Download Identification of a novel viral protein in infectious bursal disease

Journal of General Virology (1995), 76, 437443. Printed in Great Britabl
437
Identification of a novel viral protein in infectious bursal disease
virus-infected cells
Egbert Mundt, 1. J6rg Beyer I and Hermann Miiller2t
1 Federal Research Centre f o r Virus Diseases o f Animals, D-17498 Insel Riems and 2 Institut fiir Virologic,
Justus-Liebig-Universitiit Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany
Infectious bursal disease virus (IBDV), a member of the
specifies two genomic double-stranded
Birnaviridae,
RNAs, segment A and segment B. Segment A encodes a
l l 0 k D a polyprotein which is processed into virus
proteins VP2, VP3 and VP4. A second open reading
frame (ORF), designated ORF A-2, immediately preceding and partially overlapping the 110 kDa protein
gene has also been described. After prokaryotic expression of this ORF and immunization of rabbits with
the expressed protein we obtained reagents that allowed
Infectious bursal disease virus (IBDV) causes an
immunosuppressive disease in chickens by destruction of
the lymphoid cells in the bursa of Fabricius (BF).
Chickens are fully susceptible to IBDV infection between
3 and 6 weeks of age. Two distinct serotypes of IBDV,
designated as serotypes I and II, have been identified
(McFerran et al., 1980; Jackwood et al., 1982). Serotype
II viruses are avirulent for chickens (Ismail et al., 1988),
whereas serotype I viruses are virulent. However,
considerable variation exists between different isolates
(Saif et al., 1987).
IBDV belongs to the Birnavirus genus of RNA viruses,
which have a bisegmented double-stranded genome (for
review see Kibenge et al., 1988). IBDV segment A was
shown to encompass one large open reading frame
(ORF) which encodes a 110 kDa primary translation
product. This precursor polyprotein is processed into
three mature viral proteins (VP), VP2, VP3 and VP4
(Hudson et al., 1986; Spies et al., 1989). VP2 and VP3 are
the major structural proteins of IBDV, whereas VP4
most likely represents a putative virus-encoded protease
(Azad et al., 1987; Jagadish et aL, 1988). RNA segment
* Author for correspondence. Fax +49 38351 7219.
t Presentaddress:Virologie,Universit~tLeipzig,Margarete-BlankStrasse 8, D-04103 Leipzig,Germany
0001-2728 © 1995 SGM
the identification of the ORF A-2 gene product in
IBDV-infected cells. The ORF A-2 protein exhibits an
apparent molecular mass of 21 kDa which is larger than
the size of 16.5 kDa calculated from the deduced amino
acid sequence. Immunofluorescence studies demonstrated the presence of the ORF A-2 protein in bursa
samples from IBDV-infected chicken. In summary, the
IBDV ORF A-2 product represents the fifth IBDV
protein described. Therefore, we propose to designate it
as IBDV VP5.
B contains one ORF encoding a 90 kDa protein, VP1
(Azad et al., 1985). This protein is thought to represent
the putative RNA-dependent RNA polymerase (Spies et
al., 1987; Spies & Miiller, 1990). The termini of both
genome segments are circularized by a 90 kDa protein
(MiJller & Nitschke, 1987), most likely VP1.
Nucleotide sequence analysis indicated that IBDV
RNA segment A contains a second small ORF, designated ORF A-2, with a coding capacity for an approximately 16 kDa protein encompassing 145 amino acids.
ORF A-2 precedes and partially overlaps the 110 kDa
polyprotein gene (Bayliss et al., 1990; Spies et al., 1989).
However, no protein product from this gene has so far
been identified in IBDV-infected cells or IBDV virions.
