Journal
of General Virology (1998), 79, 2957–2963. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Two domains of the Borna disease virus p40 protein are
required for interaction with the p23 protein
Mikael Berg,1 Christian Ehrenborg,2 Jonas Blomberg,3 Ru$ diger Pipkorn4 and Anna-Lena Berg5
1, 5
Department of Veterinary Microbiology, Section of Immunology1 and Department of Pathology5, Swedish University of Agricultural
Sciences, Box 588, S-751 23 Uppsala, Sweden
2
Uppsala Graduate School in Biomedical Research, Uppsala, Sweden
3
Department of Clinical Microbiology, Section of Virology, University of Uppsala, Academic Hospital, Uppsala, Sweden
4
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld, Heidelberg, Germany
Borna disease virus (BDV) has five major open
reading frames, which encode the proteins p40,
p23, gp18, p57 and p190. By analogy with other
negative-strand RNA viruses, p40 is a putative
nucleoprotein and p23 is a putative phosphoprotein. These proteins are known to form com-
Introduction
Borna disease virus (BDV) is a neurotropic agent that
causes encephalomyelitis in horses, sheep, cattle and cats
(Ludwig et al., 1988 ; Lundgren et al., 1995 ; Rott & Becht,
1995). Recent reports of viral nucleic acid and proteins in
human peripheral blood mononuclear cells suggest that BDV
may also be involved in human neuropsychiatric disorders
(Bode, 1995). BDV is a non-segmented negative-strand RNA
virus and is the only member of the Bornaviridae. The genome
of 8900 bases has five major open reading frames, encoding the
proteins p40, p23, gp18, p57 and p190 (Briese et al., 1994 ;
Cubitt et al., 1994). These proteins may be functional
homologues of other proteins positioned at the same sites in
the genome of other viruses in the same order, Mononegavirales.
On the basis of this assumption, p40 may be considered to be
a nucleoprotein (NP), p23 a phosphoprotein (P), gp18 a matrix
protein (M), p57 the major glycoprotein (G) and p190 a
polymerase (L). The NP surrounds the RNA genome but is also
involved in various steps of transcription and replication. The
P protein is an important factor of the polymerase and is
necessary for RNA synthesis. Proteins NP, P and L can form
complexes with each other, and this ability to interact is
thought to play a role in their respective functions during
transcription and replication (Gao & Lenard, 1995 ; Horikami et
Author for correspondence : Mikael Berg.
Fax ­46 18 471 43 82. e-mail Mikael.Berg!bmc.uu.se
0001-5706 # 1998 SGM
plexes with each other and with the polymerase
protein in other viruses. In this paper, it is shown
that BDV p40 and p23 can form complexes with
each other in infected cells. Furthermore, the amino
acids of p40 that are necessary for formation of this
complex have been mapped.
al., 1992 ; Takacs & Banerjee, 1995). Since p40 and p23 have
been reported to copurify in chromatography and immunoprecipitation experiments, it has been proposed that these
proteins form a complex or have common epitopes (BauseNiedrig et al., 1992 ; Haas et al., 1986 ; Hsu et al., 1994 ; Kliche
et al., 1996 ; Thiedemann et al., 1992 ; VandeWoude et al.,
1990). p23 has been shown to form a dimer, the important
amino acid in this interaction being a cysteine at position 125
(Kliche et al., 1996). Other than this, little is known about BDV
p40 and p23 and their functions. In a first attempt to elucidate
the function of these proteins in infected cells, we report here
that BDV p23 and p40 form a complex with each other.
Furthermore, we map the amino acids of p40 that are necessary
for this complex to form.
Methods
+ RNA extraction and cDNA synthesis. Total RNA was extracted
from approximately 100 mg brain tissue (cerebral cortex) from a German
horse with confirmed Borna disease, by using the TRIzol reagent (Life
Technologies). One µg total RNA was reverse-transcribed to cDNA by
Superscript (Life Technologies), with oligo d(T) – as a primer.
