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J Virol, May 1998, p. 4492-4497, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Viral Proteins after Infection of
Cultured Hepatocytes with Rabbit Hemorrhagic Disease Virus
Matthias
König,
Heinz-Jürgen
Thiel, and
Gregor
Meyers*
Department of Clinical Virology, Federal
Research Centre for Virus Diseases of Animals, D-72001
Tübingen, Germany
Received 15 October 1997/Accepted 28 January 1998
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ABSTRACT |
The calicivirus rabbit hemorrhagic disease virus (RHDV), which
replicates predominantly in the livers of infected rabbits, cannot be
propagated in tissue culture. To enable the performance of in vitro
studies, rabbit hepatocytes were isolated by liver perfusion and
gradient centrifugation. After inoculation with purified RHDV, more
than 50% of the cells proved to be infected. Protein analyses led to
the detection of 13 RHDV-specific polypeptides within the infected
cells. These proteins were assigned to defined regions of the viral
genome, resulting in a refined model of RHDV genome organization.
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TEXT |
Rabbit hemorrhagic disease (RHD) is
a contagious disease often associated with liver necrosis,
hemorrhages, and high mortality. The causative agent of
RHD is a positive-stranded RNA virus which belongs to the family
Caliciviridae, a group of nonenveloped animal viruses (8). The RHD virus (RHDV) genome consists of a
polyadenylated RNA molecule of 7,437 nucleotides with a virus-encoded
protein (VPg) covalently attached to its 5' end (12, 13).
The genomic RNA contains one long open reading frame (ORF1) encoding a
hypothetical primary translation product of 257 kDa, which gives rise
to mature viral proteins by proteolytic processing. Most, if not all,
cleavages are executed by a virus-encoded trypsin-like cysteine
protease showing significant similarity to the 3C proteases of
picornaviruses (4). So far, viral protein expression has
only been studied by in vitro translation of viral RNA and
detection of RHDV-encoded proteins with specific antibodies (2,
24). Together with data obtained after bacterial expression of
RHDV proteins, these studies led to the first comprehensive model
of the organization of a calicivirus genome (23, 24).
Accordingly, the identified viral gene products are arranged in the
ORF1-encoded polyprotein in the order
NH2-p16-p23-p37-p41-p69-VP60-COOH. A second ORF (ORF2) is
located at the extreme 3' end of the genomic RNA; expression of ORF2
via a not-yet-identified mechanism leads to VP10, a component of RHDV
virions (24). In RHDV-infected cells, a 2.2-kb subgenomic mRNA which is colinear with the 3' one-third of the genomic RNA is
transcribed (13). This mRNA apparently represents the major source of the RHDV capsid protein VP60; the latter is also generated via cleavage of the ORF1-encoded polyprotein (15, 23).
Like the human caliciviruses, e.g., Norwalk virus or Southampton
virus, RHDV so far cannot be propagated in tissue culture cells. To
enable the investigation of protein synthesis and other aspects of the
RHDV life cycle in infected cells, we devised a system for in vitro
propagation of RHDV based on primary rabbit liver cells. Infected
hepatocytes were used for analysis of RHDV protein expression,
resulting in a refined model of the organization of the calicivirus
genome.
Isolation and cultivation of rabbit hepatocytes.
Infected
animals usually contain large amounts of RHDV virions in the liver and
the spleen. By immunocytochemical methods, viral antigen was detected
in hepatocytes and reticuloendothelial cells of the liver
(14). As a first step toward in vitro propagation of RHDV,
rabbit hepatocytes were isolated and maintained in vitro. Hepatocytes
have to be released carefully from their tissue environment by enzyme
digestion since mechanical mobilization will unequivocally result in
severe cell damage (20). Perfusion techniques using collagenase have been successfully applied for the isolation of hepatocytes from rabbits (22).