To look for conservation of this ORF, the deduced
amino acid sequences from ORF A-2 from different
IBDV strains and isolates (serotype I strains Cu-1, Spies
et al., 1989; 52/70, Bayliss et al., 1990; STC, Kibenge et
al., 1990; Cu-IM, Bernstein, 1993; and serotype II
strains OH, Kibenge et al., 1991 ; 23/82, Bernstein, 1993)
were compared with the strain P2 sequence (E. Mundt,
unpublished) by the program PROSIS. Conservation
was higher among serotype I strains (between 98.6 % and
100 %) than between the two serotype II strains (92.4 %).
The ORF A-2 protein sequences of serotype I strains P2
and Cu-1 were identical. By contrast, amino acid
sequence comparison between these two serotype I
strains and the serotype II strain 23/82 (Bernstein, 1993)
identified 28 amino acid changes. Both deduced proteins
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contain seven cysteine residues. The positions of most of
these cysteine residues are conserved, although differences were observed at positions 100 and 105.
The aim of this study was the identification of the
O R F A-2 protein in IBDV-infected cells in culture and in
chickens using an antiserum prepared against E. coliexpressed ORF A-2 protein. The serotype I strain P2,
isolated and attenuated in duck and chicken embryo cells
by Schobries et al. (1977) was plaque-purified three times
in chicken embryo cells (CEC) before use. IBDV strains
23/82 (Chettle et al., 1985) and Cu-1 (Nick et al., 1976)
were a kind gift from Professor H. Becht (University of
Giessen, Germany). CEC were infected with the IBDV
strain P2 at an m.o.i, of 5 and incubated at 39 °C for 8 h.
Whole cell nucleic acids were released by proteinase K
(0.2 mg/ml) digestion in 0"5 % SDS. Nucleic acids were
ethanol-precipitated after phenol-chloroform extraction,
resuspended in DEPC-treated water and used for the
amplification of the small O R F of segment A by RTPCR. Primers for the RT-PCR bracketing the O R F A-2
were selected according to the sequence of the IBDV
strain P2 large genome segment (E. Mundt, unpublished). The sense primer of 25 nucleotides
(5' C A T A T G G T T A G T A G A G A T C A G A C A A 3') contained a NdeI recognition site (bold type) and the start
codon of the small O R F (underlined) at its 5' end,
whereas the 31 nucleotide antisense primer (5' AGGATCC
A G T T C A C T C A G G C T T C C T T G G A A G 3') specified a
B a m H I cleavage site (bold type) and the virus specific
stop codon (underlined) at the 5' end. The RT-PCR was
carried out using standard procedures (Sambrook et aI.,
1989). PCR products were electroeluted after agarose gel
electrophoresis, and cloned blunt-end into SmaI-cleaved
plasmid pBluescript(+) (Stratagene) as described by
Zou & Brown (1992). The O R F A-2 fragment was
excised with B a m H I - N d e I and ligated into B a m H I NdeI-digested expression vector pET-3a (Novagen)
giving rise to recombinant plasmid pET-3aORF. The
correct orientation of the insert was verified by sequencing (data not shown). After transformation into
E. coli BL21 (DE3) pLys S (Novagen), expression of the
O R F A-2 protein was induced by addition of IPTG.
Expression of the protein in E. coli 3 h after induction
was determined by S D S - P A G E and Coomassie Brilliant
blue staining (data not shown). For the preparation of
anti-ORF A-2 antibodies whole E. coli BL21 (DE 3)
pLysS cells expressing O R F A-2 were used. After
repeated washings with 50 mN-Tris-HC1, pH 7-4 the
bacteria were heat inactivated by incubation at 56 °C for
30 min. This material was injected subcutaneously into
rabbits at intervals of 3 to 4 weeks (rabbit anit-ORF A2 serum). A second anti-ORF A-2 serum was obtained
from a mouse (mouse anti-ORF A-2 serum) by the same
procedures. Whole cell lysates of bacteria transformed
1
2
3
4
5
6
kDa
v~.
•i
~i,~ !!i~!iJ!i~:,
i
....... .......
.