"# ")
+ Construction of plasmids
(i) Glutathione S-transferase (GST)–p40. Primers were designed to
enable expression of full-length p40 in various systems. Restriction sites
for BamHI and NdeI were inserted upstream of the p40 sequence, and
a restriction site for EcoRI was inserted in the reverse primer. The p40
forward primer was 5« CCCGGATCCCATATGCCACCCAAGAGACGC and the reverse primer was 5« CGGAATTCTTAGTTTAG-
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M. Berg and others
Table 1. Peptides spanning the sequence of BDV p40
Peptides (20-mers) covering the entire sequence of BDV p40 were synthesized, each overlapping the next by
15 residues.
No.
Residues
Sequence
No.
Residues
Sequence
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1–20
6–25
11–30
16–35
21–40
26–45
31–50
36–55
41–60
46–65
51–70
56–75
61–80
66–85
71–90
76–95
81–100
86–105
91–110
96–115
101–120
106–125
111–130
116–135
121–140
126–145
131–150
136–155
141–160
146–165
151–170
156–175
161–180
166–185
171–190
176–195
MPPKRRLVDDADAMEDQDLY
RLVDDADAMEDQDLYEPPAS
ADAMEDQDLYEPPASLPKLP
DQDLYEPPASLPKLPGKFLQ
EPPASLPKLPGKFLQYTVGG
LPKLPGKFLQYTVGGSDPHP
GKFLQYTVGGSDPHPGIGHE
YTVGGSDPHPGIGHEKDIRQ
SDPHPGIGHEKDIRQNAVAL
GIGHEKDIRQNAVALLDQSR
KDIRQNAVALLDQSRRDMFH
NAVALLDQSRRDMFHTVTPS
LDQSRRDMFHTVTPSLVFLC
RDMFHTVTPSLVFLCLLIPG
TVTPSLVFLCLLIPGLHAAF
LVFLCLLIPGLHAAFVHGGV
LLIPGLHAAFVHGGVPRESY
LHAAFVHGGVPRESYLSTPV
VHGGVPRESYLSTPVTRGEQ
PRESYLSTPVTRGEQTVVKT
LSTPVTRGEQTVVKTAKFYG
TRGEQTVVKTAKFYGEKTTQ
TVVKTAKFYGEKTTQRDLTE
AKFYGEKTTQRDLTELEISS
EKTTQRDLTELEISSIFSHC
RDLTELEISSIFSHCCSLLI
LEISSIFSHCCSLLIGVVIG
IFSHCCSLLIGVVIGSSSKI
CSLLIGVVIGSSSKIKAGAE
GVVIGSSSKIKAGAEQIKKR
SSSKIKAGAEQIKKRFKTMM
KAGAEQIKKRFKTMMAALNR
QIKKRFKTMMAALNRPSHGE
FKTMMAALNRPSHGETATLL
AALNRPSHGETATLLQMFNP
PSHGETATLLQMFNPHEAID
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
181–200
186–205
191–210
196–215
201–220
206–225
211–230
216–235
221–240
226–245
231–250
236–255
241–260
246–265
251–270
256–275
261–280
266–285
271–290
276–295
281–300
286–305
291–310
296–315
301–320
306–325
311–330
316–335
321–340
326–345
331–350
336–355
341–360
346–365
351–370
TATLLQMFNPHEAIDWINGQ
QMFNPHEAIDWINGQPWVGS
HEAIDWINGQPWVGSFVLSL
WINGQPWVGSFVLSLLTTDF
PWVGSFVLSLLTTDFESPGK
FVLSLLTTDFESPGKEFMDQ
LTTDFESPGKEFMDQIKLVA
ESPGKEFMDQIKLVASYAQM
EFMDQIKLVASYAQMTTYTT
IKLVASYAQMTTYTTIKEYL
SYAQMTTYTTIKEYLAECMD
TTYTTIKEYLAECMDATLTI
IKEYLAECMDATLTIPVVAY
AECMDATLTIPVVAYEIRDF
ATLTIPVVAYEIRDFLEVSA
PVVAYEIRDFLEVSAKLKED
EIRDFLEVSAKLKEDHADLF
LEVSAKLKEDHADLFPFLGA
KLKEDHADLFPFLGAIRHPD
HADLFPFLGAIRHPDAIKLA
PFLGAIRHPDAIKLAPRSFP
IRHPDAIKLAPRSFPNLASA
AIKLAPRSFPNLASAAFYWS
PRSFPNLASAAFYWSKKENP
NLASAAFYWSKKENPTMAGY
AFYWSKKENPTMAGYRASTI
KKENPTMAGYRASTIQPGAS
TMAGYRASTIQPGASVKETQ
RASTIQPGASVKETQLARYR
QPGASVKETQLARYRRREIS
VKETQLARYRRREISRGEDG
LARYRRREISRGEDGAELSG
RREISRGEDGAELSGEISAI
RGEDGAELSGEISAIMKMIG
AELSGEISAIMKMIGVTGLN
ACCAGTCAC. These primers were used to amplify the p40 open
reading frame from the cDNA library. Products were analysed by agarose gel electrophoresis. PCR products were purified with the Wizard
PCR purification system (Promega) according to the manufacturer’s
manual. Purified PCR products were then cut with BamHI and EcoRI
and ligated into the pGEX vector (Pharmacia).
