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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APV virions are non-enveloped icosahedrons with a diameter of approximately 45 nm (Lehn & Müller, 1986 ; Müller & Nitschke, 1986
). The circular double-stranded DNA genome, consisting of 4981 bp, is transcribed bidirectionally for the expression of early and late genes (Rott et al., 1988
; Luo et al., 1995
). Marked differences from mammalian polyomaviruses have been observed in the non-coding regulatory region, the region encoding the large tumour (T) antigen, and the agnoproteins (Luo et al., 1994
, 1995
). BFDV isolates from different species of birds can be grouped within one genotype and one serotype, and it has been suggested to adopt the name APV for all known isolates (Johne & Müller, 1998
). Based on the unique molecular and biological characteristics of APV, it has been proposed to place this virus into a distinct subgenus Avipolyomavirus within the genus Polyomavirus (Stoll et al., 1993
).
The functions of agnoproteins, small proteins of the primate polyomaviruses simian virus 40 (SV40), BK virus (BKV) and JC virus (JCV), still remain unknown (Jackson & Chalkley, 1981 ; Rinaldo et al., 1998
). They are encoded by an open reading frame (ORF) located near to the 5'-end of the late bicistronic mRNA that also encodes the structural protein VP1. In APV, late gene expression is complicated by two putative promotor regions as well as partial and alternative splicing events. Seven mRNAs potentially encoding different agnoproteins have been observed during the infectious cycle (Luo et al., 1995
). Recently, the two largest agnoproteins have been designated as agnoprotein 1a and agnoprotein 1b, respectively (Li, 1996
; Liu, 1997
). Agnoprotein 1a consists of 176 amino acids with no obvious sequence similarities to agnoproteins of mammalian polyomaviruses (Luo et al., 1995
; Rott et al., 1988
). By alternative splicing the ORF for agnoprotein 1b is created, which shares a common N- and C-terminus with agnoprotein 1a but has an internal deletion of amino acids 69 to 132 (Luo et al., 1995
).
In this communication, induction of apoptosis by APV infection of chicken embryo (CE) cells, and by the expression of agnoprotein 1a and agnoprotein 1b in insect cells and in CE cells is described. The significance of these proteins for the induction of apoptosis and the production of infectious progeny was investigated by transfection of CE cells with APV full-length plasmids which either did express agnoprotein 1a and agnoprotein 1b, or did not.
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Methods |
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Expression of agnoprotein 1a and agnoprotein 1b in insect cells.
Total RNA was isolated from APV-infected CE cells 5 days post-infection (p.i.) by proteinase K treatment and subsequent phenolchloroform extraction (Sambrook et al., 1989 ). Agnoprotein 1a and agnoprotein 1b cDNAs were generated by RTPCR (Titan-RTPCR system, Boehringer Mannheim) using isolated RNA as template and primers 5' CAA CAA CAT GTC TAC TCC AGC G 3' and 5' CGG GGG ATC CTT AGC GAG CC 3'. The 548 bp (agnoprotein 1a) and 356 bp (agnoprotein 1b) products were digested using AflIII and BamHI and ligated to NcoI/BamHI-restricted baculovirus transfer vector pL546 (kindly provided by M. Pawlita, Heidelberg, Germany). The resulting plasmids were used to generate the recombinant baculoviruses agno1a-baculo and agno1b-baculo. Generation and amplification of recombinant baculoviruses was carried out using the BaculoGOLD kit (Pharmingen) and Sf9 cells as previously described (Pawlita et al., 1996
).
Transient expression of agnoprotein 1a and agnoprotein 1b in CE cells.
For transient expression in CE cells, the cDNAs of agnoprotein 1a and agnoprotein 1b were amplified as described above, but using primers 5' CCT TTG GAT CCC ATG TCT ACT C 3' and 5' CGG GGG ATC CTT AGC GAG CC 3'. The PCR products with 552 bp (agnoprotein 1a) and 360 bp (agnoprotein 1b) were digested with BamHI and ligated to the BamHI-restricted eukaryotic expression vector pSG5 (Stratagene), to generate plasmids agno1a-CE and agno1b-CE, respectively. Both plasmids were purified with the Qiagen Plasmid Midi kit (Qiagen) and used to transfect CE cells.