-- 64
"~':
: i
-- 48
i
-- 36.4
- 29-7
ORF A-2 -~
!
; •
Fig. 1. Expression of ORF A-2 protein in transformed E. coli and
IBDV-infected CEC. Proteins from E. coli transformed with pET3aORF (lane 1) or pET-3a (lane 2) and from CEC infectedwith IBDV
serotype I strains Cu-1 (lane 4) and P2 (lane 6) or serotype II strain
23/82 (lane 5), or from uninfected CEC (lane 3), were analysed in
Western blots with rabbit anti-ORF A-2 serum. The location of
molecular mass markers is indicated, as is the position of the ORF A2 protein.
with pET-3aORF were used for immunization to prevent
possible denaturation of the recombinant protein during
purification. The chicken anti-IBDV serum and rabbit
anti-chicken immunoglobulin G serum were a kind gift
from Professor H. Becht (University of Giessen,
Germany). Antisera against whole virus of strain P2 were
prepared by repeated subcutaneous injections of rabbits
with purified virus in complete Freund's adjuvant (rabbit
anti-IBDV serum).
As shown in Fig. 1 (lanes 1 and 2), in a Western blot
of bacterial lysates the antiserum reacted with a number
of proteins from both pET-3aORF- (lane 1) and pET-3atransformed bacteria (lane 2). However, a 21 kDa protein
which was absent in the pET3a-transformed cells was
recognized in the pET-3aORF-transformed bacteria. To
analyse expression of the O R F A-2 protein during IBDV
infection in cell culture, CEC were infected with the
serotype I IBDV strains P2 and Cu-1 or the serotype II
strain 23/82 and analysed in Western blots (Fig. 1, lanes
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(a)
1
VP1 --~
VP2 --~
VP3 --~
439
(b)
2
3
4
5
6
7
8
9
2
kDa kDa
97
69
97-69-
46
46--
30
3
4
5
6
7
8
9
~
30 -
VP4 --~
21-5
21-5
ORF A-2 --~
14.3
~- ORF A-2
14.3
Fig. 2. (a) Radioimmunoprecipitation of ORF A-2 protein from IBDV-infected CEC. CEC infected with IBDV strain P2 (lanes 1, 3,
5, 7) or uninfected CEC (lanes 2, 4, 6, 8) were labelled 4 h p.i. for 60 rain with [14C]leucine, lysed and immunoprecipitated with rabbit
normal serum (lanes 1, 2), rabbit anti-ORF A-2 serum (lanes 3, 4), rabbit anti-IBDV serum (lanes 5, 6) or chicken anti-IBDV serum
(lanes 7, 8). Molecular mass markers were in lane 9. The location of viral proteins VP1, VP2, VP3 and VP4 is marked, as is the location
of the ORF A-2 protein. (b) Expression kinetics of ORF A-2 in IBDV-infected CEC. CEC were infected with IBDV strain P2 at an
m.o.i, of 5 and labelled for 60 min with [14C]leucine at 1 h (lane 2), 2 h (lane 4), 4 h (lane 6) and 6 h (lane 8) p.i. Lanes 3, 5, 7 and 9
show noninfected controls. Lysates were precipitated with rabbit anti-ORF A-2 serum. Lane 1 contains molecular mass markers. The
location of the ORF A-2 protein is also indicated.
3-6). Both Cu-1- (lane 4) and P2- (lane 6) infected C E C
showed specific reactivity o f a 21 k D a protein with the
a n t i - O R F A-2 serum which was absent in serotype II
strain 23/82-infected (lane 5) and in uninfected cells (lane
3).
