The interaction of hepatitis C virus and intracellular lipid metabolism

QR355 Virology

Hubb, Jonathan Ramsay ; McCruden, E

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Hepatitis C Virus, HCV, Fatty acid biosynthesis, Polyunsaturated fatty acids, lipid metabolism, virology, fatty acid synthase;   

Others  :  http://theses.gla.ac.uk/133/1/2008hubbphd.pdf

来源: University of Glasgow

【 摘 要 】

Chronic hepatitis C virus (HCV) infection causes inflammation of the liver, whichcan lead to fibrosis and cirrhosis over time. Whether liver damage is aconsequence of viral infection or is due to an immune mediated response is notclear. Steatosis is a histopathological feature often found in HCV infectedpatients. Steatosis is the accumulation of intracytoplasmic lipid droplets withinhepatocytes. It has been linked to the progression of fibrosis (Adinolfi et al.,2001). Steatosis was found significantly more frequently in patients infectedwith 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 HCVgenotype 3 and transgenic mice studies have been restricted to genotype 1proteins.Three chimpanzees experimentally infected with HCV showed differentialregulation of genes encoding enzymes concerned with lipid metabolism.Treatment of HCV genotype 1b replicon containing cells with cerulenin, whichinhibits fatty acid synthase, reduced replication of HCV RNA in a dose dependentmanner (Su et al., 2002).Polyunsaturated fatty acids (PUFAs) have recently been shown to inhibitreplication of a genotype 1b sub-genomic replicon. PUFAs are essential and areknown to down regulate lipogenic gene expression. However, the inhibitoryeffect of PUFAs on HCV RNA levels was thought to be independent of theirinhibitory effect on fatty acid biosynthesis (Kapadia et al., 2005).To assess the effects of cerulenin and fatty acids on HCV genome replication wemeasured replication by northern blot analysis of total HCV RNA and using areplicon expressing luciferase. HCV protein production was measured bywestern blot using an antibody to the NS5A protein. To examine the effect onlong chain fatty acid synthesis, we measured incorporation of 14C acetate intototal cellular lipids. Toxicity was assayed using mitochondrial enzyme activityassays.Treating genotype 1b replicon cells with 30 μM cerulenin led to inhibition offatty acid biosynthesis and a corresponding inhibition of HCV RNA replication.However, at this level of cerulenin, only 60 % of cells were viable. Inhibition offatty acid biosynthesis was not observed at the lower non-toxic concentrationsof 10 μM and 3 μM, although HCV replication was inhibited. These experimentswere repeated using more frequent media changes and different suppliers ofcerulenin. However, similar results were obtained. When a genotype 2areplicon expressing cell line (JFH1) was treated with cerulenin it was possible toinhibit both HCV RNA levels and fatty acid biosynthesis in a dose dependantmanner. Furthermore cerulenin treatment of an alternative genotype 1bexpressing cell line led to an inhibition of fatty acid synthesis in a dosedependent manner.We have studied the effects of the PUFAs, docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) on JFH1 replicon (genotype 2) replication using bothconstitutive and transiently expressing systems. For a control, we used oleicacid, a monounsaturated fatty acid. DHA and EPA administered from 3 to 100μM concentration showed a dose responsive reduction in replication. Fatty acidbiosynthesis was also inhibited; however at the higher concentrations there werereductions in cell viability. Oleic acid did not effectively inhibit JFH1 replicationeven though, at higher concentrations, there was a small reduction in 14Cacetate incorporation. Initial immunofluorescence data indicated that NS5A fociwere not disrupted by treatment of cells with PUFAs and fluorescence recoveryafter photobleaching data indicated that PUFAs did not increase ER membranefluidity.A genotype 3 genome was amplified and sequenced using reverse-transcriptionpolymerase chain reaction (RT-PCR) from the serum of an HCV genotype 3ainfectedpatient. A majority sequence was assembled and amplificationproducts were ligated into vectors, which were sequenced and mutated back tothe majority sequence. The genotype 3 genome was modified by the exclusionof the structural genes and non-structural (NS) protein 2. A bicistronic repliconwas created in which the HCV internal ribosome entry site (IRES) controlledexpression of the selectable marker neomycin phosphotransferase and theencephalomyocarditis virus IRES controlled expression of the NS proteins. RNAreplicons were transcribed and electroporated into HuH-7 cell lines. Atransiently expressing replicon was made by replacing the neomycin gene with afirefly luciferase gene. Cells expressing neither the constitutively nor thetransiently genotype 3 replicon sustained viral replication.In conclusion cerulenin inhibited HCV replication at levels, which did not inhibitfatty acid biosynthesis and were not toxic. There was toxicity at ceruleninconcentrations, which inhibited fatty acid biosynthesis. Cerulenin inhibitedreplication but by a mechanism other than inhibition of fatty acid biosynthesis.Cells with different passage histories were shown to behave differently to eachother in their response to drugs.The PUFAs, DHA and EPA exert an inhibitory effect on HCV replicon replicationand fatty acid biosynthesis at non-toxic levels. Oleic acid did not inhibit HCVreplication at equivalent concentrations. The mechanism behind PUFA inhibitionof HCV RNA levels is still unknown.An attempt to create genotype 3 constitutively and transiently expressingreplicon HuH-7 cell lines failed.