(ii) GST–p23. The cloning of p23 was done similarly, with primers
which allowed various cloning strategies. The p23 forward primer was
5« CCCAAGCTTCGCATATGGCAACGCGACCATCG and the reverse primer was 5« CGGAATTCTTATGGTATGATGTCCCA. These
primers were used to amplify the p23 open reading frame from the
cDNA library. The PCR product was purified, cut with HindIII and
EcoRI and ligated into the pGEX vector.
+ Expression and preparation of protein extracts. GST fusion
protein vectors were transformed into E. coli strain BL21 by standard
CJFI
techniques. Single colonies were collected, and then grown in LB medium
plus ampicillin until the A was approximately 0±5. Fusion protein
'!!
expression was induced by the addition of 0±4 mM IPTG, and cells were
then grown for several hours. Bacteria were collected by centrifugation
and incubated in lysis buffer (20 % sucrose, 0±1 M NaCl, 0±1 % NP40,
2±5 mM DTT, 0±1 mg}ml lysozyme) on ice for about 30 min. Lysates
were sonicated to shear the DNA and then centrifuged. The protein
concentration was determined and proteins were analysed by SDS–
PAGE. Finally, the extracts were aliquoted and snap-frozen in liquid
nitrogen, and stored at ®70 °C until use.
+ [35S]Methionine labelling of cells. C6 and permanently infected
C6 BDV cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 5 % foetal bovine serum (FBS). About 70 %
confluent cells were starved for 30 min in methionine-free medium,
washed with medium and labelled for 6–12 h with [$&S]methionine added
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Interaction of Borna disease virus p23 and p40
to methionine-free medium supplemented with 1 % FBS. The cells were
scraped off the plate and lysed with RIPA buffer.
+ GST pull-down (GST-PD) analysis. In general, the total sample
volume was 20 µl. All protein dilutions and binding to GST beads were
done in potassium phosphate buffer (20 mM potassium phosphate,
pH 7±4 ; 0±1 M NaCl ; 1 mM EDTA ; 2±5 mM DTT ; 10 % glycerol ; 0±1 %
NP40 ; and 0±5 mg}ml BSA). GST fusion protein extracts and C6 cell
extracts were added to a total volume of 20 µl, and the mixture was
incubated at room temperature for 30 min. GST beads were added and
the total volume was increased to 100 µl. The extracts plus GST beads
were incubated on a rotating wheel for 1 h. The beads were pelleted and
washed four times. SDS loading dye was then added and the samples
were analysed by SDS–PAGE. Gels were dried and autoradiographed.
+ ELISA interaction assay. Microtitre plates (96-well ; Polysorp,
Nunc) were coated with either 20-mer overlapping peptides covering the
BDV p40 sequence or an extract containing p40 protein.
