Hubb, Jonathan Ramsay
The interaction of hepatitis C virus and intracellular lipid metabolism.
PhD thesis, University of Glasgow.
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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
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
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
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
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.
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