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Journal of Virology, December 1998, p. 9755-9762, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Two Proline-Rich Nuclear Localization Signals in
the Amino- and Carboxyl-Terminal Regions of the Borna Disease
Virus Phosphoprotein
Yuko
Shoya,1
Takeshi
Kobayashi,1
Toshiaki
Koda,1
Kazuyoshi
Ikuta,2
Mitsuaki
Kakinuma,1 and
Masahiko
Kishi1,*
Sections of Bacterial
Infection1 and
Serology,2 Institute of Immunological
Science, Hokkaido University, Sapporo 060-0815, Japan
Received 22 June 1998/Accepted 9 September 1998
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ABSTRACT |
Borna disease virus (BDV) uses a unique strategy of replication and
transcription which takes place in the nucleus, unlike other known,
nonsegmented, negative-stranded RNA viruses of animal origin. In this
process, viral constituents necessary for replication must be
transported to the nucleus from the cytoplasm. We report here the
evidence that BDV P protein, which may play an important role in viral
replication and transcription, is transported into the nucleus in the
absence of other viral constituents. This transportation is
accomplished by its own nuclear localization signals (NLSs), which are
present in both N-terminal (29PRPRKIPR36) and
C-terminal (181PPRIYPQLPSAPT193) regions of the
protein. These two NLSs can function independently and both have
several Pro residues as key amino acids.
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INTRODUCTION |
Borna disease (BD) is a naturally
occurring progressive encephalopathy of horses and sheep caused by
infection with a neurotropic enveloped virus, BD virus (BDV) (16,
26). Molecular biological analyses have shown that BDV has a
nonsegmented, negative-stranded RNA genome of 8.9 kb, which is present
in the nucleus of BDV-infected cells (3-6, 22). The BDV
genome consists of at least six open reading frames (ORFs). ORF I
encodes nucleoproteins (N) of 38 or 40 kDa (15, 17, 20). ORF
II encodes a phosphoprotein (P) of 23 to 24 kDa (12, 15,
28). ORF III encodes an 18-kDa glycoprotein (M, gp18)
(11). ORF IV encodes a protein of 56 or 64 kDa which is
glycosylated to give a glycoprotein of 84 or 94 kDa (8, 23).
ORF V encodes a predicted RNA-dependent RNA polymerase (L) of 170 to
180 kDa (3, 5). Recently, ORF ×1, which overlaps ORF II,
was reported to encode a protein (p10, or X) of 10 kDa (30).
The genomic organization of BDV is similar to those of animal
rhabdoviruses such as vesicular stomatitis virus and rabies virus
except that the BDV genome has an additional ORF ×1 which overlaps
with ORF II but uses a different reading frame. Another major
difference from the animal rhabdoviruses is that the BDV genome
replicates in the nucleus of infected cells (4-6, 22).
The function of the BDV P protein is not known, although it is
generally assumed that the BDV P associates and cooperates with the L
protein to play a pivotal role in viral transcription and replication.
P proteins with such a role have been identified in animal
rhabdoviruses (29). If BDV P is a cofactor of L polymerase, it should migrate to the nucleus, where viral transcription and replication take place. Here, we report that BDV P is transported into
the nucleus in the absence of other viral constituents and that the
transportation is accomplished by virtue of BDV P protein's own
nuclear localization signals (NLSs), which are present in both
N-terminal and C-terminal regions. The NLSs of BDV P are unique in that
both can function independently and both have several proline residues
as key amino acids.
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MATERIALS AND METHODS |
BDV and cells.
Madin-Darby canine kidney (MDCK) cells
persistently infected with BDV (MDCK/BDV) (9) were kindly
provided by R. Rott, Justus-Liebig-Universität Giessen. MDCK
cells and COS-7 cells (7) were cultured as described previously (13).
Plasmid construction.
The eukaryotic expression plasmid
pP-Wild encoding BDV P (amino acids 1 to 201) was constructed as
follows. To obtain cDNA corresponding to the products of the entire BDV
genome, total RNA extracted from MDCK/BDV cells was reverse transcribed
by using a BDV-specific primer pair as described previously
(25). BDV P cDNA was amplified by PCR from a BDV cDNA with
BDV P-specific primers 1 and 2 (Tables 1
and 2) and then cloned into the
EcoRI-KpnI sites immediately downstream of and in
frame with the influenza virus hemagglutinin (HA) 12CA5 epitope tag in
the eukaryotic expression vector pcDL-HA (13). Eukaryotic
expression plasmids encoding a series of deletion mutants of BDV P were
constructed as described above with the sets of primers listed in
Tables 1 and 2. The resultant constructs, pP-del.N18, pP-del.N40,
pP-del.N82, pP-del.C117, pP-del.C148, and pP-del.C182, encoded BDV P
lacking the N-terminal 18, 40, and 82 amino acid residues and the
C-terminal 85, 54, and 20 amino acid residues, respectively. pRSV-1/41,
pRSV-41/181, pRSV-172/201, pRSV-18/36, pRSV-172/193, and pRSV-179/201,
eukaryotic expression plasmids encoding Escherichia coli
lacZ fused with relatively large polypeptide chains from BDV
P, were constructed as described for pRSV-N.LacZ (13).
