Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, D-04103 Leipzig, Germany1
Author for correspondence: Hermann Müller. Fax +49 341 9738219. e-mail virology{at}vetmed.uni-leipzig.de
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Abstract |
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Introduction |
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Morphologically, APV is characterized by a non-enveloped particle, 4550 nm in diameter, containing a 4981 bp circular double-stranded (ds) DNA genome (Dykstra & Bozeman, 1982 ; Müller & Nitschke, 1986
; Lehn & Müller, 1986
; Rott et al., 1988
). The genome is transcribed bidirectionally for the expression of early and late genes (Luo et al., 1995
). Despite these overall similarities, remarkable differences from mammalian polyomaviruses have been found in the genome sequences, especially in the non-coding regulatory region and in the regions encoding the large tumour (T) antigen and the agnoproteins of APV (Luo et al., 1994
). The differences in the nucleotide sequences and the ability of APV to cause an acute fatal disease, unusual in mammalian polyomaviruses, have led to the suggestion that this virus should be placed in a distinct subgenus, Avipolyomavirus, within the genus Polyomavirus (Stoll et al., 1993
).
The agnoproteins of the mammalian polyomaviruses simian virus 40 (SV40), BK virus (BKV) and JC virus are small proteins encoded by an ORF located close to the 5'-end of the late mRNA, which also encodes the structural protein VP1 (Jackson & Chalkley, 1981 ; Rinaldo et al., 1998
). The functions of these agnoproteins in the virus life-cycle are still unknown. In SV40 infection, the agnoprotein is expressed several hours after the major structural protein, VP1 (Jackson & Chalkley, 1981
; Jay et al., 1981
), it accumulates in a perinuclear location (Jay et al., 1981
; Nomura et al., 1983
) and is not incorporated into the virus capsid (Jackson & Chalkley, 1981
). SV40 mutants that do not express the agnoprotein are still viable in tissue culture (Shenk et al., 1976
). Recently, it has been shown that the agnoprotein of BKV interacts with cellular proteins, but not with viral proteins (Rinaldo et al., 1998
).
In APV, late mRNAs show a high degree of heterogeneity due to two starting points of late mRNA transcription and partial as well as alternative splicing events. Based on the localization in the genome and the splicing pattern, seven ORFs have been proposed to encode agnoproteins in a subset of 18 mRNAs (Luo et al., 1995 ; Liu et al., 2000
; Johne et al., 2000
). Recently, Liu & Hobom (1999)
have concluded that only two of these agnoproteins, 2a and 2b, represent the real counterparts of SV40 agnoprotein, whereas agnoproteins 1a and 1b share few similarities, if any, with the agnoproteins of SV40.
The ORF of agnoprotein 1a encodes a protein of 176 aa. The ORF for agnoprotein 1b is created by alternative splicing. The deletion of the codons for amino acids 69132 of agnoprotein 1a results in a final protein of 112 aa. A multiple phosphorylation pattern has been shown recently for agnoproteins 1a and 1b, resulting in a complex series of electrophoretically separable subspecies. Phosphatase treatment yields the two primary proteins, with apparent molecular masses of 32 and 29 kDa (Liu & Hobom, 2000 ; Liu et al., 2000
). The expression of agnoproteins 1a and 1b of APV has been shown to be essential for virus replication in chicken embryo (CE) cells, and a function as apoptotic inducers during APV replication has been demonstrated (Johne et al., 2000
). In accordance with the observations of Liu & Hobom (1999)
, it has been concluded from these studies that the biological functions exerted by agnoproteins 1a and 1b are different from those of the agnoproteins of mammalian polyomaviruses (Johne et al., 2000
).
In this study, the intranuclear localization of agnoprotein 1a in APV-infected CE cells and its presence in purified APV particles are demonstrated. Furthermore, we demonstrate that agnoprotein 1a interacts with the major structural protein, VP1, and binds dsDNA. These observations indicate a specific function(s) of agnoprotein 1a within the APV particle as a fourth structural protein, with the proposed designation VP4.