We used an extracorporeal two-step perfusion technique to obtain large
quantities of vital hepatocytes for subsequent studies. The first
perfusion step included removal of remaining blood cells and
Ca2+ by using preperfusion buffer [140 mM NaCl, 7 mM
KCl, 10 mM HEPES, 8 mM D-(+)-glucose, 0.1 mM EGTA, pH
7.4]. Ca2+ is believed to stabilize intercellular hepatic
adhesion factors. Therefore, deprival of Ca2+ is
regarded as a prerequisite for optimal results in collagenase digestion (20). In the second step, the liver lobes were
perfused with a solution consisting of collagenase (500 mg/liter;
Sigma, Deisenhofen, Germany) in perfusion buffer [67 mM NaCl, 7 mM
KCl, 100 mM HEPES, 8 mM D-(+)-glucose, 6 mM
CaCl2, pH 7.6]. Finally, after removal of the liver
capsule, further collagenase digestion was performed in suspension,
thereby mobilizing the parenchymal cells, with a yield of about
109 viable cells per liver as determined by trypan blue
exclusion (16).
Freshly prepared cells plated on tissue culture vessels precoated with
collagen (type 1; Sigma) dissolved in 0.2% acetic acid
were
rapidly adsorbed and formed a confluent monolayer. After
24 h, the majority of the cells showed a polygonal shape resembling
that
of hepatocytes and had assembled in trabecular structures.
Hepatocytes
containing two or more nuclei were consistently observed.
Electron
microscopic analysis of cells from a 5-day culture revealed
an
ultrastructure similar to that of hepatocytes (data not shown).
Since addition of fetal bovine serum proved to be toxic to the
parenchymal cells, cultures were kept in a chemically defined
medium
(Williams medium E [Gibco] buffered with 10 mM HEPES and
supplemented
with 5 µg of insulin per ml, 2 µg of glucagon per
ml, 0.1 µg of
epidermal growth factor per ml, 0.25 µg of hydrocortisone
per ml, 5 ng of H
2SeO
3 per ml, 2 mM
L-glutamine, and 100 µg of
gentamycin per ml); the
culture medium was exchanged every other
day. Hepatocyte cultures were
maintained for 2 to 3 weeks under
the conditions described above. After
this period, hepatocytes
were increasingly overgrown by nonparenchymal
cells.
To obtain cell preparations of higher purity, low-speed isodensity
Percoll gradient centrifugation was employed (
11). More
than
90% of the gradient-purified liver cells were viable. The
proportion
of contaminating nonparenchymal cells was negligible,
since hepatocyte
cultures could be maintained for more than 4
weeks without visible
signs of overgrowth (data not shown).
Infection of cultured rabbit hepatocytes with RHDV.
To study
the ability of RHDV to infect cultured rabbit hepatocytes, the cells
were inoculated with CsCl gradient-purified virus. Infection was
monitored by the detection of the RHDV major capsid protein (VP60) in
an immunoperoxidase assay using a mixture of three monoclonal
antibodies (1H8, 5G3, and 6G2 [6]).
Examination of hepatocyte cultures 24 h after infection with RHDV
revealed cells expressing VP60 (Fig.
1).
The percentage
of infected cells increased significantly in cultures
maintained
for 48 or 72 h after infection (Fig.
1). However, even
after prolonged
incubation, a considerable proportion of cells remained
negative
in the immunostaining assay. Virus-infected cells revealed
signs
of degeneration, including condensed nuclei considerably smaller
than the ones from noninfected cells; excessive lysis of cells
was not
observed (Fig.
1).
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FIG. 1.
Infection of cultured hepatocytes with RHDV. Overnight
cultures were either mock infected (A) or inoculated with
gradient-purified virus and subsequently incubated for 24 h (B),
48 h (C, E, and F), or 72 h (D). Immunostaining was performed
with monoclonal antibodies directed against RHDV VP60 major capsid
protein and anti-mouse immunoglobulin G conjugated to horseradish
peroxidase. Cells were counterstained with hematoxylin. (E and F)
Higher magnifications of a culture 48 h after infection. Note the
noninfected cells (E, bottom region), apparently newly infected cells
(E, center), and cells showing a pronounced cytopathic effect (E, left
and right margins; F [note condensation and size of nuclei]).