Briefly, cDNA fragments encoding various polypeptides from BDV P were
amplified by PCR with pairs of primers (Tables 1 and 2) with BDV P cDNA
as a template and then cloned into the KpnI site between the
gpt and trpS sequences, in frame with the
lacZ gene in RSV-LacZ (14). Plasmids pRSV-18/28, pRSV-29/36, pRSV-181/193, and pRSV-192/201, expressing
-galactosidase fused with relatively short polypeptides from
BDV P, were constructed similarly except for the use of adapters
(Tables 1 and 2). pRSV-sub.29, pRSV-sub.31, pRSV-sub.35, and
pRSV-sub.32/33, encoding amino acid-substituted fusion proteins of
pRSV-29, -31, -35, and -32/33, respectively, and pRSV-sub.182,
pRSV-sub.186, pRSV-sub.189, and pRSV-sub.192, encoding amino
acid-substituted fusion proteins of pRSV-182, -186, -189, and -192, respectively, were constructed similarly with adapters (Tables 1 and
2). The nucleotide numbering used here follows that used previously for
a horse-derived BDV, strain V (the EMBL databank, accession number
U04608). The nucleotide sequences of recombinant constructs were
confirmed by using a 373A automatic DNA sequencer (Applied Biosystems).
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TABLE 2.
Primer pairs and adapters used to amplify BDV P and its
mutant fragments inserted into eukaryotic expression plasmids
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Antibodies.
Anti-BDV P polyclonal antiserum was prepared by
immunization of a rabbit with glutathione column-purified glutathione
S-transferase (GST)-BDV-P fusion protein expressed in
E. coli. Mouse monoclonal antibody (MAb) to the 12CA5
epitope of influenza virus HA was obtained from Boehringer Mannheim.
Mouse anti--galactosidase MAb was from Gibco BRL. Horseradish
peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG)
antibody was from Amersham. Dichlorotriazinyl amino fluorescein
(DTAF)-conjugated goat anti-rabbit IgG antibody was from Immunotech.
HRP-conjugated sheep anti-mouse IgG antibody was from Amersham.
DTAF-conjugated goat anti-mouse IgG antibody was from Immunotech.
Transfection, Western blotting, and immunofluorescence
analysis.
For eukaryotic expression of BDV P, COS-7 cells were
seeded at 2.0 × 105 cells per 35-mm-diameter
glass-bottom culture dish, and on the next day the cells were
transfected with 2 µg of plasmid DNA with Lipofectamine (Gibco BRL).
After cultivation for 48 h, the transfected cells were harvested
for detection of the expressed BDV P by Western blotting with rabbit
anti-BDV P serum (1:500 dilution) as the first antibody and
HRP-conjugated sheep anti-mouse IgG antibody (1:5,000 dilution) as the
secondary antibody. To detect the -galactosidase fusion proteins,
the cultured cells were subjected to Western blotting analysis with the
anti--galactosidase MAb (1:500 dilution) as the first antibody and
HRP-conjugated sheep anti-mouse IgG antibody (1:5,000 dilution) as the
secondary antibody. An ECL Western blotting kit (Amersham) was used to
visualize the expressed protein.
The transfected cells were also subjected to indirect
immunofluorescence assay (IFA). After culture, the cells were fixed
with 4% paraformaldehyde prior to treatment with 0.4% Triton X-100
(
32). To detect BDV P and its deletion mutants, anti-BDV P
antiserum
(1:500 dilution) was used as the first antibody, and
DTAF-conjugated
goat anti-mouse IgG antibody (1:50 dilution) was used
as the secondary
antibody. Similarly, for the detection of BDV P-fused
-galactosidase
proteins, mouse anti-
-galactosidase MAb (1:100
dilution) and
DTAF-conjugated goat anti-mouse IgG antibody (1:50
dilution) were
used. To detect the 12CA5 epitope of HA, mouse
anti-12CA5 MAb
(1:40 dilution) and DTAF-conjugated goat anti-mouse IgG
antibody
(1:50 dilution) were used. After being stained, the cells were
analyzed with a confocal laser scanning microscope (Meridian
Instruments).
MDCK/BDV cells were used as a positive control, and
noninfected
MDCK cells or nontransfected COS-7 cells served as negative
controls.
