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Journal of Virology, September 1998, p. 7697-7702, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Nucleolus Is the Site of Borna Disease Virus
RNA Transcription and Replication
J. M.
Pyper,*
J. E.
Clements, and
M. C.
Zink
Division of Comparative Medicine, Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
Received 13 February 1998/Accepted 3 June 1998
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ABSTRACT |
Borna disease virus (BDV) is a neurotropic nonsegmented
negative-strand RNA virus with limited homology to rhabdoviruses and paramyxoviruses. A distinguishing feature of BDV is that it replicates in the nucleus of infected cells. Strand-specific probes used for in
situ hybridization of infected rat brain showed that there was
differential localization of positive- and negative-strand RNAs within
the nucleus of neurons. Within nuclei, sense-strand RNAs were
preferentially localized within nucleolar regions while genomic-sense
RNAs were found in both nucleolar and nonnucleolar regions. These
results suggested a role for the nucleolus in BDV replication. Nucleoli
isolated from persistently infected neuroblastoma cells contained both
genomic and antigenomic BDV RNA species as well as an enrichment of the
39/38-kDa and gp18 BDV proteins. Since the nucleolus is the site of
rRNA transcription, we examined BDV transcription in the presence of
inhibitors of RNA polymerase I. Inhibition of RNA polymerase I did not
affect levels of BDV transcription.
| |
TEXT |
Borna disease virus (BDV) is a
neurotropic virus that causes severe neurological disease in its
natural hosts (horses, sheep, cattle, and cats) (reviewed in reference
30). The disease is rare, found mostly in central
Europe and Scandinavia, and is not readily transmitted between animals.
In rats, clinical signs of disease become apparent only when the virus
reaches the hippocampus. Hippocampal neurons appear to be most
sensitive to the effects of BDV infection and express extremely high
levels of BDV antigens and RNA (25, 29).
BDV is a nonsegmented negative-strand (NNS) RNA virus that has recently
been classified as a paramyxovirus. However, it is unique among NNS
viruses infecting animals in that it replicates in the nucleus (5,
11) and has a number of spliced mRNAs (9, 32).
Previous unpublished observations from this laboratory suggested that
BDV RNA was concentrated in the nucleoli of infected rat brain neurons.
In the present study, we extended these observations by using
strand-specific probes for in situ hybridization of sections from
infected rat brain. At early time points after infection, these probes
revealed a differential pattern of BDV RNA in and around the nucleolus.
Sense-strand RNA was detected within the nucleolus, whereas
genomic-sense RNA was detected throughout the nucleus. Although BDV RNA
and protein species were detected in isolated nucleoli, inhibition of
RNA polymerase (Pol) I did not affect synthesis of BDV RNA species.
Localization of BDV RNA in neurons of infected rats.
Previous
experiments using in situ hybridization suggested that BDV RNA
localized in the nucleoli of infected rat brain neurons (unpublished
observations). To further examine this possibility, 35S-labeled strand-specific probes were used to detect BDV
antigenomic (sense) RNA (Fig. 1a) and BDV
genomic RNA (Fig. 1b). Single-stranded probes were made from products
of single-sided PCR that specifically amplified one strand of cloned
BDV DNA. Clone RT-PCR 5'/3' was used as a template to make the
single-stranded probes; this clone contains ~300 nucleotides (nt)
from each end of the BDV genome and includes coding sequences from the
nucleocapsid and Pol genes as well as the terminal noncoding sequences
(10). To make a probe that detects sense RNA, clone RT-PCR
5'/3' was linearized with SmaI, and an SP6 primer was used
to synthesize sense-strand DNA in a one-sided PCR. This fragment was
gel purified and used as the template DNA for making a probe with
35S-dCTP (Amersham) with the Oligo Labeling kit
(Pharmacia). In a similar fashion, a probe was prepared to detect
genomic RNA. Clone RT-PCR 5'/3' was linearized with SstI,
and a T7 primer was used to synthesize an antisense DNA fragment. The
purified fragment was used as a template for making
35S-dCTP-labeled probe. Probes were labeled to a specific
activity of 109 cpm/µg and added to the tissue sections
at a concentration of 0.2 µg/ml in hybridization buffer. The sections
were incubated at 37°C overnight, washed, and dipped in radiographic
emulsion (NTB-3; Kodak). Sections were developed after 2 or 3 days of
incubation in the dark and counterstained with hematoxylin to identify
nuclear structure.