Instrumental magnification, ×100 (A to D) or ×1,000 (E and F). The
dark staining of the condensed nuclei in panel F resulted from the blue
counterstain, which cannot be distinguished from the antibody staining
evident on a black and white picture.
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In infected cultured hepatocytes, VP60 was mostly found in the
cytoplasm; only weak staining occurred in the nuclei of the
cells. This
finding stands in marked contrast to the picture observed
in liver
sections from diseased rabbits, where the nuclei of hepatocytes
were intensively stained by anti-VP60 antibodies (
1).
Transport
of VP60 into the nucleus has not been thoroughly
investigated.
Heterologous expression of VP60 in different eukaryotic
cell lines
did not result in the detection of VP60 within nuclei. In
situ
hybridization studies with liver sections from infected rabbits
revealed that viral RNA is not located in the nucleus (data not
shown).
The presence of RHDV VP60 in cultured hepatocytes demonstrated that
these cells can be infected in vitro. Furthermore, the
strong signals
observed in infected cells suggested that replication
of the viral
genome and/or transcription of the subgenomic mRNA
takes place in
cultured hepatocytes. Negative cells may represent
populations of
hepatocytes that either cannot be infected by RHDV
or,
alternatively, do not support translation and/or
replication/transcription
of viral RNA. The question of whether
infectious virus is actually
produced and released by cultured
hepatocytes remains unanswered.
Analysis of viral proteins produced in infected hepatocytes.
Characterization of calicivirus genomes has rapidly progressed in the
past few years (for a review, see reference 7). In contrast, little is known about the proteins expressed by
caliciviruses. This is mainly due to the fact that tissue culture
systems and/or antibodies specific for individual viral proteins are
not available. The only authentic calicivirus proteins demonstrated so
far are the major capsid protein (with a molecular weight of 58 to 76 kDa) (reviewed in reference 7), VP10
(24), and VPg (3, 5, 13, 19), which are all found
in virions. By in vitro translation of viral RNA and precipitation of
translation products with a set of region-specific antisera raised
against bacterial fusion proteins, the putative organization of RHDV
ORF1 has been determined (24). To check whether the data
obtained in vitro reflect the in vivo situation, the expression of RHDV
proteins in infected rabbit hepatocytes was analyzed by metabolic
labeling and immunoprecipitation. Freshly prepared hepatocytes
(106) were seeded in a 3.5-cm-diameter culture dish and
then infected with gradient-purified RHDV 24 h later. After 1 h, the virus suspension was removed; the cells were then washed twice
with labeling medium (18) and subsequently incubated in this
medium for 1 h. Afterward, the supernatant was removed
and the cells were incubated for 4 h in 0.5 ml of labeling medium
containing 125 µCi of Tran35S-label (ICN
Biochemicals, Meckenheim, Germany). The labeled cells were washed twice
with phosphate-buffered saline and processed for immunoprecipitation as
described previously (18).
After in vitro translation of the viral RNA, the amino-terminal
one-third of the polyprotein encoded by ORF1 gave rise to
three
processing products of 16, 23, and 37 kDa (
24). In addition,
a band of about 60 kDa, which presumably represented a precursor
composed of the latter two cleavage products, was sometimes detected
(
24). The analogous proteins were also found in
RHDV-infected
hepatocytes. Polypeptides of 16, 23, and 60 kDa were
identified
after precipitation with antiserum A, which is directed
against
the first 300 amino acids encoded by ORF1 (Fig.
2A). The 16-kDa
protein also reacted with
antiserum B, which was raised against
amino acids 9 to 112 of the
protein encoded in ORF1. Thus, p16
represents the
amino-terminal cleavage product of the polyprotein.
The amount of p16
precipitated with either antiserum is very small,
possibly due to
instability of this protein in the infected cells.
The polypeptides of
23 and 60 kDa were recognized not only by
antiserum A but also by
antiserum D, which is directed against
amino acids 232 to 303. p60 was
also precipitated with an antiserum
covering the region from amino
acids 393 to 702 of the polyprotein
(antiserum E). Antiserum E
precipitated a second protein of 37
kDa. Thus, as already hypothesized
after the in vitro translation
experiments, p60 represents a precursor
that is further processed
into p23 and p37.
