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

24 RESEARCH REVIEWS | Frontier Runners of Hepatitis C Virus
Viral pseudoparticle
(HCVpp)
E2
HCV E1
Retroviral
Genome
Retroviral
Capsid
Structural
proteins
HCV Complete genome
Resistence
gene
IRES
Non-structural
proteins
Non-structural
proteins
Manipulation of
HCV Genome
(1) DNA with cloned HCV Genome (2) In vitro Transcription
(4) Transfection
T7-RNA Polymerase
(3) HCV RNA Transcripts
(5) Defection infected cells
(IFM, qRT-PCF)
HCV Replicon
96 h 72 h
Infection
Model for the
HCV
Infection process
Transfection
Selection
HCV Replicon cells
Model for the
HCV
RNA Replication
Huh-7 Hepatoma cells
Manipulation of
the host cell
(e.g. RNAi)
Analysis of
Phenotypes
IFM
α-NS3
Wild type
Mutant
Fig. 2. Important HCV cell-model systems prior to development
of the JFH1 infection system. IRES, internal ribosome entry
point. Graphic: Twincore
Fig. 3. Experimental procedure for examination of HCV replication
with the aid of the JFH1 infection system. IFM, immunofluorescence;
qRT-PCR, quantitative Reverse-Transcriptase Polymerase
Chain Reaction; RNAi, RNA-interference. Graphic: Twincore
mixed-viruses (“HCV-pseudo-particles” = HCVpp), with a
retroviral core and an HCV-typical virus envelope, preferentially
infect liver cells 1 . Since the early stages of infection
are conveyed essentially by the proteins in the virus
envelope (the HCV E1 and E2 proteins, for the HCVpp case),
a system was made available that permitted analysis of the
HCV infection process by means of molecular-biological
technology (Figure 2). It was the developments of 2005 to
2006 that ultimately rendered the methods and approaches
of classic virology accessible to HCV. Together with Wakita
and other colleagues, we have been able to show that a
new HCV-isolate taken from a Japanese patient with a case
of fulminant hepatitis (“Japanese fulminant hepatitis 1” =
JFH1) could not only efficiently replicate in Huh-7 cells, but
it could also yield virus particles in the culture medium 15 .
More over, we were also able to show that these particles
(“cell culture grown HCV” = HCVcc) are infectious not only
in cell cultures, but in vivo as well. By constructing JFH1
variations with structural proteins from other virus isolates,
we substantially enhanced the efficiency level of the infection
system 13 . Further HCV chimeras have been constructed
in the mean time by us and other groups. In this manner,
comparative examinations between different isolates are
now possible as regards the route of transmission and the
mechanisms of virus formation and virus release.
With the aid of molecular-biological techniques we can now
manipulate, in a targeted manner, either the virus genome
or the host cell and subsequently characterise the influence
of these modifications on virus propagation (Figure 3). In
this fashion we gain essential knowledge concerning the
virus’ essential interactions with the host cell, its method of
propagation within the cell, and we can identify new targets
for development of a direct antiviral therapy. Moreover, new
therapy procedures can be evaluated regarding their
effectiveness. Our HCV cell-culture model represents
thereby the central key to solving clinically relevant issues
that are current in HCV research.
The pathway of the virus through the clinic An important
aspect in the management of hepatitis C is prevention,
since the mode of transmission from blood to blood should
be controllable with suitable hygienic measures. Particularly,
transmissions within the hospital environment,
the stability of the virus under variable environmental
conditions and its sensitivity to chemical disinfection
agents are in the focus of our efforts. Examinations and
experiences heretofore have been based, due to a lack of
HCV in vitro models, almost exclusively on studies with the
bovine diarrhoea Virus (BVDV), which is a relative of the
HCV and which has been cultivated for a fairly long time to
date. However, these studies with the surrogate virus for
HCV allow only a semi-dependable estimation of the
infectiousness of the HCV.