The results shown in Fig.
1C,
2C,
3C,
4C,
5C, and
6C are
representative
examples of the average expression pattern found in each
experiment.
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RESULTS |
Intracellular localization of BDV P in cells infected with BDV and
transfected with BDV-P expression plasmids.
The specificity of
rabbit anti-BDV P antiserum was verified by Western blotting of the
lysates of MDCK cells noninfected and persistently infected with BDV.
As shown in Fig. 1B, a protein of
approximately 23 to 24 kDa was detected with the anti-BDV P antiserum
in the MDCK/BDV cell lysate (lane 2) but not in the noninfected MDCK
lysate (lane 1). Anti-BDV P antiserum preabsorbed with a GST-BDV P
fusion protein expressed in E. coli did not detect this
protein in MDCK/BDV cells (data not shown), confirming that this
antiserum specifically recognized BDV P. IFA with the anti-BDV P
antiserum allowed visualization of the intracellular localization of
the BDV P. The staining pattern of MDCK/BDV cells was compatible with
the previously reported observations (9) of intense
spot-like staining in the nucleus with relatively weak and diffuse
staining in the cytoplasm (Fig. 1C, panel b). No staining was detected in noninfected MDCK cells (Fig. 1C, panel a). Neither the preabsorbed antiserum nor the preimmune rabbit serum stained the MDCK/BDV cells
(data not shown).
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FIG. 1.
Expression and subcellular localization of deletion
mutants of BDVP. (A) The cDNA fragments of BDV P inserted into pcDL-HA
are shown. The numbers indicate amino acid positions of BDV P. (B)
Expression of wild-type and deletion mutants of BDV P in COS-7 cells as
detected by Western blotting with rabbit antiserum. (C) IFA staining of
COS-7 cells expressing wild-type and deletion mutants of BDV P with
rabbit anti-BDV P. Noninfected MDCK cells (MDCK/ ) served as a
negative control (a), and MDCK/BDV cells served as a positive control
of BDV P in persistently infected cells (b). COS-7 cells were
transfected with pCDL-HA (c), pP-Wild (e), pP-del.N18 (f), pP-del.N40
(g), pP-del.N82 (h), pP-del.C117 (i), pP-del.C148 (j), or pP-del.C182
(k). Nontransfected COS-7 cells (COS7/) served as a negative
transfection control (d).
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To examine whether BDV P is transported into the nucleus in the absence
of other viral constituents, the eukaryotic expression
plasmid pP-Wild
(Fig.
1A) encoding the entire BDV P was constructed
and transfected
into COS-7 cells. Transient expression of BDV
P was examined by Western
blotting with anti-BDV P antiserum.
A protein of approximately 23 to 24 kDa was detected in the lysate
of cells transfected with
pP-Wild (Fig.
1B, lane 5). Similar results
were obtained by Western
blotting with a mouse MAb to the HA epitope
(data not shown). The
anti-BDV P antiserum did not react with
the HA 12CA5 epitope in the
lysate of cells transfected with pcDL-HA
(Fig.
1B, lane 3). Thus, the
recombinant BDV P protein which was
detectable with anti-BDV P
antiserum was transiently expressed
in COS-7 cells after transfection
with pP-Wild.
The subcellular localization of BDV P in COS-7 cells transfected with
pP-Wild was examined by IFA with anti-BDV P antiserum.
In contrast to
the IFA pattern observed in MDCK/BDV cells, the
transfected COS-7
cells showed diffuse and intense fluorescent
staining mostly in the
nucleus (Fig.
1C, panel e). This antiserum
did not react with cells
transfected with pcDL-HA as a control
(Fig.
1C, panel c). Neither the
preabsorbed antiserum nor the
preimmune rabbit serum stained
pP-Wild-transfected cells (data
not shown). IFA staining of the
pP-Wild-transfected cells with
a mouse MAb to the HA epitope gave
essentially the same results
(data not shown). These results indicated
that BDV P protein is
transported into the nucleus in the absence of
other viral
constituents.
Subcellular localization of a series of deletion mutants of BDV
P.
To map the regions involved in the nuclear targeting activity
within BDV P, eukaryotic expression plasmids encoding a series of
deletion mutants of BDV P, i.e., pP-del.N18, pP-del.N40,
pP-del.N82, pP-del.C117, pP-del.C148, and pP-del.C182, were constructed
(Fig. 1A) and transfected into COS-7 cells. Transient expression of the
P deletion mutants was confirmed by Western blotting (Fig. 1B,
lanes 6 to 11). On IFA, all of the deletion mutants of BDV P tested
were localized to the nucleus (Fig. 1C, panels f to k). These
results suggested that the nuclear targeting activity of BDV P
is associated with the central region comprised of amino acid residues
83 to 116. An alternative possibility was that BDV P has more than one
NLS, one of which is located in the N-terminal region and the other is
located in the C-terminal region.