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FIG. 1.
In situ hybridization of BDV-infected rat brain 12 days
postinfection. Sections were hybridized with single-stranded DNA probes
that detect sense-strand RNA (a) and genomic RNA (b). (a) The nucleolus
is densely labeled, while there are few grains in the remainder of the
nucleus. (b) The nucleolus is spared, but grains are densely
distributed at the periphery of the nucleolus and found more diffusely
in the nucleoplasm of the neuron. The figure was prepared with
Photoshop.
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Sagittal sections of brains from pairs of neonatal rats infected for
periods of time ranging from 3 days to 7 weeks were examined
(total of
18 infected rats). For the earliest two time points
(3 and 8 days
postinfection), no in situ hybridization signal
could be detected. At
12 days postinfection, silver grains could
be detected in some of the
neurons. Hematoxylin counterstaining
was used to reveal the subcellular
architecture of these neurons,
allowing us to categorize the pattern of
grains. Representative
cells hybridized with strand-specific probes are
shown in Fig.
1. Figure
1a is an example of detection of sense-strand
RNA, and
Fig.
1b is an example of detection of genomic RNA. At later
time
points, the density of silver grains was so high that it was
impossible
to discriminate subnuclear regions in the cells. Therefore,
the
quantitative analysis was limited to the two animals sacrificed
12 days postinoculation. This time point, when grains were first
visible,
is expected to represent the earliest stage in replication
and thus may
more clearly reveal differential subcellular localization
of
replicating viral RNAs. For each probe and each rat, 100 cells
with
identifiable nucleoli were examined. Cells were evaluated
for the
distribution of grains by examining the relative number
of grains over
the nucleus and the nucleolus.
Although individual cells showed different distributions, overall
trends did emerge from an analysis of cells that showed
predominantly
nucleolar or predominantly nuclear (nonnucleolar)
density of grains
(Table
1). For both animals, sense RNAs
were
present at higher levels in the nucleolus than in the nucleus.
In
contrast, the genomic RNAs tended to be equivalently represented
in
both nucleolar and nonnucleolar regions, although grains were
often
concentrated in perinucleolar regions. This examination
strongly
suggested that the nucleolus was involved in viral RNA
synthesis. It
further suggested that there was differential retention
of antigenomic
and genomic RNA species within the nucleolus. The
higher levels of
antigenomic RNA within the nucleolus suggest
its retention as a
template for synthesis of the genomic RNA while
the higher nuclear (and
perinucleolar) levels of genomic RNA suggest
that it is being exported
from the site of synthesis.
Detection of BDV RNA species in nucleolar fractions.
The in
situ hybridization data strongly suggested an association of BDV RNA
with the nucleolus. To extend this observation, RNA was prepared from
subcellular fractions of BDSK cells (28), which are
persistently infected human neuroblastoma SKNSH cells (2).
The nucleolar isolation protocol of Bolla et al. (3) was
used with aliquots saved from intermediate stages of purification. All
solutions contained 10 mM vanadyl ribonucleoside complex (Life Sciences) to inhibit RNases during the fractionation procedure. The
crude nucleoli were washed with TE (1 mM Tris-HCl [pH 8.0], 1 mM
EDTA), digested with RQ1 DNase (Promega), and finally washed with 2 M
NaCl to remove any contaminants. RNA was prepared from each fraction
(7) and recovered from cytoplasmic, nuclear, and nucleolar
fractions as well as from the TE wash of the crude nucleoli but was not
recovered from the nonnucleolar nuclear fraction despite the presence
of vanadyl ribonucleoside complex in all solutions during the
fractionation procedure.