Microtitre plates were coated at 4 °C overnight with solutions of
overlapping peptides at 20 or 200 ng}ml in carbonate buffer (0±16 %
Na CO , 0±24 % NaHCO ). After washing the plates (three times in
# $
$
0±9 %, w}v, NaCl ; 0±05 %, v}v, Tween 20), a 45 min blocking step was
performed at 37 °C with 0±5 % BSA in PBS. The plates were washed three
times and then incubated for 90 min at 37 °C with GST or full-length
GST–p23 (0±5 µg}ml in PBS). The plates were then washed, and rabbit
anti-p23 antibody, diluted 1 : 500 in serum diluent (50 mM Tris–HCl,
pH 7±4 ; 0±35 M NaCl ; 0±1 % Tween 20 ; 5 % normal swine serum), was
added to the wells. The plates were incubated with the primary antibody
at 37 °C for 1 h, and then washed three times and incubated with
peroxidase-conjugated swine anti-rabbit antibody (DAKO), diluted
1 : 1500 in serum diluent. After incubation with the secondary antibody
at 37 °C for 1 h, the plates were washed three times and the chromogenic
substrate (0±5 mg}ml O-phenylenediamine, 0±33 µl}ml 30 % H O , in
# #
citric acid buffer) was added to the wells. The reaction was stopped after
20 min with 3 M H SO , and the absorbance at 492 nm was read in an
# %
ELISA reader.
p40 was coated onto 96-well ELISA plates at three different
concentrations. As a control antigen, another GST fusion protein, GST–P
(Berg et al., 1992), was used to coat the plates. After blocking and
washing of the plates, p23 was added at two different concentrations. The
proteins were incubated together for 1 h at 37 °C. After washing, plates
were incubated with an antibody against p23 for 1 h. After extensive
washing, a secondary HRP-conjugated antibody was added, and plates
were incubated for 1 h. The chromogenic substrate was added, and the
absorbance at 492 nm was measured.
+ Peptides. Peptides covering the complete p40 sequence were
synthesized according to Gausepohl et al. (1992). Purity was at least 95 %,
as determined by HPLC. Each peptide (20-mer) overlapped the next by
15 residues (see Table 1).
Results and Discussion
BDV p40 and p23 are the viral proteins which are expressed
most abundantly in infected cells. By analogy to other nonsegmented negative-strand RNA viruses, it can be assumed
that p40 forms a nucleocapsid enveloping the viral genomic
RNA, and that p23 together with the L protein forms a
polymerase complex. The exact functional significance of this
interaction is still not fully understood. However, the ability of
these proteins to interact is likely to be important for their
functions during replication and transcription.
Since p40 and p23 were found to copurify in chromatography and immunoprecipitation experiments, it has been
proposed that these proteins form a complex or have common
epitopes (Bause-Niedrig et al., 1992 ; Haas et al., 1986 ; Hsu et
al., 1994 ; Kliche et al., 1996 ; Thiedemann et al., 1992 ;
VandeWoude et al., 1990). The aim of the present study was to
clarify whether an interaction between p40 and p23 takes place
and, if so, to map which amino acids of p40 are necessary for
the interaction to occur.
Interaction between p40 and p23
To test the hypothesis that p40 and p23 interact with each
other in infected cells, we tagged one of the proteins to allow
it to be purified, labelled the other protein with [$&S]methionine,
and assayed for copurification of the two proteins. To this end,
we labelled C6 cells persistently infected with BDV (Carbone
et al., 1993), as well as non-infected C6 cells, with [$&S]methionine. BDV p40 and p23 were cloned as GST fusion proteins
(the tagged proteins) and expressed in E. coli. GST fusion
proteins and labelled extracts were used in a GST-PD assay to
demonstrate protein–protein interaction. As Fig. 1 (a) shows,
GST–p40 interacted with a protein of approximately 23 kDa
in infected C6 cells (lanes 3–4), but not in non-infected C6 cells
(lanes 1–2). This result indicates that BDV p40 interacts with a
viral protein of 23 kDa. The very small difference between the
ability of the two concentrations of GST–p40 to pull down
p23 may indicate that the concentrations of GST–p40 used in
this experiment were close to saturating the amount of p23 in
the cell extract.