Nuclear targeting activity associated with the N- and
C-terminal regions of BDV P.
To discriminate between the two
alternative possible explanations for the localization of the
NLS(s) in BDV P, eukaryotic expression plasmids encoding
-galactosidase fused with the N-terminal region (pRSV-1/41), the
middle region (pRSV-41/181), or the C-terminal region (pRSV-172/201) of
BDV P were constructed (Fig. 2A), and their nuclear targeting activities were examined. Western blotting analysis with anti--galactosidase MAb showed that the recombinant proteins of expected sizes were expressed in lysates of cells transfected with each plasmid construct (Fig. 2B, lanes 4, 5, and 6).
No protein reactive with the MAb was detected in lysates of
nontransfected cells (Fig. 2B, lane 3) or those transfected with
pRSV-HA (Fig. 2B, lane 2). COS-7 cells transfected with pRSV-LacZ served as a positive control for staining with the MAb used (Fig. 2B,
lane 1). On IFA with the anti--galactosidase MAb (Fig. 2C), the
cells transfected with pRSV-41/181 showed cytoplasmic staining (panel
e), while those transfected with either pRSV-1/41 (panel d) or
pRSV-172/201 (panel f) showed nuclear staining. -Galactosidase, by
itself, was resident in the cytoplasm (Fig. 2C, panel a). Thus, BDV P
may have two independent NLSs located in the N-terminal as well as the
C-terminal region.
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FIG. 2.
Expression and subcellular localization of
-galactosidase fused with the N-terminal, C-terminal, and middle
regions of BDV P. (A) Fragments of BDV P fused to -galactosidase are
shown schematically. The numbers indicate the amino acid positions of
BDV P. (B) Expression of the fusion protein in COS-7 cells as detected
by Western blotting with mouse anti--galactosidase MAb. (C) IFA
staining of COS-7 cells expressing fusion proteins with
anti--galactosidase MAb. COS-7 cells were transfected with pRSV-LacZ
(a), pRSV-HA (b), pRSV-1/41 (d), pRSV-41/181 (e), or pRSV-172/201 (f).
Nontransfected COS-7 cells (COS7/) served as a negative control
(c).
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Analysis of the NLS in the N-terminal region of BDV P.
The
N-terminal region of BDV P contains a basic amino-acid-rich sequence,
22RRERSGSPRPRK33, which resembles the
bipartite NLS, for example, KRKIEEPEPEPKKAK found in
Xenopus laevis protein factor xnf7 (18). In view
of these facts, we constructed pRSV-18/36, which expressed
-galactosidase fused with a peptide, including the previously
suggested NLS (peptide 22/33) of BDV P (28) and tested for
nuclear targeting activity of the expressed fusion protein. The fused
protein was observed as a single protein band on Western blotting with
anti--galactosidase MAb (Fig. 3B, lane 2) and was targeted to the nucleus (Fig. 3C, panel a). To map the NLS
within this region in more detail, pRSV-18/28 expressing -galactosidase fused with peptide 18/28 and pRSV-29/36 expressing -galactosidase fused with peptide 29/36 of BDV P were transfected into COS-7 cells. Western blotting analysis confirmed the expression of
these fusion proteins in transfected COS-7 cells (Fig. 3B, lanes 3 and
4). On IFA with anti--galactosidase MAb (Fig. 3C), the protein
expressed from pRSV-18/28 was found solely in the cytoplasm (panel b),
but the -galactosidase fused with peptide 29/36 of BDV P expressed
from pRSV-29/36 was targeted to the nucleus (panel c). Thus, the NLS
present in the N-terminal region of BDV P was not a bipartite NLS, but
rather the short sequence 29PRPRKIPR36. As the
N-terminal NLS, 29PRPRKIPR36, was rich in Pro
residues, we constructed the substitution mutants pRSV-sub.29,
pRSV-sub.31, and pRSV-sub.35 from pRSV-29/36 and transfected them
into COS-7 cells in which each of the Pro residues in
29PRPRKIPR36 was replaced by Ala
(Fig. 4A). Expression of these mutant
proteins was confirmed by Western blotting with anti--galactosidase
MAb (Fig. 4B, lanes 2, 3, and 4). On IFA with anti--galactosidase
MAb (Fig. 4C), all mutant fusion proteins transiently expressed
by transfection with pRSV-sub.29, pRSV-sub.31, and
pRSV-sub.35 were found in the cytoplasm (panels a, b, and c). These
results indicated that all three Pro residues in
29PRPRKIPR36 are indispensable for the nuclear targeting activity. In contrast, a mutant sequence,
29PRPQQIPR36 in which
32RK33 was replaced with
32QQ33, retained nuclear targeting activity
(Fig. 4C, panel d).