RNA species present in each fraction were identified by Northern
blotting analysis with probes specific for genomic and antigenomic
BDV
RNA as well as a probe specific for 18S rRNA (Fig.
2). Genomic
RNA was present at high
levels in the nucleus, nucleolus, and
the TE wash of the crude
nucleoli, with lower levels detected
in the cytoplasmic and whole-cell
preparations. Hybridization
with a probe that detects mRNAs and
antigenomic RNAs showed that
full-length antigenomic RNA was present in
all fractions, with
high levels detected in the cytoplasm, nuclei, and
nucleoli. The
0.85- and 2.1-kb mRNA bands are clearly detected in the
total
and cytoplasmic RNA lanes. However, in the nuclear, nucleolar,
and TE wash fractions there is a broad band of ~0.85 to 1 kb in
size;
the specific mRNA bands are not clearly discernible. Hybridization
with
an 18S rRNA-specific probe clearly showed the presence of
rRNA
precursor molecules in the nuclear and nucleolar fractions
as well as
the TE nucleolar wash. Only mature 18S rRNA was detected
in the
whole-cell and cytoplasmic RNA preparations.
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FIG. 2.
Detection of RNA species in subcellular fractions of
BDV-infected cells. RNA isolated from subcellular fractions was
subjected to Northern blot analysis with probes specific for BDV
genomic RNA (A), BDV sense RNA (B), and 18S rRNA (C). The locations of
the full-length genomic and antigenomic RNAs are indicated in panels A
and B. Panel B also shows the location of the abundant 0.85-kb mRNA.
The location of the mature 18S rRNA species is indicated in panel C;
asterisks show the precursor species detected in the nuclear and
nucleolar fractions and in the TE wash of nucleoli. The figure was made
from scanned fluorographs with Photoshop and Illustrator.
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Detection of BDV proteins in nucleolar fractions.
The
detection of BDV RNA species in the nucleolar fraction of persistently
infected cells led to an examination of the viral protein species
present in nucleoli. Many studies have documented punctate nuclear
immunofluorescence in BDV-infected cells, but this signal has not been
shown to colocalize with nucleoli. Metabolically labeled BDSK cells
were subjected to the same fractionation protocol except that a
cocktail of protease inhibitors was used instead of the vanadyl
ribonucleoside complex. BDSK cells were metabolically labeled overnight
with [35S]methionine and [35S]cysteine (700 µCi/ml; TransLabel; ICN) prior to isolation of nucleoli. All
solutions used in the fractionation procedure contained a cocktail of
protease inhibitors (aprotinin, 10 µg/ml; leupeptin, 10 µg/ml;
pepstatin, 1 µg/ml; TLCK
[N
-p-tosyl-L-lysine chloromethyl ketone], 40 µg/ml; TPCK [tolylsulfonyl phenylalanyl chloromethyl ketone], 40 µg/ml; phenylmethylsulfonyl fluoride, 10 µg/ml).
Purified nucleoli were suspended in 1× IP lysis buffer (1%
Zwittergent, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl
sulfate, 25 mM Tris-HCl [pH 7.5], 10 µg of phenylmethylsulfonyl
fluoride per ml), and samples from intermediate steps in the
purification were also adjusted to 1× IP lysis buffer. Lysates were
immunoprecipitated with equivalent levels of incorporated
radioactivity. Proteins were analyzed from the whole-cell extract, the
cytoplasm, the nuclei, the nucleoli, the nonnucleolar nuclear fraction,
the TE wash of the crude nucleoli, and the final 2 M NaCl wash of the nucleoli. Pooled sera from infected rats were used to analyze BDV
proteins in the different fractions. Additional antisera directed against cellular proteins were used to detect cellular nucleolar and
nonnucleolar nuclear proteins. B23 is a protein involved in shuttling
components to the nucleolus and is concentrated in the nucleolus but
not strictly localized there (4, 8, 12, 37). Anti-B23
antibody was purchased from Santa Cruz Biotechnology. SC-35 is a
nuclear spliceosomal protein that is excluded from the nucleolus
(6, 34). Anti-SC-35 antibody was purchased from Sigma.
