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

Figure 4. Loss of nuclear import of DHBV core protein
after mutational changes in its nuclear import signal. (A)
Schematic representation of the GPF-fusion used and the
mutation introduced (215RRRKVK220 → RGGEVK). (B)
Cellular distribution of wild type (RRK; left panel) and
mutant (GGE; right panel) in transfected HuH7 cells. GFP
fluorescence in live cells was analyzed using a confocal
microscope, at day 1 post transfection.
the infected cell, thereby preventing abortive nuclear
accumulation of capsids as observed in chronic HBV
patients and in HBV transgenic mice. On the other
hand, we have observed DHBc accumulation in distinct
nuclear dots (particularly early in infection).
These dots also accumulate pregenomic DHBV RNA,
suggesting a role in RNA maturation and nuclear
export.
V. Envelope-independent membrane-binding
preceeds budding and secretion of mature
hepadnaviral nucleocapsids
H. Mabit
Hepadnaviruses are DNA viruses, but as pararetroviruses,
their morphogenesis initiates with the encapsi-
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dation of a RNA pregenome and these viruses have
therefore evolved mechanisms to exclude immature
nucleocapsids from participating in budding and secretion.
Using the duck virus model and a flotation assay,
we provide evidence that binding of cytoplasmic core
particles to their target membrane is a distinct step
in morphogenesis, discriminating different populations
of intracellular capsids. The membrane-associated
subpopulation contained largely mature, double
stranded DNA genomes and was devoid of detectable
core protein phosphorylation, both features characteristic
for secreted virions. Against expectation, however,
the selective membrane attachment observed did
not require the presence of the large DHBV envelope
protein, whose interaction has been implicated to be
crucial for selective nucleocapsid-membrane interaction.
Furthermore, removal of surface-exposed phosphate
residues from non-floating capsids, did by itself
not suffice to confer membrane affinity. Collectively,
these observations argue for a model in which nucleocapsid
maturation, involving the viral genome, the
phosphorylation state, and the structure of the capsid,
leads to the exposure of a membrane-binding signal as
an initial step crucial for selecting nucleocapsids destined
to be enveloped and secreted.
VI. Recombinant hepadnaviruses as vectors
for hepatocyte-directed gene transfer
U. Protzer, U. Klöcker, A. Frank, in collaboration
with M. Nassal, Med. Uniklinik Freiburg
Being hepatropic and non-cytopathic viruses, the
hepadnaviruses have been envisaged for many years
to be potential candidates for the development of livertargeted
viral vectors. However, due to the compact
organization of the viral genome (3 kb with overlapping
genes and numerous cis-acting sequence ele-
ments), realization of this potential proved to be rather
difficult. We have now demonstrated that, by appropriate
design (i.e. replacement of the S-gene), recombinant
replication-defective HBVs and DHBVs can be
generated which specifically infect hepatocytes, and
transduce and express foreign genes under control of
an endogenous viral promoter. Exclusive expression
of the transgenes in hepatocytes, but not in non-parenchymal
liver cells, confirmed the predicted tissue- and
cell-type specificity.
Being produced at rather high titers (ca.10 8 /ml),
recombinant hepadnaviruses are useful tools for experimental
gene transfer in hepatocyte cultures and for
the study of hepadnaviral infection and its therapeutic
treatment. For example, genetically marked DHBVs
were used to demonstrate superinfection of hepatocytes
with an established hepadnaviral infection. Furthermore,
expression of a type I interferon, a secreted
protein of therapeutic use, from such a vector interfered
with the replication of the resident virus (Fig.5).
These data introduce the use of local cytokine produc-
Figure 5. Therapeutic effect of a recombinant DHBV transducing
an interferon gene. Preinfected duck hepatocytes
were superinfected at various ratios (moi) with rDHBV-
IFN or with rDHBV-GFP as a negative control. The time
course of progeny virus release is shown.
tion by HBV-mediated gene transfer as a new concept
for the treatment of acquired liver diseases, including
chronic hepatitis B. Finally, by developing genetically
marked hepadnaviruses, provide a simple assay to the
identification of infectable cells, and the opportunity
for gene transfer approaches in searching for the missing
DHBV-coreceptor or other missing co-factors.
VII. A spring-loaded topology of the large
envelope protein of Duck hepatitis B
virus
In collaboration with E. Grgacic, Melbourne
The structure and fusion potential of the duck hepatitis
B virus (DHBV) envelope proteins was examined
by treating viral particles with deforming agents
known to release envelope proteins of viruses from
a metastable to a fusion-active state. Exposure of
DHBV particles to low pH triggered a major structural
change in the large envelope protein (L) resulting in
exposure of a new trypsin cleavage site within its
S domain, but without affecting the same region in
the small surface protein (S) subunits. This conformational
change was associated with increased hydrophobicity
of the particle surface most likely arising
from surface exposure of the hydrophobic first transmembrane
domain. In the hydrophobic conformation,
DHBV particles were able to bind to liposomes and
intact cells, while in their absence these particles
aggregated, resulting in viral inactivation. These results
suggest that some L molecules are in a spring-loaded
metastable state which, when released, expose a previously
hidden hydrophobic domain, a transition potentially
representing the fusion active state of the envelope.
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