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FIG. 3.
Expression and subcellular localization of
-galactosidase fused with short peptides from the N-terminal region
of BDV P. (A) Amino acid sequences of short peptides fused to
-galactosidase. (B) Expression of the fusion proteins in COS-7 cells
as detected by Western blotting with mouse anti--galactosidase MAb.
(C) IFA staining of COS-7 cells expressing fusion proteins with
anti--galactosidase MAb. COS-7 cells were transfected with
pRSV-18/36 (a), pRSV-18/28 (b), or pRSV-29/36 (c).
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FIG. 4.
Expression and subcellular localization of
-galactosidase fused with substitution mutants of
29PRPRKIPR36 derived from BDV P. (A) The mutant
peptides fused to -galactosidase are shown. The Pro residue (P) at
position 29, 31, or 35 was replaced with Ala (A, boldface), and Arg and
Lys residues at positions 32 and 33 were replaced with Gln (Q,
boldface). (B) Expression of fusion proteins in COS-7 cells as detected
by Western blotting with mouse anti--galactosidase MAb. (C) IFA
staining of COS-7 cells expressing fusion proteins with
anti--galactosidase. COS-7 cells were transfected with pRSV-sub.29
(a), pRSV-sub.31 (b), pRSV-sub.35 (c), or pRSV-sub.32/33 (d).
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Analysis of NLS in the C-terminal region of BDV P.
According
to the experiments presented in Fig. 2, another NLS, peptide 172/201,
may be present in the C-terminal region of BDV P. To map the candidate
NLS in this region, four recombinant plasmids (pRSV-172/193,
pRSV-179/201, pRSV-181/193, and pRSV-192/201) were constructed and
transfected into COS-7 cells. These plasmids encoded -galactosidase
fused with peptides 172/193, 179/201, 181/193, and 192/201 of BDV P,
respectively (Fig. 5B). On IFA (Fig. 5C),
-galactosidase fused with peptides 172/193 (panel a), 179/201 (panel
b), or 181/193 (panel c) was targeted to the nucleus, whereas the
fusion protein with the peptide 192/201 was retained in the
cytoplasm (panel d). These results indicated that the NLS
activity was associated with the amino acid sequence
181PPRIYPQLPSAPT193 of BDV P. To
analyze the features of the NLS present in the C-terminal region of BDV
P, four substitution mutants of pRSV-181/193
pRSV-sub.182, pRSV-sub.186, pRSV-sub.189, and pRSV-sub.192were
constructed (Fig. 6A). Each plasmid
encoded a Pro-Ala substitution at the indicated position. As shown
in Fig. 6B, fusion proteins of the expected sizes were expressed in the
transfected COS-7 cells. IFA showed that all four substitution mutant
proteins were retained in the cytoplasm (Fig. 6C). Therefore,
Pro residues also play an important role in the C-terminal NLS of BDV
P.
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FIG. 5.
Expression and subcellular localization of
-galactosidase fused with short peptides from the C-terminal region
of BDV P. (A) Amino acid sequences of short peptides fused to
-galactosidase. (B) Expression of the fusion proteins in COS-7 cells
as detected by Western blotting with mouse anti--galactosidase MAb.
(C) IFA staining of COS-7 cells expressing fusion proteins with
anti--galactosidase MAb. COS-7 cells were transfected with
pRSV-172/193 (a), pRSV-179/201 (b), pRSV-181/193 (c), or pRSV-192/201
(d).
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FIG. 6.
Expression and subcellular localization of
-galactosidase fused with substitution mutants of
181PPRIYPQLPSAPT193 derived from the C-terminal
region of BDV P. (A) Mutant peptides fused to -galactosidase. The
Pro residue (P) at position 182, 186, 189, or 192 was replaced with Ala
(A, boldface). (B) Expression of fusion proteins in COS-7 cells as
detected by Western blotting with mouse anti--galactosidase MAb. (C)
IFA staining of COS-7 cells expressing fusion proteins with
anti--galactosidase. COS-7 cells were transfected with pRSV-sub.182
(a), pRSV-sub.186 (b), pRSV-sub.189 (c), or pRSV-sub.192 (d).