Similarly, GST–p23 interacted with two proteins of around
40 kDa in infected cells (lanes 7–8), but not in non-infected
cells (lanes 5–6). Previous studies have shown that BDV p40
often consists of two species, designated p40}p38 or p39}p38
(Ludwig et al., 1988 ; Pyper & Gartner, 1997). Therefore, it is
likely that GST–p23 interacts with the virus-encoded p40. As
shown in lanes 9–12, GST alone does not interact with either
protein. The band of about 27 kDa in all lanes can be considered
as background. Taken together, the results from the GST-PD
assay indicate that BDV p40 and p23 can interact with each
other in infected cells. This interaction was shown both ways ;
GST–p40 interacts with p23 and GST–p23 interacts with p40.
To confirm these results and to demonstrate that the
interaction observed was due to direct protein–protein
interaction between BDV p40 and p23, and was not an indirect
interaction via nucleic acids or some other viral or cellular
factors, we used a modified ELISA technique. Proteins to be
analysed were expressed as fusion proteins in E. coli, cleaved
from GST by digestion with thrombin, and partially purified.
As shown in Fig. 1 (b), the absorbances for wells containing the
combination p23–p40 were significantly higher than for those
containing the p23–control antigen. Taken together, these
experiments clarify the previous uncertainties concerning
whether p40 and p23 have common epitopes or form a
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M. Berg and others
(a)
GST-p40
C6
GST-p23
C6-BDV
C6
GST
C6-BDV
C6
C6-BDV
kDa
52
p40
36
27
p23
19
1
2
3
4
5
6
7
8
9
10
11
12
(b)
A492
1·4
p40 5 µg/ml
1·2
p40 0·5 µg/ml
1·0
p40 0·05 µg/ml
Control 5 µg/ml
0·8
Control 0·5 µg/ml
0·6
Control 0·05 µg/ml
0·4
0·2
p23 0·5 µg/ml
p23 0·05 µg/ml
Fig. 1. (a) Full-length BDV p40 and p23 interact with each other in a GST-PD assay. Radioactive cell extracts from BDVinfected or non-infected C6 cells were incubated together with GST–p40 or GST–p23. GST fusion proteins were retained by
the addition of GST–Sepharose beads. Accompanying (interacting) proteins were analysed by SDS–PAGE followed by
autoradiography. The concentration of cell extracts was not altered ; GST–p40, GST–p23 and GST were used at about 100 ng
(lanes 1, 3, 5, 7, 9 and 11) or about 10 ng (lanes 2, 4, 6, 8, 10 and 12). Lanes 1–4 represent GST–p40 incubated with noninfected C6 cells (1–2) or BDV-infected C6 cells (3–4). Lanes 5–8 show GST–p23 incubated with non-infected C6 cells (5–6)
or BDV-infected C6 cells (7–8). Lanes 9–12 are controls with GST only. (b) Full-length BDV p40 and p23 interact with each
other in ELISA. p40 and negative-control antigen were coated at the bottom of wells of ELISA plates at three different
concentrations as shown. p23 was added to the coated wells at 0±5 and 0±05 µg/ml, and the proteins were then incubated
together for 1 h. An antiserum against p23 was added to the wells, followed by a secondary HRP-conjugated antibody and
substrate. Absorbances for interactions are shown.
protein–protein complex. The results of the ELISA provide
further evidence of an interaction between p40 and p23.
Mapping the p40 amino acids required for interaction
with p23
We wanted to investigate the nature of the interaction
between p40 and p23 in more detail, and chose to map the
domains of p40 that were required for the interaction with p23.
Peptides covering the complete p40 sequence were syn-
CJGA
thesized (Table 1). To screen the complete peptide library for
interaction with p23, individual peptides, at different concentrations, were coated at the bottom of the wells of an ELISA
plate. Different concentrations of p23 were then added to the
wells and incubated with the peptides. Detection of an
interaction was performed with anti-p23 antibody, secondary
HRP-conjugated antibody and substrate, as described earlier.
