Research Report 2010 2011 - Helmholtz-Zentrum für ...

26 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus
with higher doses of prednisolone and other steroids
increased HCV infectivity. By means of time-kinetic
experiments, we were able to prove that HCV under
treatment of steroids can penetrate the liver cells more
quickly. With the aid of retroviral pseudotypes, we could
show that the increase in infectiousness was specific for
HCV. Further mechanistic studies revealed that prednisolone
treatment increased the mRNA and the protein expression
of SR-BI and occludin, two proteins that are essential
components of the HCV receptor complex.
It was possible to ablate this steroid effect through the use
of the glucocorticoid receptor inhibitor RU-486, which
indicates that this signalling cascade is responsible for the
influence on the receptor expression and the HCV cell entry.
Moreover, we noted that prednisolone not only facilitated
virus cell entry and spread in the human hepatoma-cell-line
Huh-7.5: the incubation with prednisolone also increased
viral dissemination in dimethyl-sulphoxide-differentiated
liver cells and primary human hepatocytes. This result
correlates with the clinical experience in the patient:
Various clinical studies had reported that administration of
high dosages of steroids, for example in the context of acute
organ rejection treatment, was associated with increased
viral load and augmented liver inflammation 8 . Correspondingly,
avoidance of highly-dosed steroids could contribute to
an improvement in clinical course after HCV-induced liver
transplant.
Hepatitis C – not only in the liver? To date research on
HCV has been primarily focussed on the liver – an obvious
focus, since the symptoms can be attributed predominantly
to damage of this organ. Upon more precise investigation
however, one can see that infection with HCV generates a
series of other symptoms as well, and these symptoms are
not necessarily in direct relation to the liver infection.
Patients with a chronic HCV infection often times present
with various nervous system-associated symptoms. Chronic
fatigue syndrome, depression and cognitive disorders
frequently appear. It is however unclear whether these
symptoms can be attributed to a direct HCV replication in
the brain and the peripheral neuronal cells, or to indirect
effects upon the central and peripheral nervous system. For
this reason we investigated whether host cells from these
tissues – among them human neuroblastoma and glioblastoma
cell-lines and microglial cells – are susceptible to HCV
infection and can propagate the virus 4 . In this context we
identified the human peripheral neuroblastoma cell-line
SKNMC, which expresses all four essential HCV-entry
factors and was susceptible to cell entry by our HCV
pseudo-particles (HCVpp). Our investigations did however
show as well that the virus cannot efficiently propagate in
these cells. Nevertheless, our results point to the fact that
HCV can for all intents and purposes penetrate into
non-hepatic cell types. These interactions may in turn
influence the development of the disease and particularly
extra-hepatic disease manifestations.
Small molecules up against an unsolved problem The
options available for therapy against HCV are currently still
insufficient. Although a series of direct antiviral substances
are in clinical development, undesirable side-effects,
resistance development and genotype-specific antiviral
activity still pose substantial challenges. For this reason, the
on-going search for appropriate therapy measures continues.
The focus is now on combinations of well-tolerated active
compounds that act at different steps within the HCV
replication cycle. In this manner, a combination therapy
with drugs acting via distinct mode of action should prevent
rapid development of viral resistance.
In order to identify substances that disturb the various
steps in the HCV replication cycle, we have developed a new
dual-reporter-gene assay, and adapted it in collaboration
with scientists at the HZI to a high-through-put-screening
procedure (Figure 4). This system encompasses the entire
HCV life-cycle and, therefore, enables identification of
inhibitors against each individual step in viral propagation 10 .
The assay is based on an HCV reporter virus that carries a
gene for a luciferase-enzyme of the firefly (F-Luc; firefly
luciferase). The host cells, which are used for propagation of
HCV, were equally furnished by us with a luciferase gene
from the deep-sea crab gaussia (G-Luc; gaussia luciferase).
Since the two luciferase enzymes use different substrates
and do not influence one another, propagation of the HCV
and the vitality of the cells are simultaneously determined.
In this manner we are able to differentiate between
antiviral molecules and inhibitors of cell growth and
cytotoxic compounds, i.e. separate the wheat from the chaff.
Subsequently we validated the system with the aid of
known HCV inhibitors for cell entry, replication and virus