Immunoprecipitation with anti-BDV antisera showed that BDV proteins
were differentially represented in the subcellular fractions
(Fig.
3A). Detection of the 24-kDa protein was
relatively constant
among the fractions although slightly lower in the
supernatants
from the TE and salt washes. In contrast, the level of the
39/38-kDa
nucleocapsid protein was slightly elevated in the nuclear
fraction
compared to that in the cytoplasmic fraction and the
whole-cell
extract but markedly elevated in the nucleolar fraction and
in
proteins eluted by the final 2 M NaCl wash of nucleoli. Proteins
washed from the crude nucleoli with TE prior to the salt wash
contained
reduced levels of nucleocapsid protein. Interestingly,
gp18 (previously
called the 14.5-kDa protein [
31]) shows a
fractionation
pattern similar to that of the nucleocapsid protein.
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FIG. 3.
Detection of protein species in subcellular fractions of
BDV-infected cells. Equivalent levels of 35S-incorporated
radioactivity were used for immunoprecipitations of subcellular
fractions by using antisera specific for BDV proteins (A) and for
markers of nucleolar proteins (B23) and nonnucleolar nuclear proteins
(SC-35) (B and C). (A) Immunoprecipitation of BDV proteins. (B)
Immunoprecipitation of B23. (C) Immunoprecipitation of SC-35. The
figure was made from scanned fluorographs with Photoshop and
Illustrator.
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The efficiency of the fractionation protocol was assessed by
immunoprecipitation of the cellular proteins, B23 and SC-35,
that
served as markers of nucleolar and nonnucleolar nuclear fractions,
respectively (Fig.
3B and C). B23 was detected in all fractions
but was
observed at higher levels in the nuclear, nonnucleolar,
and nucleolar
fractions, with the highest concentration in nucleoli
(Fig.
3B).
Fractions were also immunoprecipitated with anti-SC-35.
Little or no
SC-35 was detected in immunoprecipitations from the
whole-cell lysate,
the cytoplasmic fraction, or the salt wash.
SC-35 was present in the
immunoprecipitations from the nuclear
and nonnucleolar fractions as
well as the TE supernatant, with
highest levels detected in the
nonnucleolar fraction (Fig.
3C).
Although all immunoprecipitations were
subjected to the same stringent
washing conditions, numerous background
bands were detected only
in the nucleolar and nonnucleolar lanes.
Because of the background
in the nucleolar lane, it is impossible to
state unequivocally
that there is no SC-35 present in the nucleolar
preparation, but
it is clear that the level is much lower than that in
the nonnucleolar
fraction. Additionally, the salt wash contains much
less SC-35
than does the TE wash, also suggesting that SC-35 has been
removed
during the nucleolar isolation.
Resistance of BDV RNA synthesis to inhibitors of cellular RNA
Pols.
The detection of BDV RNA and protein species in the
nucleolus suggested a nucleolar functional role in BDV RNA synthesis. One possibility was that RNA Pol I or its cofactors might be involved in BDV RNA synthesis either directly or indirectly. To test this hypothesis, RNA species synthesized in the presence of inhibitors of
cellular transcription by RNA Pol I (25 µM camptothecin or 0.04 µg
of actinomycin D per ml) or RNA Pol I and Pol II (4 µg of actinomycin
D per ml) were analyzed in an RNase protection assay (Fig.