We reasoned that only interaction of a range of peptides
spanning an important domain would demonstrate a true
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Interaction of Borna disease virus p23 and p40
0·5
0·45
0·4
A492
0·35
0·3
0·25
0·2
0·15
0·1
0·05
Peptide
Fig. 2. Two ranges of peptides from p40 interact with p23 in ELISA. Individual peptides were used to coat wells of ELISA
plates. p23 was added to the wells and allowed to interact with the peptides. An antiserum against p23 was then added,
followed by a secondary HRP-conjugated antibody and substrate. Absorbances are shown on the y-axis and individual peptides
(labelled 1–71) on the x-axis. The sequences of the peptides are shown in Table 1.
interaction. In other words, the importance of a domain would
be confirmed by interaction with p23 of at least two
overlapping peptides. As shown in Fig. 2, two stretches of
overlapping peptides interacted strongly with p23 (peptides
14–18 and 27–28). These stretches correspond to amino acid
positions 56–100 and 131–155, respectively. Other peptides
showed weaker interaction (peptides 11, 32, 35, 42 and 59).
This demonstrates that the important amino acids which confer
the interaction with p23 are present within the following
sequences of p40 : KDIRQNAVALLDQSRRDMFHTVTPSLVFLCLLIPGLHAAFVHGGVPRESY and LEISSIFSHCCSLLIGVVIGSSSKI. Based on a computer hydrophobicity analysis,
both of these domains are relatively hydrophobic (not shown).
It would be interesting to determine whether p23 also has a
hydrophobic region that is important for interaction with p40.
If this was the case, it would suggest that p23 and p40 form a
complex via hydrophobic interactions.
To study the p40–p23 interaction further, we used the
peptides from the previous experiment in a competition
experiment. Combinations of peptides were used to compete
with the interaction demonstrated in Fig. 1 (a, b). The experiment followed the earlier procedure, except that p23 was
incubated for 1 h with the peptides before being added to a
plate coated with full-length p40. As shown in Fig. 3 (a), the
two ranges of peptides which interacted in the previous
experiment (peptides 14–18 and 27–28) competed for the
interaction between p40 and p23. In contrast, the single
peptides identified in the previous experiment (peptides 11, 12,
32, 35, 42 and 59) were unable to displace the p40–p23
interaction. The reason why these peptides did not displace the
p40–p23 interaction even though they did interact with p23 is
probably that they represent only a small part of the whole
interacting domain. Peptides 27 and 28 seemed to be the most
effective competitors, indicating that this domain of p40 may
confer the strongest interaction with p23.
To investigate the contribution of individual peptides, we
used all the peptides within the two stretches 14–18 and 27–28
individually in another competition experiment. As shown in
Fig. 3 (b), all the peptides from the first domain, except 17 and
18, competed individually for the p40–p23 interaction. This
indicates that the strongest interaction is derived from a part of
this stretch of peptides, from the C-terminal part of peptide 14
to the N-terminal part of peptide 18, and that some, but not
all, of the important amino acids are missing from the latter
peptides (17 and 18). For example, a cysteine is present in
peptides 14, 15 and 16 but missing from peptides 17 and 18.
Despite the fact that peptide 17 interacted strongly in the
previous experiment, it represents only a small portion of the
interacting domain ; the remainder interacts strongly enough
to keep the p40–p23 complex stable. Based on these results,
one could possibly define the amino acids of this domain of
p40 that are important for interaction with p23 as those from
around residue 70 to residue 90. It is also possible that
peptides 14–16 are more able to resemble the native structure
than peptides 17 and 18, and are therefore more effective
competitors.
Fig. 3 (b) also shows that competition by either of the
peptides from the second domain, 27 and 28, completely
blocks the interaction, indicating that the interaction mediated
by this domain of p40 is shared by both of these peptides but
not by the flanking peptides.
Although the other peptides that interacted with p23 in the
original screen failed to block the p40–p23 interaction, it is still
possible that they may interact with p23 in vivo. However,
these interactions may be too weak to compete for the strong
interaction mediated by the two domains of p40 that we have
identified.