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DISCUSSION |
Transcription and replication of the BDV genome take place in the
nucleus of BDV-infected cells. Both N and P proteins have been found in
the nucleus of BDV-infected cells (3). Recently, an
additional viral protein X was also found in the nucleus of infected
cells (24, 30). The BDV L polymerase is thought to be a
constituent of a viral ribonucleoprotein complex required for the
replication and transcription of BDV genome in the nucleus (3,
5). The mechanisms by which these proteins are imported from the
cytoplasm into the nucleus are not well understood. L polymerase has
basic amino acid clusters which are good candidates as NLS (3,
5), but the activities of these sequences have not been tested as
yet. Protein X may pass through nuclear pores due to its low molecular
weight or by association with P (24). N protein has an NLS
consisting of a basic amino acid cluster in the N-terminal region, the
activity of which has been established experimentally (13,
19). An NLS candidate in P protein was suggested by Thierer et
al. (28), but the activity of this sequence has not been
studied. Since P associates with N, P (self-association), X
(24), and probably with L, P may play a key role in viral replication and transcription.
We have identified two NLSs within the P protein:
29PRPRKIPR36 in the N-terminal
region and 181PPRIYPQLPSAPT193 in the
C-terminal region. Each NLS was concluded to function independently
based on the following observations. The deletion mutants lacking
either N-terminal or C-terminal regions were still targeted to the
nucleus (Fig. 1). On recombinant protein analysis, either the
sequence from the N-terminal region or the sequence from the C-terminal region conferred nuclear targeting activity on -galactosidase, which
is normally resident in the cytoplasm (Fig. 2). In contrast, the
sequence derived from the middle part of BDV P protein did not cause
-galactosidase to move to the nucleus. The reason why BDV P protein
has two independent NLSs is not clear at present.
A cluster of basic amino acid residues of BDV P
22RRKRSGSPRPRK33 has been proposed
as an NLS (28), but its activity has not yet been confirmed
experimentally. Although the BDV P clone in that study was derived from
MDCK/BDV, the sequence was different from those of many other clones
reported in the literature. The BDV P clone used in the present study,
which was also cloned from MDCK/BDV, has the sequence
22RRERSGSPRPRK33; i.e., the Lys at position 24 was replaced by Glu. Horse-derived strains, including strains V
(GenBank accession number U04608; see also reference 1) and C6BV
(5), also had Glu at position 24. It remains to be
determined whether the 22RRKRSGS28 sequence of
Thierer's BDV P has NLS activity. Irrespective of this issue, however,
the 22RRERSGS28 sequence that is present in
many BDV P proteins is not an NLS, but the neighboring
29PRPRKIPR36 sequence did show NLS activity.
The most intriguing findings of the present investigation are the
unique features of the NLSs present in the BDV P protein. Unlike those
of many nucleoproteins, the NLSs of the BDV P protein lack basic amino
acids. In particular, the C-terminal NLS,
181PPRIYPQLPSAPT193, contained only one
basic amino acid residue. Instead, both NLSs are rich in Pro
residues. Pro has been shown to conform unfolded structures and has
been reported to be important for the conformation of NLS in
combination with several successive basic amino acid residues
(2). For example, PKKKRK in simian virus 40 large T antigen
(10), PNKKKRK in simian virus 40 VP2 (31), and
PKKARED in the polyomavirus large T antigen (21) are all
composed of a Pro succeeded by basic-amino-acid-rich sequences. In
these NLSs, the roles of basic-amino acids have been analyzed
extensively, leaving Pro unchanged. At a glance, the N-terminal NLS of
BDV P, 29PRPRKIPR36, resembles the NLSs of
viral proteins as cited above. However, substitution of any Pro residue
within this sequence abolished the nuclear targeting activity (Fig. 4).
In contrast, replacement of 32RK33 with
32QQ33 within
29PRPRKIPR36 did not abrogate the NLS activity
(Fig. 4C, panel d). The NLS present in the C-terminal region of BDV P
was also a Pro-rich sequence. Again, the replacement of any of these
Pro residues with Ala abrogated the NLS activity (Fig. 6). NLS composed
of Pro-rich residues may therefore represent a novel functional element.
According to a computer simulation model of BDV P (12), both
N and C termini are extruded from the folded compact structure, presumably forming random coils due to their high contents of Pro
residues. The NLSs found in the present study are located in these
random-coiled structures.
Recently, the regions of BDV P critical for interactions with N, P, and
X proteins were mapped by a two-hybrid system by using deletion mutants
of BDV P (24). The sites for interactions with X, P, and N
proteins are present in the regions 33 to 115, 135 to 172, and 197 to
201, respectively. The two NLSs identified here do not overlap with
these binding sites, except that the 33-to-36 region of the N-terminal
NLS was included in the border of the X-binding region. As the
X-binding site has not been narrowed, the N-terminal NLS of BDV P may
be distinct from the X-binding site. Even if the N-terminal NLS
overlapped the X-binding site, N-X heterodimers may target the nucleus
by virtue of the NLS in the C-terminal region of BDV P.