4). For this experiment, the RNA species
synthesized during the drug treatments were metabolically labeled with
[32P]orthophosphate, and the probes were unlabeled RNA
species generated in vitro. Four T-25 flasks of BDSK cells were
metabolically labeled in the presence of inhibitors of cellular RNA
polymerases. One flask of cells was labeled without drug treatment
(control). The other flasks were treated with drugs: 25 µM
camptothecin (Pol I inhibition), 0.04 µg (low) of actinomycin D (Pol
I inhibition) per ml, or 4 µg (high) of actinomycin D (Pol I and Pol
II inhibition) per ml. The flasks were incubated with the drugs in
complete medium for 1 h. They were then washed with phosphate-free
RPMI medium (Gibco). The labeling medium contained the appropriate drug
in phosphate-free RPMI supplemented with 10% dialyzed fetal calf serum
(Gibco) and 2 mCi of [32P]orthophosphate (New England
Nuclear) per ml. Cells were maintained in labeling medium for 5 h,
and then RNA was isolated (7). Both the total yield of RNA
and the incorporation of radioactivity were determined for each sample.
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FIG. 4.
RNase protection analysis of RNA species synthesized in
the presence of RNA Pol inhibitors. 32P-labeled RNA was
isolated from cells that were metabolically labeled during treatment
with Pol inhibitors. The labeled RNA was hybridized with unlabeled
antisense transcripts generated in vitro. Following digestion with
RNase A, the protected fragments were resolved by gel electrophoresis.
The specific full-length protected band is indicated by an asterisk in
each panel. (A) BDV probe. (B) Actin probe. (C) 18S rRNA probe. For
panel C, 1 µg of total labeled cellular RNA was used for
hybridization. For panels A and B, 10 µg of total labeled cellular
RNA was used for hybridization. The figure was made from scanned
fluorographs with Photoshop and Illustrator. campto, camptothecin; Act
D, actinomycin D.
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To detect RNA species transcribed during the drug treatments, unlabeled
antisense transcripts were hybridized to the labeled
RNA isolated from
BDSK cells followed by RNase A digestion and
gel analysis. Unlabeled
probes specific for BDV RNA, actin mRNA,
and 18S rRNA were transcribed
in vitro. The BDV 3' RACE clone
(
10) contains 314 nt of BDV
sequence from the 3' end of the
genome, including a portion of the
nucleocapsid gene. It was linearized
with
HindIII, and
RNA was transcribed with T7 RNA Pol. Actin 104
(
18) contains
a 104-nt fragment of the human gene. The plasmid
DNA was linearized
with
BamHI, and T7 RNA Pol was used to transcribe
antisense
RNA. An 18S rRNA clone was made by amplifying a 200-nt
fragment (nt 761 to 960 from the human sequence) and inserting
the fragment into pGEM4Z
(Promega) with
KpnI and
BamHI. The cloned
DNA was
linearized with
EcoRI, and antisense RNA was transcribed
with T7 RNA Pol.
Hybridizations with the actin and BDV probes contained 10 µg of
32P-labeled total RNA isolated from BDSK cells and 2 µg
of unlabeled
antisense RNA probe. One microgram of total labeled RNA
was hybridized
to 2 µg of the 18S rRNA probe to ensure that there was
excess
probe in the reaction. Multiple probes could not be hybridized
in a single tube because of high background levels arising from
the
digested labeled cellular RNA. Hybridization and digestion
conditions
have been described previously (
27), except that
the
digestion buffer contained 100 µg of RNase A per ml and the
digestion
was allowed to proceed for 1 h.
The level of BDV RNA species synthesized was constant despite the drug
treatments (Fig.
4A). In contrast, the drug treatments
affected
transcription of cellular RNA by RNA Pol I and Pol II
(Fig.
4B and C).
RNA Pol II activity was assessed with an actin-specific
probe (Fig.
4B). The camptothecin treatment caused a slight reduction
in expression
of actin mRNA, but the low concentration of actinomycin
D did not.