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FIG. 2.
Immunoprecipitation of proteins from RHDV-infected
hepatocytes with a set of antisera raised against bacterial fusion
proteins containing defined regions of the polypeptides encoded by the
RHDV genome (24). The upper portion of each panel shows the
precipitated proteins separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Each lane is labeled with a
letter indicating the antiserum used for precipitation. On the right
side of each gel, bands discussed in the text are marked with arrows
and the designations of the precipitated proteins. The positions of
size marker proteins (14C-labeled molecular mass markers;
Amersham, Braunschweig, Germany) are indicated on the left (in
kilodaltons). A scheme below each gel indicates the reactivities of the
different antisera. The ORF1-encoded polyprotein is symbolized by a
white bar marked by a scale (in 100-amino-acid increments). Within the
polyprotein, the locations of VPg and the RHDV protease (Pro) as well
as the positions of amino acid motifs common for RNA virus polymerases
(Pol) and helicases (2C-like) are shown. The protein encoded by ORF2 is
shown below the ORF1 product. Gray bars labeled with letters indicate
the protein segments contained in the different fusion proteins used
for generation of the antisera. The exact features of the antisera have
been described previously (24). In the lower part of each
panel, the putative locations of the precipitated proteins (black bars)
with respect to the polyprotein are shown. Please note that the gels of
all three panels have been mounted from one protein gel. In panels B
and C, the last lane of panel A and the last lane of B, respectively,
are included to facilitate comparison of the electrophoretic mobilities
of the individual proteins. (A) Proteins precipitated with antisera
directed against regions within the amino-terminal one-third of the
ORF1-encoded polyprotein. (B) Precipitation products obtained with
antisera recognizing different segments of the central region of the
polyprotein. Further processing of p29 into p23/2 and X is hypothetical
and could occur in different ways, as indicated in the box at the
bottom. (C) Proteins precipitated with antisera directed against the
carboxy-terminal half of the ORF1-encoded polyprotein and the ORF2
product. Ns, rabbit preimmune serum.
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In addition to the four proteins detected in vitro and in vivo,
products of 26 and 65 kDa were identified in infected cells.
With
regard to their reactions with the different antisera, these
two
proteins are related to p23 and p60, respectively. Since there
are no
indications of alternative processing reactions, e.g.,
shorter versions
of p16 or p37, it is assumed that p23 and p60
have the same protein
backbones as p26 and p65, respectively.
The increase in molecular
weight could be due to posttranslational
modifications, which are
apparently not observed in vitro. In
addition to the above-described
products, antisera A, D, and E
precipitated a protein of about 28 kDa.
The nature of this weak
band is not known.
After coupled in vitro transcription-translation of RHDV cDNA
constructs, Alonso et al. detected a band of 80 kDa, derived
from the
region corresponding to the 5' part of the genome, which
was apparently
not further processed (
2). This finding stands
in marked
contrast to our data; the reason for this discrepancy
is not known.
Antisera F, H, and I are directed against bacterial fusion proteins
encompassing different parts of the central part of the
ORF1-encoded
polyprotein; the region covered by antiserum H (amino
acids 875 to
1023) represents the C-terminal part of the larger
region F (amino
acids 704 to 1023), whereas the region recognized
by antiserum I is
located further downstream (amino acids 1023
to 1170) (
24).
After in vitro translation of viral RNA, a product
of 41 kDa was shown
to react with these antisera. A product of
similar size (43 kDa) was
detected after coupled in vitro transcription-translation
of cDNA
constructs (
2). Using extracts of infected hepatocytes,
precipitation with antiserum F resulted in the detection of four
bands
of 41, 29, 23, and 13 kDa. The proteins of 41, 29, and 23
kDa were also
recognized by antiserum H. In addition, after prolonged
exposure, bands
corresponding to proteins of 13 and 14 kDa were
identified with this
antiserum (data not shown). The latter two
proteins were also
precipitated with antiserum I, together with
p41 (Fig.