The results presented here were obtained with COS-7 cells, which are
resistant to BDV infection. In addition, interactions between BDV P and
other viral constituents and/or cellular factors have not been studied
in the context of the nuclear targeting activity of BDV P. Despite
these limitations, the presence of two potential NLSs in BDV P would
provide a fundamental basis for elucidation of the biology of BDV infection.
| |
ACKNOWLEDGMENTS |
We thank Rudolf Rott (Justus-Liebig-Universität Giessen,
Giessen, Germany) for providing the MDCK/BDV cells.
This work was partly supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture and by The
Akiyama Foundation.
| |
FOOTNOTES |
*
Corresponding author. Present address: Central
Laboratories, Kyoritsu Shoji Corporation, 2-9-22, Takamihara,
Kukizaki-Machi, Inashiki-Gun, Ibaraki 300-1252, Japan. Phone:
81-298-72-3361. Fax: 81-298-72-1916. E-mail: JDY00475{at}nifty.ne.jp.
| |
REFERENCES |
| 1.
|
Binz, T.,
J. Lebelt,
H. Niemann, and K. Hagenau.
1994.
Sequence analysis of the p24 gene of Borna disease virus in naturally infected horse, donkey and sheep.
Virus Res.
34:281-289[Medline].
|
| 2.
|
Boulikas, T.
1993.
Nuclear localization signals (NLS).
Crit. Rev. Eukaryot. Gene Expr.
3:193-227[Medline].
|
| 3.
|
Briese, T.,
A. Schneemann,
A. J. Lewis,
Y. S. Park,
S. Kim,
H. Ludwig, and W. I. Lipkin.
1994.
Genomic organization of Borna disease virus.
Proc. Natl. Acad. Sci. USA
91:4362-4366[Abstract/Free Full Text].
|
| 4.
|
Cubitt, B., and J. C. de la Torre.
1994.
Borna disease virus (BDV), a nonsegmented RNA virus, replicates in the nuclei of infected cells where infectious BDV ribonucleoproteins are present.
J. Virol.
68:1371-1381[Abstract/Free Full Text].
|
| 5.
|
Cubitt, B.,
C. Oldstone, and J. C. de la Torre.
1994.
Sequence and genomic organization of Borna disease virus.
J. Virol.
68:1382-1396[Abstract/Free Full Text].
|
| 6.
|
de la Torre, J. C.
1994.
Molecular biology of Borna disease virus: prototype of a new group of animal viruses.
J. Virol.
68:7669-7675[Free Full Text].
|
| 7.
|
Gluzman, Y.
1981.
SV40-transformed simian cells support the replication of early SV40 mutants.
Cell
23:175-182[Medline].
|
| 8.
|
Gonzalez-Dunia, D.,
B. Cubitt,
F. A. Grässer, and J. C. de la Torre.
1997.
Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry.
J. Virol.
71:3208-3218[Abstract].
|
| 9.
|
Herzog, S., and R. Rott.
1980.
Replication of Borna disease virus in cell culture.
Med. Microbiol. Immunol.
168:153-158[Medline].
|
| 10.
|
Kalderon, D.,
W. D. Richardson,
A. F. Markham, and A. E. Smith.
1994.
Sequence requirements for nuclear location of simian virus large-T antigen.
Nature
311:33-38.
|
| 11.
|
Kliche, S.,
T. Briese,
A. Henschen,
L. Stitz, and W. I. Lipkin.
1994.
Characterization of a Borna disease virus glycoprotein, gp18.
J. Virol.
68:6918-6923[Abstract/Free Full Text].
|
| 12.
|
Kliche, S.,
L. Stitz,
H. Mangalam,
L. Shi,
T. Binz,
H. Niemann,
T. Briese, and W. I. Lipkin.
1996.
Characterization of the Borna disease virus phosphoprotein, p23.
J. Virol.
70:8133-8137[Abstract].
|
| 13.
|
Kobayashi, T.,
Y. Shoya,
T. Koda,
I. Takashima,
P. K. Lai,
K. Ikuta,
M. Kakinuma, and M. Kishi.
1998.
Nuclear targeting activity associated with Borna disease virus nucleoprotein.
Virology
243:188-197[Medline].
|
| 14.
|
Koda, T.,
S. Hasan,
A. Sasaki,
Y. Arimura, and M. Kakinuma.
1994.
Regulatory sequences required for hst-1 expression in embryonal carcinoma cells.
FEBS Lett.
342:71-75[Medline].
|
| 15.
|
Lipkin, W. I.,
G. H. Travis,
K. M. Carbone, and M. C. Wilson.
1990.
Isolation and characterization of Borna disease cDNA clones.