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Methods |
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Sf9 cells were cultivated at 27 °C in TNM-FH insect medium (Sigma) supplemented with 10% foetal calf serum and used for the propagation of recombinant baculoviruses. E. coli XL-1 Blue MRF' cells (Stratagene) were used for DNA cloning and expression of recombinant proteins.
Construction of plasmids.
For the expression of agnoproteins 1a and 1b in E. coli and subsequent purification by affinity chromatography, total RNA of APV-infected CE cells was isolated 4 days p.i. by proteinase K treatment and phenolchloroform extraction (Sambrook et al., 1989 ). RTPCR was performed by using the Titan RTPCR system (Boehringer Mannheim) with the isolated RNA as template and the primers 5'-CAACAACATGTCTACTCCAGCG-3' and 5'-GTGCAGATCTATAGCGAGCCG-3'. The PCR products, of 545 bp (agnoprotein 1a) and 350 bp (agnoprotein 1b), were digested with AflIII and BglII and the resulting fragments were ligated to NcoI/BglII-restricted vector pQE-60 (Qiagen). The resulting plasmids, p1aHis and p1bHis, contain the coding sequences for agnoproteins 1a and 1b with additional codons for a C-terminal tail with the sequence YRSHHHHHH (Fig. 1
).
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The plasmid pAPVnc, containing nucleotides 1202 of the non-coding regulatory region of the APV genome (numbering according to Rott et al., 1988 ), was constructed for the generation of a DNA probe to be used in mobility-shift DNA-binding assays. Plasmid pAPVinf was cleaved with NcoI and HinfI and a 202 bp fragment was isolated. After a fill-in reaction with Klenow enzyme (AGS), the fragment was cloned into the HincII site of the vector pBluescript SK(+) (Stratagene).
Expression and purification of His-tailed proteins.
Saturated cultures of E. coli transformed with the appropriate expression plasmid were diluted 1:100 in 1 litre fresh medium (LB broth supplemented with 100 µg ampicillin per ml). Cells were grown until the OD600 reached 0·8 and expression was induced with 1 mM IPTG. After 5 h, bacteria were harvested by centrifugation, resuspended in sonication buffer (10 mM TrisHCl, pH 7·8, 50 mM KH2PO4, 300 mM NaCl, 10 mM -mercaptoethanol) and subsequently lysed by sonication on ice for six 30-s bursts. The lysate was centrifuged at 10000 g for 20 min and the supernatant was used for purification of the His-tailed proteins with Ni2+NTA affinity resin (Qiagen).
All purification steps were performed at room temperature. The resin (4 ml) was added to a 20 ml column (Qiagen) and equilibrated with 20 ml sonication buffer. The cell lysates were loaded on the column, which was then washed with 20 ml washing buffer (40 mM TrisHCl, pH 7·5, 20% glycerol, 100 mM KCl, 1 mM -mercaptoethanol) supplemented with 20 mM imidazole. The proteins were eluted in fractions of 4 ml containing 50, 80, 120, 200, 300 and 500 mM imidazole in washing buffer. Aliquots of each fraction were analysed by SDSPAGE and immunoblotting. Fractions with large amounts of recombinant protein were pooled and the concentration of total protein was measured by the Pierce BCA protein reagent assay. The purity of the recombinant proteins was determined by densitometric analysis of Coomassie brilliant blue-stained gels after SDSPAGE by using Gel-Pro Analyzer software (Media Cybernetics).
Expression of proteins in insect cells.
Generation of recombinant baculovirus expressing agnoprotein 1a has been described recently (Johne et al., 2000 ). The APV structural proteins VP1 and VP2 were expressed by using the baculovirus transfer vector pL546 (kindly provided by M. Pawlita, Heidelberg), with the C-terminal undecapeptide KPPTPPPEPET of the SV40 large T antigen as a tag.
