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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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 438 Short communication 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 Short communication (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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 440 Short communication 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- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 Short communication 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 442 Short communication 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42 Short communication We thank Dr T. Mettenleiter for his critical reading of the manuscript. The authors appreciate the excellent technical assistance of Ms Dietlind Kretzschmar. References AZAD, A. A., BARRETT, S. A. & FAIIEY, K. J. 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Identification of sequence elements containing signals for replication and encapsidation of the reovirus M1 genome segment. Virology 186, 377-388. (Received 20 June 1994; Accepted 23 September 1994) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 15:52:42