Proc. Natl. Acad. Sci. USA
87:4184-4188[Abstract/Free Full Text].
|
| 16.
|
Ludwig, H.,
L. Bode, and G. Gosztonyi.
1988.
Borna disease: a persistent virus infection of the central nervous system.
Prog. Med. Virol.
35:107-151[Medline].
|
| 17.
|
McClure, M. A.,
K. J. Thibault,
C. G. Hatalski, and W. I. Lipkin.
1992.
Sequence similarity between Borna disease virus p40 and a duplicated domain within the paramyxovirus and rhabdovirus polymerase proteins.
J. Virol.
66:6572-6577[Abstract/Free Full Text].
|
| 18.
|
Miller, M.,
B. A. Reddy,
M. Kloc,
X. X. Li,
C. Dreyer, and L. D. Etkin.
1991.
The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development.
Development
113:569-575[Abstract].
|
| 19.
|
Pyper, J. M., and A. E. Gartner.
1997.
Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus.
J. Virol.
71:5133-5139[Abstract].
|
| 20.
|
Pyper, J. M.,
J. A. Richt,
L. Brown,
R. Rott,
O. Narayan, and J. E. Clements.
1993.
Genomic organization of the structural proteins of Borna disease virus revealed by a cDNA clone encoding the 38-kDa protein.
Virology
195:229-238[Medline].
|
| 21.
|
Richardson, W. D.,
B. L. Roberts, and A. E. Smith.
1986.
Nuclear localization signals in polyomavirus large-T.
Cell
44:77-85[Medline].
|
| 22.
|
Schneemann, A.,
P. A. Schneider,
R. A. Lamb, and W. I. Lipkin.
1995.
The remarkable coding strategy of Borna disease virus: a new member of the nonsegmented negative strand viruses.
Virology
210:1-8[Medline].
|
| 23.
|
Schneider, P. A.,
C. G. Hatalski,
A. J. Lewis, and W. I. Lipkin.
1997.
Biochemical and functional analysis of the Borna disease virus G protein.
J. Virol.
71:331-336[Abstract].
|
| 24.
|
Schwemmle, M.,
M. Salvatore,
L. Shi,
J. Richt,
C. H. Lee, and W. I. Lipkin.
1998.
Interaction of the Borna disease virus P, N, and X proteins and their functional implications.
J. Biol. Chem.
273:9007-9012[Abstract/Free Full Text].
|
| 25.
|
Shoya, Y.,
T. Kobayashi,
T. Koda,
P. K. Lai,
H. Tanaka,
T. Koyama,
K. Ikuta,
M. Kakinuma, and M. Kishi.
1997.
Amplification of a full-length Borna disease virus (BDV) cDNA from total RNA of cells persistently infected with BDV.
Microbiol. Immunol.
41:481-486[Medline].
|
| 26.
| Stitz, L., T. Bilzer, J. A. Richt, and R. Rott. 1993. Pathogenesis of Borna disease. Arch. Virol.
7(Suppl.):135-151.
|
| 27.
|
Thiedemann, N.,
P. Presek,
R. Rott, and L. Stitz.
1992.
Antigenic relationship and further characterization of two major Borna disease virus-specific proteins.
J. Gen. Virol.
73:1057-1064[Abstract/Free Full Text].
|
| 28.
|
Thierer, J.,
H. Reihle,
O. Grebenstein,
T. Binz,
S. Herzog,
N. Thiedemann,
L. Stitz,
R. Rott,
F. Lottspeich, and H. Niemann.
1992.
The 24K protein of Borna disease virus.
J. Gen. Virol.
73:413-416[Abstract/Free Full Text].
|
| 29.
|
Wagner, R. R.
1990.
Rhabdoviridae and their replication, p. 867-882.
In
B. N. Fields, et al. (ed.), Fields virology, 2nd ed. Raven Press, New York, N.Y.
|
| 30.
|
Wehner, T.,
A. Ruppert,
C. Herden,
K. Frese,
H. Becht, and J. A. Richt.
1997.
Detection of a novel Borna disease virus encoded 10 kilodalton protein in infected cells and tissues.
J. Gen. Virol.
78:2459-2466[Abstract].
|
| 31.
|
Wychowski, C.,
D. Benichou, and M. Girard.
1987.
The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.
J. Virol.
61:3862-3869[Abstract/Free Full Text].
|
| 32.
|
Ye, Z.,
D. Robinson, and R. R. Wagner.
1995.
Nucleus-targeting domain of the matrix protein (M1) of influenza virus.
J. Virol.
69:1964-1970[Abstract].
|
Journal of Virology, December 1998, p. 9755-9762, Vol. 72, No. 12
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Top
Abstract
Introduction
