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

the HBV genome is expressed only in a particular
subset of hepatocytes, again demonstrating the importance
of liver architecture and of hepatocyte microenvironment.
Analysis of virus replication in HBVtransgenic
mice lacking the large envelope protein do
not support a prominent role of the L protein for regulating
the levels of intracellular replication intermediates
in HBV, in contrast to evidence for such a mechanism
for the DHBV system.
II. Hepadnavirus replication after transfer of
hepadnaviral genomes mediated by adenoviral
vectors
M. Sprinzl, H. Oberwinkler, B. Zachmann-Brand,
J. Dumortier, U. Protzer
To bypass the species barrier that precludes animal
studies with the human HBV, we have established
recombinant adenoviruses that transfer HBV and
DHBV genomes into heterologous hepatocytes, or
into cell lines which are not infectable but capable
of supporting virus replication. In contrast to transfection,
this approach allows to introduce a defined
number of hepadnavirus genomes, thereby simulating
natural initiation of hepadnavirus replication from
extrachromosomal DNA templates; it should enable
us to study in more detail in vitro and in vivo the influence
of the state of the host hepatocyte, and the use of
immune mediators on hepadnavirus replication. Thus,
we have started to use adenoviral genome transfer into
cultured mouse hepatocytes to test for the influence of
defined immune mediators (from the mouse) on hepatitis
B virus replication in vitro. Furthermore, transduction
of hepadnavirus genomes was shown to efficiently
establish HBV and DHBV replication in the
mouse liver, yielding serum titers comparable to those
from HBV-transgenic animals. In these mice, viral
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gene expression and replication was maintained for
up to three months, provided that an (adenovirusdirected)
immune response was suppressed.
III. A Golgi-resident protein participates as
an uptake receptor in duck hepatitis B
virus infection
K. Breiner, B. Glass, S. Urban
Major efforts through two decades have produced a
growing list of HBV receptor candidates, mostly only
defined as virus binding proteins, but have failed to
characterize any of these functionally. More progress
has been made in the DHBV animal model which
allows infection experiments with primary hepatocytes.
Including this essential complement for analysis,
we have now identified and characterized in detail
a bona fide DHBV receptor molecule, which plays
a crucial role in a conceptually new mechanism of
viral entry. This protein, a cellular carboxypeptidase
(CPD, historically termed gp180), had been initially
only characterized as a virus binding protein - and met
with scepticism since it lacked the expected tissue tropism,
species specificity, and since is predominantly
concentrated in the trans-Golgi network and not at the
plasma membrane where conceptually viruses must
interact with host cell receptor(s). Our conclusion that
gp180 is nevertheless a functional DHBV receptor
is based on a comprehensive series of experimental
observations: (i) gp180 is the only duck protein that
binds to recombinant DHBV preS, a ligand on the
virus surface previously shown to be essential for
infection, (ii) a preS subdomain, functionally defined
in infection competition experiments, coincides with
the domain determining physical gp180 binding, (iii)
anti-gp180 antibodies, as well as recombinant gp180/
CPD, efficiently block DHBV infection of cultured
duck hepatocytes, (iv) expression of gp180 in a
human hepatoma cell line mediates cellular attachment
and subsequent internalization of fluorescently
labeled DHBV particles into vesicular structures;
gp180 expression does, however, not render these
heterologous cells permissive for productive DHBV
infection. Furthermore, gp180/CPD is down-regulated
in DHBV-infected cells through intracellular interaction
with the preS ligand, a finding matching our
observation that DHBV infection reduces subsequent
superinfection about 20-fold. Taken together, these
and further data support the model that gp180/CPD
acts as the uptake receptor in a multistep process
(Fig.3). Although predominantly concentrated intracellularly,
the very limited cell surface exposition of
gp180/CPD (varied experimentally by gp180-transducing
adenoviruses) was found to be sufficient to
mediate virus particle uptake by coendocytosis, while
fusion at an endosomal membrane is predicted to
require an (yet unknown) species-specific fusion coreceptor.
Thus, gp180/CPD represents a first example
for a receptor that is virtually absent from the cell surface,
but nevertheless recognized and utilized for virus
Figure 3. Model of cellular gp180 traffic and DHBV entry.
DHBV-complexed gp180 is arrested in the endosome to
allow interaction with a second receptor postulated to trigger
membrane fusion (Breiner et al., 2000).
internalization leading to productive infection. These
new insights into hepadnaviral entry from the DHBV
model are now being used to re-design the strategies
in our search for the receptor of the medically relevant
human HBV.
IV. Signals for bidirectional cyto-nucleoplasmic
transport in the DHBV capsid protein
H. Mabit, K. Breiner, A. Knaust, B. Zachmann-
Brand
Like other viruses that replicate in the nucleus, the
hepadnaviruses rely on the the cellular cyto-nucleoplasmic
transport machinery to bring the infecting
nucleocapsid to the nucleus. To get insight into the
mechanism operating and the signals used, we have
started to study this process in the DHBV system. By
analyzing the subcellular localization of various segments
of the DHBV core protein (DHBc) fused to
the green fluorescent protein, and by further mutational
analysis, we mapped a single nuclear localization
signal (NLS) in the DHBc sequence. Mutational
inactivation of this sequence prevented DHBc nuclear
targeting (Fig. 4), and in the context of the complete
virus genome, it blocked virus production from
transfected cells. The mutant genome was still capable
of directing the early steps of DHBV infection in
duck hepatocytes, however, compared to wild type,
with delayed gene expression and without infecting
neighbouring cells, both results supporting the interpretation
that the NLS is essential for intracellular
genome amplification and virus production.
Finally, evidence from heterokaryon experiments suggests
that the DHBc sequence contains, in addition to
its NLS, also a nuclear export signal. We propose
that this NES, or a bidirectional shutling signal, counterbalances
NLS function in the productive state of
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