T o further test reactivity of different sera with the
21 k D a O R F A-2 protein, infected and noninfected C E C
proteins were labelled with [14C]leucine ( A m e r s h a m ) and
analysed by r a d i o i m m u n o p r e c i p i t a t i o n after incubation
with n o r m a l rabbit serum, rabbit a n t i - O R F A2-serum,
rabbit a n t i - I B D V serum and chicken a n t i - I B D V serum,
respectively. I m m u n o p r e c i p i t a t i o n s of radiolabelled proteins f r o m IBDV-infected (Fig. 2a, lanes 1, 3, 5, 7) or
noninfected cells (Fig. 2a, lanes 2, 4, 6, 8) were
performed. C o m p a r e d to the n o r m a l rabbit serum (lanes
1, 2), the rabbit a n t i - O R F A-2 protein serum (lanes 3, 4),
specifically precipitated a 21 k D a protein f r o m I B D V infected cell lysates (lane 3). Precipitation o f a protein of
similar size f r o m IBDV-infected cell lysates was also
observed using the rabbit a n t i - I B D V serum (lane 5). T h e
chicken a n t i - I B D V serum (lanes 7, 8) specifically precipitated VP1, VP2, VP3 and VP4 f r o m IBDV-infected
cells. One protein with a molecular mass slightly larger
than 21 k D a could also be precipitated with the chicken
a n t i - I B D V serum f r o m IBDV-infected cells (lane 7).
However, since a protein of similar size was also found
after precipitation of lysates of noninfected cells (lane 8)
it is unclear at present whether this protein is related to
the 21 k D a O R F A-2 product. In s u m m a r y , the rabbit
a n t i - O R F A-2 and the rabbit a n t i - I B D V serum exhibited
reactivity with a novel IBDV-specific protein o f 21 k D a
in infected CEC.
To analyse the expression kinetics of the 21 k D a O R F
A-2 protein, C E C were infected at an m.o.i, o f 5, pulselabelled for 1 h at 1, 2, 4 and 6 h after infection and
proteins were i m m u n o p r e c i p i t a t e d with rabbit a n t i - O R F
A-2 protein serum. As shown in Fig. 2 (b), expression of
the O R F A-2 protein was first detectable 3 h postinfection (p.i.) (lane 4), increased in a m o u n t at 5 h p.i.
(lane 6), and was found in decreased a m o u n t s at 7 h p.i.
(lane 8). N o protein f r o m uninfected C E C was specifically precipitated at any time point (lanes 3, 5, 7, 9).
To determine localization of the O R F A-2 protein in
IBDV-infected cells, immunofluorescence studies o f
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Fig. 3. Demonstration of IBDV ORF A-2 protein expression in CEC by immunofluorescence.CEC were infected with the serotype 1
strains P2 (c, d) or the serotype lI strain 23/83 (e,f), respectively. Uninfected CEC were used as a negative control (a, b). Acetonefixed CEC cultures were incubated with rabbit anti-ORF A-2 serum (a, c, e) and rabbit anti-IBDV serum (b, d, f). Antigemantibody
complexes were demonstrated by incubation with swine fluorescein-conjugatedanti-rabbit immunoglobulin G serum (Dako).
infected and uninfected CEC were performed. After
acetone fixation, specific fluorescence was not observed
in uninfected cells with either the rabbit anti-ORF A-2
protein serum (Fig. 3 a) or rabbit anti-IBDV serum (Fig.
3b). In contrast cells infected with IBDV serotype I
strain P2 (Fig. 3 c, d) were recognized by both the antiO R F A-2 serum and the anti-IBDV serum. Surprisingly,
despite the lack of recognition of a protein in Western
blots of serotype II strain 23/82-infected CEC proteins
(see Fig. 1, lane 5), 23/82-infected CEC also exhibited
fluorescence with both antisera (Fig. 3e, f ) . O R F A-2
specific fluorescence was uniformly distributed in the
cytoplasm. In contrast, fluorescence using the rabbit
anti-IBDV serum was associated with large intensely
stained aggregates within the cytoplasm as described by
Lukert & Davis (1974).