2B). Because of
its size and reaction pattern, p41 is
likely to represent a fusion of
p29 and a second protein of about
12 to 14 kDa. This second protein
could be p13 or p14. It is not
clear whether p13 and p14 represent
different processing products
or one of these polypeptides is a
posttranslationally modified
version of the other. With regard to size
and location in the
polyprotein, p13/p14 represents VPg (
13,
23,
24). Analogously
to VPg-pU and VPg-pUpU of picornaviruses
(
21), RHDV VPg could
be covalently linked to nucleotides in
the course of RNA replication.
Accordingly, one of the detected bands
may represent the original
cleavage product whereas the other is
modified by addition of
one or more nucleotides. Further experiments
are needed to verify
this hypothesis. It is not clear at present why
antiserum F precipitates
p13 but not p14.
According to the processing scheme indicated above, the polypeptide of
23 kDa (p23/2) should represent a product generated
by cleavage of p29
(Fig.
2B). The hypothetical second cleavage
product of about 6 kDa
could not be detected, possibly due to
a lack of specific antibodies in
our sera or instability of the
cleavage product. Alternatively, p23/2
could represent the product
of a distinct route of p41 processing. This
hypothesis would also
imply the existence of at least one additional
protein. However,
the reaction pattern of p23/2 with the different
antisera and
the failure to detect further cleavage products make the
latter
alternative unlikely.
In addition to the products described above, a band of 37 kDa was
visible with antisera F, H, and I, as well as with antiserum
D (Fig.
2A
and B). It is not clear whether this band is specific
since the
precipitate formed with the preimmune serum contained
a protein of the
same size.
After in vitro translation of the viral RNA, a variety of products
derived from the carboxy-terminal half of the ORF1-encoded
polyprotein
was detected. Among these products was a protein of
69 kDa that was
proposed to cover the region between p41 and the
capsid protein VP60
(
24). p69 encompasses the viral 3C-like
protease and the
putative RNA-dependent RNA polymerase. Others
identified a product of
73 kDa that was derived from the corresponding
region of a cDNA
construct (
2). Earlier studies based on bacterial
expression
had demonstrated the presence of a processing site
between the protease
and the polymerase which was cleaved by the
viral protease with a
rather low efficiency (
23). This processing
was apparently
not observed for the polypeptides derived from
in vitro translation,
since products with molecular masses below
69 kDa were not found in the
in vitro experiments (
2,
24).
Interestingly, RHDV-infected
hepatocytes contained not only a
protein of about 72 kDa but also
polypeptides of 58 and 15 kDa
(Fig.
2C). This was shown by analyses
using antisera J, which
is directed against amino acids 1172 to 1332, and antiserum K,
which is directed against residues 1332 to 1727. While
p72 is
recognized by antisera J and K, p15 is precipitated with
antiserum
J but not with antiserum K. After longer exposure times, a
weak
band comigrating with p15 was also visible after precipitation
with antiserum I (data not shown). Taken together, the data imply
that
p15 represents the RHDV protease, which is known to consist
of 143 amino acids located in the ORF1 polyprotein between residues
1109 and
1251 (
24). Consequently, p58 must be regarded as the
RHDV
polymerase and p72 must represent the uncleaved protease-polymerase
precursor. The C-terminal cleavage product encoded by ORF1 is
VP60,
which is detected by antiserum M.
The genome of every calicivirus contains at its 3' end a small ORF with
the capacity to encode a protein of 100 to 200 amino
acids (reviewed in
reference
7). It was shown for RHDV that
the
product of this ORF represents a structural protein of 10
kDa which was
termed VP10 (
24). After precipitation with antiserum
N, which is directed against part of the ORF2-encoded
protein,
VP10 could be demonstrated in the extract from the
infected hepatocytes
(Fig.
2C). The generation of VP10 was also
observed after in vitro
translation (data not shown); the mechanism
responsible for expression
of this protein has not yet been elucidated.