To determine the efficiency of transfection, the plasmid pGREEN LANTERN-1 (Life Technologies), which expresses the green fluorescent protein (GFP), was co-transfected in each reaction. The transfection mixture was prepared from 5 µg of the appropriate DNA, 1 µg of the GFP-expressing reporter plasmid, and 8 µl of the LipofectAMINE reagent (Life Technologies) in serum-free Dulbeccos modified Eagles medium. Pre-confluent monolayers of CE cells grown on glass coverslips were incubated for 2 h at 38 °C with the transfection mix. After this time, the transfection mix was removed and the cultures were covered with medium containing 5% foetal calf serum. Twenty-four hours after transfection, the percentage of fluorescent cells was determined by fluorescence microscopy, counting 200 cells in each experiment. The same cultures were used for immunoblotting and TUNEL assays.
Generation of an infectious APV full-length plasmid.
Cloning of genomic BFDV-1 DNA has been described recently (Johne & Müller, 1998 ). In the resulting plasmid, designated as pAPV, the viral genome had been cloned into pBluescript II SK(+) (Stratagene) via the BamHI site located within the coding region of VP1. As this led to a disruption of this gene, plasmid pAPV was digested with HpaI and Ecl136II. The resulting 3852 bp fragment was circularized, linearized using BamHI, and ligated with a 4981 bp pAPV BamHI fragment to generate plasmid pAPVinf, capable of expressing all viral genes.
Site-directed mutagenesis.
The megaprimer PCR technique (Barik, 1997 ) was used for site-directed mutagenesis at the initiation codon of agnoprotein 1a and agnoprotein 1b. The megaprimer was synthesized by PCR, with pAPVinf as template and primers 5' GCC CTG AGA TCT GAA CAT TTC ACT TGT AA 3' and 5' TGG AGT AGA CCA GCT GTT GCT AGG G 3' (mutated nucleotides underlined), and used in a second PCR, together with primer 5' GTG CAG ATC TAT AGC GAG CCG 3' and the same template. Both reactions were carried out with Pwo DNA polymerase (PeqLab). The PCR product was cleaved with Ecl136II and EheI. The resulting 670 bp cleavage product was ligated to an 8164 bp fragment of Ecl136II/EheI-digested pAPVinf, in order to generate pAPVinf.mut. DNA sequencing of the inserted PCR product revealed this plasmid to be identical to the APV full-length plasmid pAPVinf, with the exception that GAT at position 289291 was replaced by CTG. With this mutation the initiation codon ATG for agnoprotein 1a and agnoprotein 1b is exchanged for TGG.
Immunoblotting.
Expression of viral structural proteins and agnoproteins was demonstrated by immunoblotting with antibodies elicited in rabbits immunized with APV strain BFDV-1 particles (Stoll et al., 1993 ). The antibodies cross-react with the structural polypeptides of SV40 (Stoll et al., 1993
).
These antibodies can also be used for the detection of agnoprotein 1a and agnoprotein 1b, as these proteins have been observed in APV particles (R. Johne & H. Müller, unpublished data), in addition to the structural proteins VP1, VP2 and VP3 (Müller & Nitschke, 1986 ; Stoll et al., 1994
). Their reactivity with agnoprotein 1b, however, is low, as only large amounts of agnoprotein 1b expressed in Esherichia coli can be detected (R. Johne & H. Müller, unpublished data).
Antibodies directed against a -galactosidaselarge T antigen fusion protein of BFDV-1 (Stoll et al., 1993
) were used for the detection of large T antigen and small T antigen of APV.
DNA fragmentation analysis and TUNEL assay.
Fragmented DNA indicating apoptosis was demonstrated by electrophoresis of isolated DNA or in situ using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay (Gavrieli et al., 1992 ). For DNA isolation, cells were treated with proteinase K at various times after infection. Cellular DNA was extracted with phenolchloroform (Sambrook et al., 1989
), treated with RNase A (Pharmacia), and subjected to electrophoresis on ethidium bromide-stained 2% agarose gels. For the TUNEL assay, CE cells were fixed with acetone for 15 min and 2% paraformaldehyde for 15 min at 4 °C. This was followed by permeabilization with 0·1% Triton X-100 for 30 min at 4 °C. DNA fragmentation was analysed using the in situ Cell Death Detection kit (Boehringer Mannheim). The percentage of apoptotic cells was determined by counting 200 cells in each experiment and averaging the results of three tests.