The VP1-encoding region was amplified by PCR with pAPVinf as template and the primers 5'-GGCTACATGTCCCAAAAAGGAAAAGGAAGC-3' and 5'-TAGTCAGATCTGCGGGGAGCTTTGGGGGGCAT-3'. The PCR product was digested with AflIII and BglII and ligated to NcoI/BamHI-restricted vector pL546. The VP2-encoding region was amplified by PCR with the primers 5'-CTAAGCCATGGGAGCTATCATTTCGGCTATAGC-3' and 5'-GTTGTTGGATCCTCTGGACCTGACTTTACGTC-3' and pAPVinf as template. The PCR product was digested with NcoI and BamHI and ligated as above. The resulting plasmids were used to generate recombinant baculoviruses for the production of the tagged structural proteins VP1tag and VP2tag. Generation and amplification of recombinant baculoviruses were carried out by using the BaculoGOLD kit (Pharmingen) and Sf9 cells as described previously (Pawlita et al., 1996 ).
Preparation of antisera.
His-tailed proteins agno 1aHis, agno 1bHis and agno 1a69132His, purified by affinity chromatography on Ni2+NTA resin, were mixed with Freunds incomplete adjuvant (Sigma) and used for the intracutaneous immunization of rabbits. Two subcutaneous booster immunizations were performed at 2-week intervals. Sera were collected 2 weeks after the last immunization.
Analysis of proteins.
Proteins were analysed by SDSPAGE and immunoblotting as described previously (Stoll et al., 1993 ). The monoclonal antibody KT3 (kindly provided by M. Pawlita) was used to identify proteins tagged with the C-terminal undecapeptide of the SV40 large T antigen (MacArthur & Walter, 1984
). The low molecular mass markers calibration kit (Pharmacia) and biotinylated low molecular mass markers (Bio-Rad) were used as standards.
Immunofluorescence.
For the subcellular localization of proteins by immunofluorescence, CE cells grown on glass coverslips were fixed in acetonemethanol for 30 min at -20 °C. Primary antibodies were used at dilutions of 1:200 to 1:400 and FITC-conjugated secondary antibodies (Sigma) were used at a dilution of 1:100.
Co-immunoprecipitation assay.
Sf9 cells were co-infected with different recombinant baculoviruses and maintained for 4 days at 27 °C. Thereafter, the cells were lysed by incubation for 1 h on ice in 1 ml lysis buffer [20 mM TrisHCl, pH 7·4, 137 mM NaCl, 10% glycerol, 0·01% Triton X-100, 2 mM EDTA, 50 mM -glycerophosphate, 20 mM sodium pyrophosphate, 2 mM Pefabloc protease inhibitor (Boehringer Mannheim)]. Cellular debris was removed by centrifugation and the supernatant was incubated with the specific antibodies for 1 h at 4 °C. After the addition of 20 µl protein Aagarose (Sigma), the mixture was shaken gently at 4 °C for 1 h. Thereafter, the agarose beads were pelleted by centrifugation, washed five times with lysis buffer and subsequently analysed by immunoblotting.
Mobility-shift DNA-binding assay.
The DNA probes were prepared by restriction endonuclease digestion of plasmid DNA and subsequently labelled with [-32P]dCTP (Hartmann Analytic) by using the Klenow enzyme (AGS) as described previously (Buratowski & Chodosh, 1996
).
In the mobility-shift DNA-binding assay, the DNA probes (about 20000 c.p.m.) and purified proteins were incubated for 5 min at room temperature in binding buffer (40 mM TrisHCl, pH 7·4, 10 mM HEPESNaOH, 50 mM KCl, 5 mM MgCl2, 0·1 mM EDTA, 0·8 mM DTT, 12% glycerol). ProteinDNA complexes were resolved on 6% polyacrylamide gels under non-denaturating conditions (40:1 acrylamide:bisacrylamide, 0·25 M Tris base, 1·9 M glycine, 10 mM EDTA) by electrophoresis at 20 mA until bromophenol blue, added in a separate lane, approached the bottom of the gel. The gel was then dried and the DNA bands were localized by autoradiography.