Genetic map of RHDV.
Analysis of the proteins present in
RHDV-infected primary hepatocytes allowed the establishment of a
genetic map for RHDV (Fig. 3). According
to this proposed map, the translation product of ORF1 is initially
cleaved into products p16, p60/65, p41, p72, and VP60. Further
processing of the p60/65 region leads to p23/26 and p37, while products
of 15 and 58 kDa can be derived from the part of the polyprotein
comprising p72. It is not known whether p60/65 and p72 represent
incompletely processed precursors or stable proteins with specific
functions in virus replication, as described, e.g., for 3AB or 3CD of
picornaviruses (17). Also, p41 is further processed into
several polypeptides (see below).
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FIG. 3.
Schematic representation of the RHDV-encoded proteins.
The upper bars indicate the hypothetical primary translation products
of ORF1 and ORF2; the scale represents amino acid numbers. Regions
which contain known sequence motifs or already-identified viral
proteins are indicated. EG and ET, processing sites determined so far
(2, 23, 24). The gray-shaded bars below represent VP10 and
the products resulting from processing of the ORF1-encoded polyprotein.
The designations of proteins which could not be demonstrated after in
vitro translation are written in boldfaced letters. The polypeptides
generated by the hypothetical cleavage of p29 are enclosed in a box.
The hypothesized further processing of p29 has not been proven; two
alternative ways for this processing to occur are indicated. Protein
"X" has not been demonstrated.
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The conclusions drawn from the in vitro translation experiments and the
results obtained by analysis of the infected hepatocytes
led to the
establishment of similar genetic maps. However, the
existence of
several important differences must be stressed. Expression
of p15 and
p58 as well as the putative modification of p60 and
p23, leading to p65
and p26, respectively, could only be demonstrated
in vivo. Moreover,
there were obvious differences between the
in vitro and the in vivo
results with regard to the processing
of p41. After in vitro
translation, only p41 was detected. Since
it was known that the VPg
gene is located in the part of the genome
coding for p41, further
processing of p41 had to be postulated.
Nevertheless, the detection of
four different polypeptides in
vivo, namely p13, p14, p23/2, and p29,
with a hypothetical fifth
cleavage product of about 6 kDa was
unexpected. Even though the
scheme presented in Fig.
2 and
3 represents
a consistent model
for the processing of this part of the polyprotein,
a considerable
effort that includes the generation of additional
serological
reagents, heterologous expression, and pulse-chase labeling
of
RHDV proteins will be necessary to elucidate the exact cleavage
cascade. Such approaches will also help to elucidate the nature
of some
bands visible in Fig.
2 that to date cannot be explained.
With regard to the arrangement of the genes coding for nonstructural
proteins, the genomic organization of RHDV exhibits obvious
similarities to that of picornaviruses. In a schematic alignment
of
their polyproteins, p16 would correspond to poliovirus 2A,
p23 would
correspond to 2B, and p37 would correspond to 2C (
24).
The
arrangement of the protease and putative RNA-dependent RNA
polymerase
at the end of the nonstructural region of the polyprotein
fits with the
organization of all members of the picornavirus
superfamily
(
10). Interesting differences are found for the
regions containing p41 of RHDV and the similarly positioned 3AB
of
picornaviruses. With the exception of foot-and-mouth disease
virus,
only two processing products of 3AB are known (
9,
17);
in
contrast, p41 apparently gives rise to at least four different
polypeptides (Fig.
4). Since the
carboxy-terminal processing product
in both cases is VPg, the
differences concern 3A and the amino-terminal
two-thirds of p41,
which gives rise to RHDV p29. 3A of poliovirus
is a small hydrophobic
protein of about 10 kDa. RHDV polypeptide
p29 contains a highly
hydrophobic region at its carboxy terminus,
whereas the rest
of the protein is moderately hydrophilic. Further
processing of
p29 could result in a hydrophobic carboxy-terminal
product,
exhibiting similarities to poliovirus 3A, and an amino-terminal
polypeptide, for which a counterpart is lacking in
picornaviruses.