To analyse expression of viral antigens in infected
animals, bursal tissue from a 4-week-old SPF chicken
infected with strain P2 was removed 2 days after
infection. Bursal tissue from a non-infected chicken
served as a negative control. Cryosections were assayed
by indirect immunofluorescence using the mouse anti-
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441
Fig. 4. Expressionof ORF A-2 protein in IBDV-infectedbursal cells. Cryosectionsof the bursa of Fabricius (BF) of a chickeninfected
with strain P2 2 days after infection(c, d) and cryosectionsof BF of a noninfectedchicken(a, b) were incubated with rabbit anti-IBDV
serum (b, d) and mouse anti-ORF A-2 serum (a, c), respectively.Reactivity'of the antibodies with the antigen weredemonstrated after
incubation with swine fluorescein-conjugatedanti-rabbit immunoglobulin G or goat fluorescein-conjugatedanti-mouse immnnoglobulin G (Dako).
O R F A-2 protein serum (Fig. 4a, c) or rabbit anti-IBDV
serum (Fig. 4b, d), respectively. Mouse anti-ORF A-2
serum was used because the rabbit anti-ORF A-2 serum
gave a strong background in bursal cells. Neither serum
reacted with non-infected bursal tissue (Fig. 4a, b).
However, both sera exhibited fluorescence with bursal
tissue from strain P2-infected animals (Fig. 4c, d). A
limited number of cells with cytoplasmic fluorescence
was demonstrated in the medulla of the bursal follicles
after incubation with the mouse anti-ORF A-2 protein
serum (Fig. 4 b), whereas a large number of cells exhibited
fluorescence after incubation with the rabbit anti-IBDV
serum (Fig. 4a). Histological examinations (data not
shown) of the infected bursal tissue showed alterations
due to virus infection (severity 2a to 2b according to
Muskett, 1976). In summary, the O R F A-2 protein is
expressed in the bursa of Fabricius during infection of
chicken by IBDV.
The existence of a second open reading frame on
segment A of IBDV, preceding and partially overlapping
the gene for the 110 kDa polyprotein, and having a
coding capacity for an approximately 16 kDa protein,
has previously been suggested (Spies et al., 1989; Bayliss
et al., 1990; Kibenge et al., 1990). Indeed, after
immunoprecipitation of in vitro translation products of
genomic R N A with chicken reconvalescence serum the
presence of a 16 kDa protein was demonstrated (Azad et
al., 1985). It has also been shown that segment A of
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various IPNV strains, i.e. strain Jasper (Duncon &
Dobos, 1986) and N1 (Havarstein et al., 1990), possess a
second ORF partially overlapping the polyprotein gene
(Havarstein et al., 1990) and a 17 kDa protein has been
demonstrated by SDS PAGE of [a~S]methionine-labelled
purified IPNV. However, no correlation of these products with ORF A-2 has so far been shown, although it
has been suggested that this protein is probably virusspecific. Comparison between the amino acid sequences
of ORF A-2 proteins of different strains of IBDV and
IPNV has indicated only limited amino acid homology,
although, interestingly three cysteine residues are located
at conserved positions within the respective IBDV and
IPNV proteins (Havarstein et al., 1990). Taken together,
the similar positions on the genomes, the similar
molecular masses as well as the conservation of cysteine
residues which may be important for the secondary/
tertiary structure of the proteins could be indicative of
similar functions for these proteins.
In this study, we demonstrate the existence of the ORF
A-2 protein in IBDV-infected chicken cells in tissue
culture and in bursal tissue of infected animals. Since this
represents the fifth IBDV protein identified, we propose
to designate it IBDV VP5. The discrepancy between the
calculated (16.5 kDa) and the apparent molecular mass
in SDS-PAGE (21 kDa) of IBDV VP5 is still puzzling.
Nucleotide sequence analysis confirmed the correct
integration of the ORF A-2 gene in the expression
vector. Post-translational alterations may account for
this difference, although the nature of any modification is
unclear. Alternatively, extensive higher order structuring
may lead to aberrant migration in SDS-PAGE.