However, as expected, the high concentration of actinomycin
D reduced
actin mRNA expression to undetectable levels. An 18S
rRNA-specific
probe was used to assess the drugs' effects on RNA
Pol I synthetic
activity (Fig.
4C). The 18S rRNA probe protects
a 200-nt fragment,
although an additional band of ~100 nt shows
similar responses to the
drugs. Both camptothecin and the low
concentration of actinomycin D
markedly reduced expression of
the 18S rRNA species but did not
completely inhibit it. However,
no 18S rRNA was detected when cells
were treated with the high
concentration of actinomycin D.
From analysis of 18S rRNA and actin mRNA synthesis, it is clear that 4 µg of actinomycin D per ml inhibited cellular transcription
by both
RNA Pol I and RNA Pol II. The conditions expected to specifically
inhibit cellular transcription by RNA Pol I were not completely
effective in blocking 18S rRNA synthesis, although transcription
was
significantly reduced. However, none of these drug treatments
(including the high dose of actinomycin D) affected the level
of BDV
RNA synthesis, indicating that inhibition of the cellular
RNA Pols
neither increased nor decreased transcription of BDV
RNA.
Conclusions.
This study demonstrates that the nucleolus is the
site of BDV RNA synthesis. In situ hybridization of newly infected
neurons showed a concentration of BDV RNA in and around the nucleolus, with a concentration of antigenomic RNA within the nucleolus. The
antigenomic species are expected to be retained in the nucleolus, while
some of the genomic species would be exported to form viral particles.
The observation of genomic species at the periphery of the nucleolus
suggests export from the site of synthesis. Differential localization
of viral RNA species was not detectable at later times of infection in
neurons in the rat brains, nor was it detectable by Northern blotting
analysis of the persistently infected cells. It is likely that high
levels of RNA expression obscure subtle quantitative differences. In
the persistently infected cells, both positive- and genomic-sense 9-kb
viral RNAs were found in the nucleolus. The presence of both genomic
and antigenomic BDV RNAs in the nucleolus is consistent with BDV
replicative processes occurring in the nucleolus.
A subset of BDV proteins was also concentrated in the nucleolus. If
viral RNA synthesis occurs in the nucleolus, the presence
of certain
BDV proteins would be required in the nucleolus. Viral
Pol activity
must be present at the site of synthesis, but the
protein is presumably
present at such low levels as to be undetectable
by
immunoprecipitation. By analogy with other NNS viruses, the
24-kDa
phosphoprotein is expected to play a role in BDV replication.
Although
nucleoli did not contain enriched levels of the 24-kDa
protein, it was
present at significant levels within nucleoli.
Its presence in multiple
cellular compartments may reflect a multifunctional
role for this
phosphoprotein in addition to its presumed role
in replication. The
nucleocapsid proteins are expected to be present
at high levels at the
site of viral RNA replication since genomic
and antigenomic viral RNAs
of NNS viruses are encapsidated at
the time of synthesis. The 39/38-kDa
nucleocapsid proteins are
indeed present at high levels in the
nucleolus.
Curiously, we also observed that the level of the gp18 glycoprotein
(formerly identified as the 14.5-kDa protein [
31]) was
elevated in the nucleolus. gp18 is encoded by the third open reading
frame of BDV (
21). For other NNS viruses, the third open
reading
frame encodes the matrix protein. The matrix proteins of NNS
viruses
are believed to form a bridge between the nucleocapsid protein
and the viral envelope. In rhabdovirus infections, M binds to
the
nucleocapsid core. The nucleocapsid core-M protein complexes
migrate to
regions of the plasma membrane where G protein is concentrated
and
where the virus buds from the cell (reviewed in reference
35). In contrast, in the classic paramyxoviruses the
M protein
associates with the viral glycoprotein at the cell surface
prior
to arrival of the nucleocapsid (reviewed in reference
22).
The nucleolar concentration of gp18 suggests that it associates with
newly synthesized nucleocapsids before their export from
the nucleolus.