The polypeptide p23/2 most likely represents a
processing product
of p29; a corresponding second cleavage product
could not be identified.
Elucidation of the processing of p41 will also
be interesting
with regard to the relationship between caliciviruses
and picornaviruses.
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FIG. 4.
Comparison of the genomic regions encoding part of the
nonstructural proteins of RHDV and poliovirus. The genomes are shown as
bars, and the locations of individual genes are indicated. The regions
shown in gray symbolize those parts of the genomes for which the
experiments indicate the existence of a major difference between RHDV
and picornaviruses (see also the text and the legend to Fig. 3).
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The method for isolation and cultivation of primary rabbit hepatocytes
described here allowed for the first time the study
of RHDV after
infection of cells in vitro. It is assumed that
transcription and
replication of viral RNA take place within the
infected hepatocytes
even though we have not been able to prove
that viral replication in
the cultured cells is completed by the
release of infectious virus. The
amount of viral protein detected
in the infected cells is most likely
not solely attributable to
translation of RNA derived from the input
virus. Furthermore,
detection of efficient processing at sites which
are not cleaved
in vitro and of products which are either not generated
or not
stable after in vitro translation is also indicative of viral
replication. In any case, the infection of cultured hepatocytes
represents a means of analyzing RHDV gene expression on the basis
of
infected cells. Even though the method described here is laborious
and
expensive, it has opened a door to work on selected topics
of RHDV
biology. As outlined above, this approach has provided
the most
authentic view of the organization of a calicivirus genome
available
thus far. This is remarkable since some other family
members, e.g.,
feline calicivirus and San Miguel sea lion virus,
can be easily
propagated in tissue culture cells.
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ACKNOWLEDGMENTS |
We thank Silke Esslinger and Petra Wulle for excellent technical
assistance and L. Cappucci for providing monoclonal antibodies 1H8,
5G3, and 6G2.
This work was supported by grants Th 298/3-1, Me 1367/1-2, and Me
1367/1-3 from the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Virology, Federal Research Centre for Virus Diseases of
Animals, P.O. Box 1149, D-72001 Tübingen, Germany. Phone: 49 7071-967207. Fax: 49 7071-967303. E-mail:
gregor.meyers{at}tue.bfav.de.
Present address: Institut für Virologie, FB
Veterinärmedizin, Justus-Liebig-Universität Giessen,
D-35392 Giessen, Germany.
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REFERENCES |
| 1.
|
Alexandrow, M.,
R. Peshev,
I. Yanchev,
S. Bozhkov,
L. Doumanova,
T. Dimitrov, and S. Zacharieva.
1992.
Immunohistochemical localization of the rabbit haemorrhagic disease viral antigen.
Arch. Virol.
127:355-363[Medline].
|
| 2.
|
Alonso, J. M. M.,
R. Casais,
J. A. Boga, and F. Parra.
1996.
Processing of rabbit hemorrhagic disease virus polyprotein.
J. Virol.
70:1261-1265[Abstract].
|
| 3.
|
Black, D. N.,
J. N. Burroughs,
T. J. R. Harris, and F. Brown.
1978.
The structure and replication of calicivirus RNA.
Nature
274:614-615[Medline].
|
| 4.
|
Boniotti, B.,
C. Wirblich,
M. Sibilia,
G. Meyers,
H.-J. Thiel, and C. Rossi.
1994.
Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus.
J. Virol.
68:6487-6495[Abstract/Free Full Text].
|
| 5.
|
Burroughs, J. N., and F. Brown.
1978.
Presence of a covalently linked protein on caliciviral RNA.
J. Gen. Virol.
41:443-446[Abstract/Free Full Text].
|
| 6.
|
Capucci, L.,
G. Frigoli,
L. Rønshold,
A. Lavazza,
E. Brocchi, and C. Rossi.
1995.
Antigenicity of rabbit haemorrhagic disease virus studied by its reactivity with monoclonal antibodies.
Virus Res.