To rule out the possibility that the peptides blocked the
p40–p23 interaction via non-specific ‘ stickiness ’, we performed
a control experiment using another negative-strand RNA
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CJGB
M. Berg and others
(a)
2·5
11+12
14 –18
27+28
32+35
42
59
A492
2·0
1·5
1·0
0·5
0·1
0·2
0·4
0·8
1
5
Concentration of peptides (µg/ml)
(b)
1·2
14
15
16
17
18
27
28
7
1·0
A492
0·8
0·6
0·4
0·2
1
2
3
4
5
Concentration of peptides (µg/ml)
Fig. 3. (a) Two groups of peptides can block interaction between fulllength p40 and p23. Experimental procedures were similar to those
described in Figs 1 (b) and 2, except that p23 was incubated with various
combinations of peptides for 1 h before being added to wells of ELISA
plates coated with full-length p40. As shown by the decreasing
absorbances, the combinations 14–18 and 27–28 can compete with the
interaction between p23 and p40. (b) Individual peptides can block
interaction between full-length p40 and p23. Experimental procedures
were identical to those in (a), except that p23 was incubated with
individual peptides belonging to the two groups 14–18 and 27–28.
Peptides 14–16 and 27–28 were able individually to compete with the
interaction between p40 and p23, as shown by decreasing absorbances.
For comparison, a non-interacting peptide (no. 7) was included in the
experiment.
virus, the porcine rubulavirus La-Piedad-Michoacan-Mexico
virus (Berg et al., 1992). We used the putative functional
homologues NP and P, which also interact with each other in
GST-PD analysis (unpublished results). We tested the NP and
P interaction in the ELISA assay and performed a similar
competition analysis. The peptides which strongly inhibited
the BDV p40–p23 interaction had no effect on NP–P
interaction (not shown).
Another possibility would be that the peptides block the
antibody binding site, inhibiting the detection of the interaction but not the interaction itself. However, since we used
a polyclonal antiserum, and also tested other sera against p23,
we find this unlikely. To rule out this possibility conclusively,
we coated p23 on ELISA plates and then added peptides and
anti-p23 antibodies. The peptides could not compete in this
control ELISA experiment (not shown).
It should be noted that previous studies (Ludwig et al.,
CJGC
1988 ; Hsu et al., 1994 ; Pyper & Gartner, 1997) and our own
data presented here show p40 to be present in two forms,
referred to as p40}p38 or p39}p38. In a recent paper by Pyper
& Gartner (1997), it was suggested that these two forms differ
by the addition of 13 amino acids at the N terminus. In the
same study, the authors suggested that this may explain why
the 39 kDa form localizes to the nucleus while the 38 kDa form
is found both in the nucleus and cytoplasm, implying different
functions for the two forms of p40 (Pyper & Gartner, 1997).
According to Hsu et al. (1994), only the 40 kDa form and not
the 38 kDa form interacts with p23. In our initial GST-PD
experiment with GST–p23 (Fig. 1 a, lanes 7–8), it appears that
at higher GST–p23 concentrations both forms of p40 bound
equally well. However, at a lower concentration of GST–p23
only the larger form bound, indicating that this interaction was
stronger or more specific. We cannot, at this point, rule out that
the two forms of p40 interact with each other, and that the
38 kDa form is detected through its interaction with the
40 kDa form, and not directly with p23. On the other hand, the
mapping of the important amino acids of p40 indicates that
both forms of p40 should have the capacity to complex with
p23, since both forms contain the amino acids required for
formation of this complex.
In summary, the results of our study confirm previous
reports that p40 and p23 form a protein–protein complex. In
addition, we have now identified the domains of BDV p40 that
are required for interaction with BDV p23. These two domains
are present in both forms of p40 (p40}p38 or p39}p38) (Pyper
& Gartner, 1997), but it appears that p23 interacts slightly
more strongly with the larger form of p40. Further studies will
be necessary in order to elucidate the functional significance of
this observation.
Thanks are due to Dr W. Ian Lipkin for the C6 cells, C6 BDV cells and
the anti-p23 rabbit serum. The horse brain sample was kindly provided
by Dr Wolfgang Zimmermann. This work was supported by a grant from
the Swedish Council for Forestry and Agricultural Research (SJFR).
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Received 22 May 1998 ; Accepted 17 August 1998
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