In the competition mobility-shift DNA-binding assay, unlabelled competitor DNA was mixed with the DNA probe in binding buffer before the purified protein was added. For specific competition, unlabelled DNA probe was used. Unspecific competitors were poly(dAdT) (Boehringer Mannheim), poly(dA) (Boehringer Mannheim) and 16S and 23S rRNA from E. coli (Boehringer Mannheim). The concentration of DNA was determined by measuring the A260 with a DU-64 spectrophotometer (Beckman).
Software.
Sequence data were compared by using the Lasergene System software (DNASTAR). Calculation of the apparent molecular mass of a protein and determination of its relative abundance in a protein mixture were performed by densitometric analysis of Coomassie brilliant blue-stained gels using the Gel-Pro Analyzer software (Media Cybernetics).
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Results |
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Detection and localization of agnoprotein 1a in APV-infected CE cells
The antisera described above were used for the detection of the agnoproteins in APV-infected CE cells by immunoblotting. A double band with an apparent molecular mass of 32/33 kDa, corresponding to agnoprotein 1a, was detected by all of the antisera; however, none of the antisera detected a protein band corresponding to agnoprotein 1b. As expected, the control antiserum APV recognized additional bands representing the structural proteins VP1, VP2 and VP3 of APV (Fig. 3a
; 3 days p.i.). No proteins were detected in mock-infected cells (not shown). The presence of the agnoproteins was also determined in a time-course experiment by immunoblot analysis of constant volumes taken from individual infected CE cell cultures at various times after infection. Increasing amounts of agnoprotein 1a were detected between 24 and 72 h p.i. with the antiserum
1a (Fig. 3b
). A similar pattern was observed when the antisera
1b and
1a69132 were used (not shown). Again, agnoprotein 1b could never be detected.
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Detection of agnoprotein 1a in purified virus particles
APV particles as well as SV40 particles purified by repeated CsCl gradient centrifugation were analysed by SDSPAGE and Coomassie brilliant blue staining. The structural proteins VP1, VP2 and VP3 and bands with smaller apparent molecular masses, representing cellular histones, were detected in both virus preparations (Fig. 5a). Remarkably, however, an additional double band with an apparent molecular mass of 32/33 kDa was present in the preparation of APV particles. These bands represented about 13% of the total protein (Fig. 5b
).
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Interactions between the viral structural proteins and agnoprotein 1a
Sf9 cells were infected with recombinant baculoviruses expressing agnoprotein 1a and the tagged proteins VP1tag and VP2tag and were analysed by immunoblotting 4 days p.i. (data not shown). Agnoprotein 1a, VP1tag and VP2tag were detected by the antiserum APV. A minor band was present in VP1tag-expressing cells, presumably representing a degradation product of VP1tag. An additional band with an apparent molecular mass of 31 kDa, representing VP3, was found in cells infected with the VP2tag-expressing baculovirus. The monoclonal antibody KT3, which detects the tag peptide, detected VP1tag, VP2tag and the 31 kDa protein, but not agnoprotein 1a.
Sf9 cells were then co-infected with baculovirus expressing agnoprotein 1a and baculoviruses expressing either VP1tag or VP2tag, lysed after 4 days and subjected to immunoprecipitation followed by immunoblotting. Cells infected with baculoviruses expressing VP1tag, VP2tag or agnoprotein 1a only served as controls.