Interestingly, antibodies to gp18 have some neutralizing
activity
against BDV (
20). This neutralizing activity may be
directed
against M-coated nucleocapsids rather than complete virions.
BDV-infected cells produce very low levels of infectious particles
even
though they express high levels of BDV RNA and proteins.
Additionally,
it has proven difficult to conclusively identify
viral particles either
in cell culture or in infected animals.
This suggests that assembly of
complete particles is very inefficient.
Immunofluorescent detection of
newly infected tissue culture cells
usually shows that there are
discrete foci of intensely labeled
cells surrounded by cells that are
labeled less intensely, suggesting
cell-to-cell spread of infection.
Probably, infection by M-coated
nucleocapsids is relatively
inefficient, and this infection can
be blocked by anti-gp18 antibodies.
However, once one cell is
infected, adjacent cells can be infected by
cell-to-cell transmission
of incomplete particles.
The apparent nucleolar involvement suggested by the in situ experiments
led us to investigate whether RNA Pol I might be involved
in BDV RNA
synthesis. BDV RNA synthesis was not affected under
conditions that
inhibited cellular RNA Pol I, indicating that
BDV does not use cellular
Pols or cofactors for transcribing and/or
replicating its RNA. However,
it is possible that other nucleolar
proteins are involved, possibly by
providing a structural environment
for RNA synthesis or a mechanism for
export of ribonucleoprotein
particles.
The nucleolus is the site of extremely high levels of biosynthetic
activity: rRNA species are transcribed and processed, and
ribosomal
proteins are recruited to the nucleolus for assembly
of preribosomes.
BDV appears to be one of a small group of unrelated
viruses that are
known to have coopted certain aspects of nucleolar
activity. There are
only a few known examples of viral replication
in nucleoli. Plant
viroids replicate in the nuclei of infected
cells in association with
nucleoli (
33), and minute virus of
mice, a parvovirus,
replicates its DNA in host cell nucleoli (
24,
36).
The lentiviruses have made use of nucleoli in a different fashion. The
viral proteins Rev and Rex accumulate in nucleoli;
these proteins have
been shown elsewhere to be responsible for
transporting unspliced mRNAs
to the cytoplasm (
13,
15,
17,
19,
23,
26). The nucleolar
localization signal of human
T-cell leukemia virus type 1 Rex and human
immunodeficiency virus
type 1 Rev binds specifically to the nucleolar
shuttle phosphoprotein
B23 (
1,
14). B23 is a major nucleolar
protein that is found
associated with preribosomal particles, although
its subcellular
localization changes during the cell cycle (
4,
8,
12,
37,
38). Its importance in ribosomal biosynthesis is
suggested
by the fact that it is highly abundant in transformed cells
and
in actively growing cells in comparison to resting cells
(
16).
Our data strongly suggest that nucleoli are the sites for BDV
replication and/or transcription. However, since BDV, like the
lentiviruses, generates both spliced and unspliced mRNAs that
must be
transported from the nucleus, BDV may use a mechanism
that subverts
nucleolar transport functions. Although no Rev response
element-like
elements or Rev-like proteins have been described
for BDV, it is
possible that the virus uses nucleolar transport
mechanisms to deliver
viral RNA to appropriate cellular compartments.
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ACKNOWLEDGMENTS |
We thank Michele Nealen, Jeff Spelman, and Kevin Maughan for
technical assistance and Kishna Kalicharran, Barry Morse, and other
members of the Retrovirus Biology Laboratory for helpful discussions.
This work was supported by grant NS31908 from the National Institutes
of Health.
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FOOTNOTES |
*
Corresponding author. Present address: Laboratory of
Viral Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bldg. 9, Rm. 1E127, MSC 0930, Bethesda, MD 20892-0930. Phone: (301) 435-6019. Fax: (301) 435-6021. E-mail:
jpyper{at}atlas.niaid.nih.gov.
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Journal of Virology, September 1998, p. 7697-7702, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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