Although expression of the ORF A-2 protein was
detected by immunofluorescence analyses using cells
infected with serotype I and serotype II strains, only in
serotype I-infected cell lysates could ORF A-2 protein be
detected in immunoblots. This might be due to the
presence of a number of amino acid differences between
the ORF A-2 proteins of serotype I and serotype II
strains which could influence the antigenic properties of
the respective proteins. The conservation of most cysteine
residues might be indicative of a conserved tertiary
and/or secondary structure with a possible conservation
of conformation-dependent epitopes. In contrast, linear
epitopes might be different between ORF A-2 proteins of
serotype I and II strains, respectively, due to the amino
acid differences. Therefore, after reduction of the possible
disulphide-bridges and denaturation in S D ~ P A G E ,
only linear epitopes might still be present which might be
recognized in a serotype-specific fashion by the antiserum
directed against the serotype I ORF A-2 protein.
Immunofluorescence analyses demonstrated the presence of the ORF A-2 protein in infected CEC as well as
in bursal tissue of an infected chicken. Whereas with the
anti-IBDV serum punctate aggregates in the cytoplasm
were preferentially labelled, fluorescence with the antiORF A-2 serum was more generally distributed. The
aggregates probably represent accumulations of viral
proteins or virus particles as described by K~iufer &
Weiss (1976). Kinetic analyses in cell culture demonstrated a transient expression of the ORF A-2 protein
during viral replication, which probably explains the
lower number of fluorescent cells labelled with the antiORF A-2 serum as compared to the anti-IBDV serum.
The ORF A-2 protein could be detected after
precipitation with rabbit anti-ORF A-2 serum and rabbit
anti-IBDV serum of labelled proteins from infected CEC
culture. In contrast, a chicken anti-IBDV serum apparently failed to recognize the ORF A-2 gene product
although it efficiently reacted with viral structural
proteins VP1 to VP4. In Western blot analyses using
purified IBDV virions, none of the sera showed any
specific reactivity with a protein of the size of the ORF
A-2 product. This leads to the assumption that the ORF
A-2 protein probably represents a nonstructural viral
protein. A protein of this size has not been previously
described after immunoprecipitation of [35S]methioninelabelled infected cell proteins with anti-IBDV serum
(Mfiller & Becht, 1982; Jackwood et al., 1984). Since the
ORF A-2 protein contains only one methionine residue,
encoded by the start codon, it is likely that insufficient
label was incorporated into the mature protein to allow
its detection after immunoprecipitation. To alleviate this
problem, in this study labelling was performed using
[14C]leucine. The ORF A-2 protein contains 11 leucine
residues.
A 17 kDa protein which is probably virion-associated
has been found in [35S]methionine labelled IPNV virions
(Havarstein et al., 1990). In this case, detection might
have been successful due to the presence of a second
methionine residue within the putative ORF A-2 protein
of IPNV.
The function of the ORF A-2 protein is still unclear, as
are functional aspects of other proteins of similar size
encoded by RNA viruses. For example, Koonin et al.
(1991) propose an RNA/DNA-binding activity for the
small cysteine-rich proteins of plant viruses. These
proteins presumably carry out regulatory functions
during virus replication. Whether the IBDV ORF A-2
protein is involved in similar steps in the viral replicative
cycle remains to be investigated.
In summary, we demonstrate for the first time the
protein product of the ORF A-2 reading frame in IBDVinfected cells. Since this ORF A-2 protein represents the
fifth viral protein of IBDV, we designate it as IBDV VP5.
The function of this protein is unknown but further
investigations are in progress to clarify its role in IBDV
infection.
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We thank Dr T. Mettenleiter for his critical reading of the
manuscript. The authors appreciate the excellent technical assistance of
Ms Dietlind Kretzschmar.
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(Received 20 June 1994; Accepted 23 September 1994)
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