In a first series of experiments, the monoclonal antibody KT3 was used for immunoprecipitation and the antiserum APV was used for immunoblotting. As shown in Fig. 6(a)
, the tagged proteins were precipitated and the expected proteins, VP1tag and VP2tag, became visible by immunoblotting. In the VP1tag-expressing cells, the putative degradation product mentioned above was again observed (lanes 1 and 4). As before, a protein band corresponding to VP3 was present in the VP2tag-expressing cells (lanes 2 and 5). An additional, faint band in the position of agnoprotein 1a was visible in cells that had been co-infected with baculoviruses expressing VP1tag and agnoprotein 1a (lane 1). In a second series of experiments, the antiserum
1a was used for immunoprecipitation and the monoclonal antibody KT3 for immunoblotting. It is evident from Fig. 6(b)
that a band corresponding to VP1tag was only visible in cells that had been co-infected with baculoviruses expressing VP1tag and agnoprotein 1a (lane 1).
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Agno 1aHis and the polypeptide agno 1a69 132His, but not agno 1bHis, bind dsDNA
Binding of agnoproteins expressed in E. coli to DNA was tested in mobility-shift DNA-binding assays. As specific interactions with the regulatory region of the APV genome had been expected, the XhoI/HindIII-digested DNA fragment of plasmid pAPVnc containing 202 bp of the non-coding region was used as a probe in the first experiments. Aliquots of 5 µl containing 0·6 µg purified agno 1aHis, 2·0 µg purified agno 1bHis, 0·4 µg purified agno 1a69132His or 0·02 µg of a negative control, purified from E. coli transformed with the vector pQE-60, were used in binding reactions with 32P-labelled DNA. The autoradiograph presented in Fig. 7(a) shows slowly migrating bands in lane 1 with agno 1aHis and in lane 3 with agno 1a69132His, indicative of proteinDNA complexes. Only the free DNA probe was visible in the case of agno 1bHis (lane 2) or the negative control (lane 4).
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Specificity of DNA-binding activity of agno 1aHis
The specificity of DNA binding was analysed by using specific and unspecific competitors (Fig. 7c). The incubation of the labelled probe with 300 ng agno 1aHis in the presence of a 100-fold excess of the unlabelled DNA probe as a specific competitor resulted in an increase in free labelled probe (lanes 3 and 4), whereas a 1000-fold excess prevented the band shift totally (lane 5). Competition with a 1000-fold excess of poly(dAdT) as an unspecific dsDNA competitor also resulted in an increase in free labelled probe (lane 6). Incubation with a 1000-fold excess of poly(dA) as a single-stranded DNA competitor did not alter the banding pattern (lane 7), whereas incubation with E. coli 16S and 23S rRNA as single-stranded RNA competitors increased the amount of free labelled probe slightly (lane 8). When 300 ng agno 1aHis was incubated with each of the 32P-labelled dsDNA probes, prepared from different plasmids as described in the legend of Fig. 7
, a mobility shift was observed with all of these probes, indicating sequence-unspecific binding of this protein (Fig. 7d
).
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Discussion |
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Specific antisera have been prepared that are directed against agnoproteins 1a and 1b and a polypeptide consisting of 64 aa not present in agnoprotein 1b. As expected, all of these antisera reacted with agnoprotein 1a in immunoblots with APV-infected CE cells as antigen (Fig. 3a). Remarkably, however, although the antibodies directed against agnoprotein 1b reacted strongly with the recombinant protein used for its induction (Fig. 2c
), no reactivity was observed with a protein of the corresponding size in immunoblots of APV-infected CE cells (Fig. 3a
). This may be because of inefficient translation of the abundant mRNA present in infected cells or rapid proteolytic degradation of agnoprotein 1b.
The intranuclear localization of agnoprotein 1a was determined by immunofluorescence studies with APV-infected CE cells. Strong intranuclear fluorescence was observed with the antisera 1a and
1a69132. Antiserum
1b, however, showed intranuclear as well as moderate cytoplasmic fluorescence. It may be speculated that
1b reflects the intranuclear location of agnoprotein 1a and the intracytoplasmic location of agnoprotein 1b. It must be taken into account, however, that the presence of agnoprotein 1b in APV-infected CE cells has not been confirmed by immunoblotting. A potential nuclear localization signal, not present in agnoprotein 1b, has been recognized at aa 7077 of agnoprotein 1a (HRRRPYDR; Fig. 8
). Another mechanism of nuclear transport could be the co-transport of agnoprotein 1a together with other viral proteins, in a manner similar to that described for APV VP1, which is co-transported into the nucleus together with VP2 (An et al., 1999
). The intranuclear localization of APV agnoprotein 1a is in marked contrast to the perinuclear localization of the agnoproteins of SV40 and BKV (Nomura et al., 1983
; Rinaldo et al., 1998
).
