis in a dose dependant manner. Furthermore cerulenin treatment of an alternative genotype 1b expressing cell line led to an inhibition of fatty acid synthesis in a dose dependent manner. We have studied the effects of the PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on JFH1 replicon (genotype 2) replication using both constitutive and transiently expressing systems. For a control, we used oleic acid, a monounsaturated fatty acid. DHA and EPA administered from 3 to 100 μM concentration showed a dose responsive reduction in replication. Fatty acid biosynthesis was also inhibited; however at the higher concentrations there were reductions in cell viability. Oleic acid did not effectively inhibit JFH1 replication even though, at higher concentrations, there was a small reduction in 14C acetate incorporation. Initial immunofluorescence data indicated that NS5A foci were not disrupted by treatment of cells with PUFAs and fluorescence recovery after photobleaching data indicated that PUFAs did not increase ER membrane fluidity. A genotype 3 genome was amplified and sequenced using reverse-transcription polymerase chain reaction (RT-PCR) from the serum of an HCV genotype 3ainfected patient. A majority sequence was assembled and amplification products were ligated into vectors, which were sequenced and mutated back to the majority sequence. The genotype 3 genome was modified by the exclusion of the structural genes and non-structural (NS) protein 2. A bicistronic replicon was created in which the HCV internal ribosome entry site (IRES) controlled expression of the selectable marker neomycin phosphotransferase and the encephalomyocarditis virus IRES controlled expression of the NS proteins. RNA replicons were transcribed and electroporated into HuH-7 cell lines. A transiently expressing replicon was made by replacing the neomycin gene with a firefly luciferase gene. Cells expressing neither the constitutively nor the transiently genotype 3 replicon sustained viral replication. In conclusion cerulenin inhibited HCV replication at levels, which did not inhibit fatty acid biosynthesis and were not toxic. There was toxicity at cerulenin concentrations, which inhibited fatty acid biosynthesis. Cerulenin inhibited replication but by a mechanism other than inhibition of fatty acid biosynthesis. Cells with different passage histories were shown to behave differently to each other in their response to drugs. The PUFAs, DHA and EPA exert an inhibitory effect on HCV replicon replication and fatty acid biosynthesis at non-toxic levels. Oleic acid did not inhibit HCV replication at equivalent concentrations. The mechanism behind PUFA inhibition of HCV RNA levels is still unknown. An attempt to create genotype 3 constitutively and transiently expressing replicon HuH-7 cell lines failed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR355 Virology