37:221-238[Medline].
|
| 7.
|
Clarke, I. N., and P. R. Lambden.
1997.
The molecular biology of caliciviruses.
J. Gen. Virol.
78:291-301[Medline].
|
| 8.
| Cubbitt, D., D. W. Bradley, M. J. Carter, S. Chiba, M. K. Estes, L. J. Saif, F. L. Schaffer, A. W. Smith, M. J. Studdert, and H.-J. Thiel. 1995. Family Caliciviridae. Arch. Virol.
10(Suppl.):359-363.
|
| 9.
|
Forss, S., and H. Schaller.
1982.
A tandem repeat gene in a picornavirus.
Nucleic Acids Res.
10:6441-6450[Abstract/Free Full Text].
|
| 10.
|
Goldbach, R., and J. Wellink.
1988.
Evolution of plus-strand RNA viruses.
Intervirology
29:260-267[Medline].
|
| 11.
|
Kreamer, B. L.,
J. L. Staecker,
N. Sawada,
G. L. Sattler,
M. T. S. Hsia, and H. C. Pitot.
1985.
Use of a low-speed, iso-density Percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.
In Vitro Cell. Dev. Biol.
22:201-211.
|
| 12.
|
Meyers, G.,
C. Wirblich, and H.-J. Thiel.
1991.
Rabbit hemorrhagic disease virus: molecular cloning and nucleotide sequencing of a calicivirus genome.
Virology
184:664-676[Medline].
|
| 13.
|
Meyers, G.,
C. Wirblich, and H.-J. Thiel.
1991.
Genomic and subgenomic RNAs of rabbit hemorrhagic disease virus are both protein-linked and packaged into particles.
Virology
184:677-686[Medline].
|
| 14.
|
Park, J. H., and C. Itakura.
1992.
Detection of haemorrhagic disease virus antigen in tissues by immunohistochemistry.
Res. Vet. Sci.
52:299-306[Medline].
|
| 15.
|
Parra, F.,
A. J. Boga,
M. S. Marin, and R. Casais.
1993.
The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin.
Virus Res.
27:219-228[Medline].
|
| 16.
|
Paul, J.
1972.
In
Cell and tissue culture, 4th ed.
Livingstone, Edinburgh, Scotland.
|
| 17.
|
Porter, A. G.
1993.
Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions.
J. Virol.
67:6917-6921[Free Full Text].
|
| 18.
|
Rümenapf, T.,
G. Meyers,
R. Stark, and H.-J. Thiel.
1989.
Hog cholera virus characterization of specific antiserum and identification of cDNA clones.
Virology
171:18-27[Medline].
|
| 19.
|
Schaffer, F. L.,
D. W. Ehresmann,
M. K. Fretz, and M. E. Soergel.
1980.
A protein, VPg, covalently linked to 36S calicivirus RNA.
J. Gen. Virol.
47:215-220[Abstract/Free Full Text].
|
| 20.
|
Seglen, P. O.
1976.
Preparation of isolated rat hepatocytes.
Methods Cell Biol.
13:30-83.
|
| 21.
|
Takeda, N.,
R. J. Kuhn,
C.-F. Yang,
T. Takegami, and E. Wimmer.
1986.
Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells.
J. Virol.
60:43-53[Abstract/Free Full Text].
|
| 22.
|
Walpole, H. E.,
W. M. Lee,
T. Walle,
U. K. Walle,
M. J. Wilson, and J. W. Kennedy.
1990.
Rabbit hepatocytes in primary culture: preparation, viability and use in studies of propanolol metabolism.
Hepatology
11:394-400[Medline].
|
| 23.
|
Wirblich, C.,
M. Sibilia,
M. B. Boniotti,
C. Rossi,
H.-J. Thiel, and G. Meyers.
1995.
3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.
J. Virol.
69:7159-7168[Abstract].
|
| 24.
|
Wirblich, C.,
H.-J. Thiel, and G. Meyers.
1996.
Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies.
J. Virol.
70:7974-7983[Abstract].
|
J Virol, May 1998, p. 4492-4497, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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