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The presence of an almost constant amount [13% of total structural proteins (Fig. 5b); 15% in Müller & Nitschke (1986)
] of agnoprotein 1a in the APV particle suggests an interaction(s) of this protein with other components of the virion. By precipitating either VP1 or agnoprotein 1a followed by immunoblotting, co-immunoprecipitation of agnoprotein 1a and VP1 was observed, indicating a specific interaction between these viral proteins. It must taken into account, however, that the experiments were performed with tagged proteins, which does not represent natural conditions. The interaction of agnoprotein 1a with the viral DNA was investigated by using proteins expressed in E. coli. In band-shift assays, agno 1aHis and the peptide agno 1a69132His were shown to bind DNA, whereas no DNA-binding activity of agno 1bHis was observed. The presence of multiple bands (Fig. 7b
) suggests binding of increasing numbers of agno 1aHis molecules to the DNA probe. It became evident from further experiments that binding was not dependent on the nucleotide sequence of the DNA probe, notwithstanding the fact that a low-affinity sequence specificity was observed, as competition with a specific probe was more efficient than competition with the unspecific DNA probes.
Agnoprotein 1a consists of 176 aa, of which 25 are proline residues (Fig. 8a). The molecular mass deduced from the nucleotide sequence is 18·6 kDa, whereas the molecular mass apparent from SDSPAGE was 32/33 kDa. A detailed sequence analysis of amino acids 69132 of agnoprotein 1a, deleted in agnoprotein 1b, reveals a potential leucine zipper motif (Landschulz et al., 1988
) that might be responsible for DNA-binding activity. A region of basic amino acids at positions 7077, necessary for the interaction with DNA, is followed by leucine residues at positions 84, 98, 105 and 112, which could enhance DNA binding through dimerization of the protein.
The suggested functions of the agnoproteins of mammalian polyomaviruses remain controversial. In the case of SV40, agnoprotein-mediated effects have been suggested on virus assembly (Carswell & Alwine, 1986 ; Margolskee & Nathans, 1983
), maturation (Hou-Jong et al., 1987
) and regulation of transcription (Alwine, 1982
; Hay & Aloni, 1985
). For the agnoproteins of SV40 and BKV, a function in the release of mature virus is favoured (Resnick & Shenk, 1986
; Rinaldo et al., 1998
). The function of agnoprotein 1a of APV seems to be completely different. Recently, induction of apoptosis has been described as one function of agnoproteins 1a and 1b of APV (Johne et al., 2000
). The presence of agnoprotein 1a in the virus capsid suggests a second function, as a fourth structural protein, in addition to VP1, VP2 and VP3 observed regularly in polyomaviruses.
Because of the presence of two initiation codons, the region encoding the agnoproteins of APV reveals two sets of agnoproteins. As mentioned above, no structural or functional homologies can be observed between agnoprotein 1a or 1b of APV and the agnoproteins of mammalian polyomaviruses. Agnoproteins 2a and 2b (Fig. 8b), however, show distinct similarities with regard to structure (basic and hydrophobic proteins), function (non-essential genes) and subcellular localization (Liu & Hobom, 1999
). These observations and the presence of agnoprotein 1a within the APV particle justify the renaming of this protein as VP4, indicating its function as a fourth structural protein of APV, instead of the misleading designation agnoprotein 1a.
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Acknowledgments |
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References |
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Received 20 September 2000;
accepted 1 December 2000.