ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

Heinz Schaller
Regulation of Hepatitis B Virus Replication
Hepatitis B viruses (also hepadnaviruses, HBVs) are
small, enveloped DNA viruses which cause acute and
chronic liver infections in mammals and birds. Their
prototype, the human HBV, is the causative agent of
a world-wide major public health problem with about
5 % of the world population being chronic HBV carriers
and at high risk of developing liver cirrhosis and
hepatocellular carcinoma. As ‘pararetroviruses’, these
viruses are related to the retroviruses by genome organization
and replication strategy, but differ in major
features, e.g. by containing a DNA genome in the
extra-cellular state, or by producing transcripts from
a non-integrated, circular DNA template. The hepadnaviruses
are characterized by a tissue tropism and
a very narrow host range restricting them to their
respective natural hosts and close relatives. These features
have been an obstacle to the establishment of an
HBV cell culture infection system, which is urgently
needed for the development of targeted therapy.
Despite these experimental difficulties with the HBV
prototype, the hepadnavirus infection cycle has been
worked out in considerable detail from molecular
analysis of HBV replication in cells transfected with
cloned viral DNA, as well as from infection studies
in the duck Hepatitis B virus (DHBV) animal system.
The resulting replication model (Fig. 1) is well estab-
Figure 1. The HBV replication cycle. Individual steps are indicated: (1,2) attachment to, and endocytosis into the host cell;
(3,4) cytosolic release of the capsid and transport to the nucleus; (5) conversion of the viral genome into ccc-DNA, the
template for transcription; (6) synthesis of genomic RNA, its maturation and transport to the cytoplasm, and (7) its translation
into the core and polymerase gene products, followed by (8), coassembly into RNA-containing nucleocapsids, ((8‘)
cyto-nucleoplasmic shuttling of core protein subunits, postulated to participate in genomic RNA maturation); (9) reverse
transcription of the RNA genome into DNA, and (10) nucleocapsid maturation and export from the cell as enveloped virion.
Alternatively, nucleocapsids import the mature DNA genome into the nucleus for replenishment of the ccc-DNA pool (11).
For simplicity, synthesis and assembly of the envelope proteins from subgenomic RNA are omitted.
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lished with respect to the basic mechanisms leading to
the production of progeny virions (steps 7-10), but still
uncertain as to the mechanisms and cellular components
involved in determining receptor-mediated virus
uptake (steps 2-4), and also as to those that control the
establishment and persistence of productive infection
(primarily steps 4, 6, and 11). These latter processes
are difficult to study under controlled conditions
since no infectable cell lines exist even in case of
the available animal models. Nevertheless, we have
been increasingly focussing our research onto these
research areas, as their better understanding appears
to be crucial for a rational approach towards our longterm
goal, which is to establish an infection system for
studying (and to interfere with) all steps of HBV replication
cycle of the human virus in cultured cells, and
in a small test animal.
I. Parameters influencing the establishment
and maintenance of hepadnavirus replication
in primary hepatocyte cultures
U. Klöcker, B. Zachmann-Brand, B. Glass, C.
Kuhn, K. Rothmann, U. Protzer
Hepatitis B viruses use a well-balanced replication
strategy and an intimate cross talk with the host to
establish productive, but non-cytotoxic, long-term persistent
infections. As part of this strategy, virus replication
and gene expression vary greatly in response
to environmental changes of the state of the cell. For
example, transfer of preinfected duck hepatocytes out
of the tissue structure into cell culture results in an initial
reduction of virus production followed by upregulation
(Fig. 2). Furthermore, extracellular stimuli such
as cytokines, the peptide hormone glucagon, or endotoxin
from gram negative bacteria inhibit the establishment
of DHBV replication in vitro. We have now
shown that endotoxin acts indirectly by activating liver
Figure 2. Changes in virus production from DHBV-infected
primary duck hepatocytes in response to environmental
changes. After plating, preformed virus is released, followed
initially by decreased rates of production. From
day 4 on, virus production increases, reaching constant
levels around day 8. This process is inhibited by endotoxin-induced
cytokines produced from liver macrophages
(Klöcker et al., 2000).
macrophages to release cytokines which act at inhibitory
an early step of DHBV replication (Fig. 2).
Another example for cross talk with the host is our
finding that the large envelope protein, although a
structural protein, is varibly phosphorylated in cytosolic
preS domains by ERK-type MAP kinases in
response to to the state of the cell. In turn, changes in
preS-phosphorylation correlated closely with the ability
of DHBV L to activate gene expression in trans,
in vitro and in vivo. Furthermore, a pathogenic phenotype
with severe growth retardation and pathologic
liver histology was observed in ducklings infected
with a variant mimicking constitutive L phosphorylation.
The above observations from experimental DHBV
infection are complemented by data obtained with
HBV-transgenic mice, which produce HBV from a
chromosomally integrated HBV genome in titers comparable
to chronically infected humans. In this system,
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