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Results |
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Agnoprotein 1a and agnoprotein 1b induce apoptosis in Sf9 insect cells
Sf9 cells were infected with the recombinant baculoviruses agno1a-baculo and agno1b-baculo, respectively, at an m.o.i. of 10 p.f.u. per cell. Sf9 cells infected with either wild-type baculovirus or recombinant baculovirus expressing structural protein VP1 of APV (R. Johne & H. Müller, unpublished data) served as controls. Cytopathic changes observed in Sf9 cells infected with agno1a-baculo or agno1b-baculo were different from those observed in VP1-expressing and wild-type baculovirus-infected cells (not shown). Shrinkage and premature detachment of agno1a-baculo- and agno1b-baculo-infected cells from the surface were reminiscent of observations described for apoptotic cells. DNA fragmentation was not detected in cells infected with VP1-expressing baculovirus (Fig. 2A). Only weak DNA fragmentation was found in wild-type baculovirus-infected cells (Fig. 2C
). However, distinct DNA laddering was observed in cells 4 days p.i. with agno1a-baculo (Fig. 2B
) and 3 days p.i. with agno1b-baculo (Fig. 2D
).
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Further transfection experiments were performed to investigate induction of apoptosis by these plasmids. pBluescript II SK(+) was used as a negative control. Viral gene expression was determined by immunoblotting (data not shown) and induction of apoptosis was investigated by TUNEL, as described above, 4 days after transfection. As compared to CE cells transfected with pAPVinf.mut (Fig. 7A), nuclear fluorescence was observed in a considerably higher number of cells transfected with pAPVinf (Fig. 7B
) (10·8±2·8% and 29·3±3·8%, respectively). The lowest number of apoptotic cells was observed in cells transfected with the negative control (8·3±0·8%; Fig. 7C
). The transfection efficiency was 8·3±0·8 % in the case of pAPVinf.mut, 7·8±1·2% in the case of pAPVinf, and 7·8±0·8% in the case of the negative control.
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Discussion |
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After transfection of expression plasmids into CE cells, as well as after the infection of Sf9 cells with recombinant baculoviruses, agnoprotein 1b was not detected by immunoblotting, whereas agnoprotein 1a was regularly observed. As mentioned above, the antibodies used in this study only detect large amounts of agnoprotein 1b, for unknown reasons. As only low expression levels of agnoprotein 1a have been observed, it has to be taken into account that the amounts of agnoprotein 1b expressed in the experiments described are not sufficient to be detected by immunoblotting.
The data reported here demonstrate that these proteins induce apoptosis in Sf9 cells as well as in CE cells, the cell type commonly used for APV propagation. The amino acid sequence of agnoprotein 1b is identical to that of agnoprotein 1a, but with an internal deletion of amino acids 69 to 132 (Luo et al., 1995 ). Apoptosis is, therefore, induced by amino acids 1 to 68 and amino acids 133 to 176 of agnoprotein 1a. An extensive database search analysis with these sequences did not reveal significant similarities to currently known apoptotic inducers. Therefore, no conclusions can be drawn from the amino acid sequence as to the pathways by which apoptosis is induced. Transfection experiments employing cell lines with well-characterized mutations should provide some insight into these pathways.
As compared to plasmid agno1a-CE, apoptosis in CE cells was considerably stronger after transfection of plasmid agno1b-CE. When Sf9 cells were infected with agno1b-baculo, DNA fragmentation was observed about 24 h earlier as compared to infection with agno1a-baculo. The reasons for these differences are not known, but it may be speculated that amino acids 69 to 132, present in agnoprotein 1a and deleted in agnoprotein 1b, are involved. This region contains a potential nuclear localization signal (HRRRPYDR; Li, 1996 ) which might result in a decrease in apoptotic activity due to the transport of agnoprotein 1a to the nucleus.
There is increasing evidence that apoptosis plays a key role in the life-cycle of numerous viruses (Shen & Shenk, 1995 ; Teodoro & Branton, 1997
). It has been reported recently that apoptosis is induced in SV40 T antigen-transformed fibroblasts (Tsao et al., 1998
). To our knowledge, however, induction of apoptosis in the replicative cycle of mammalian polyomaviruses has not been described. In this study we observed no distinct DNA fragmentation in Vero cells infected with SV40, but instead degraded DNA, which is more indicative of necrotic than apoptotic processes. In contrast, apoptosis was clearly induced in APV-infected CE cells. It might be speculated that these conflicting observations could result from the different cell types used; they could, however, also be due to different properties of these viruses. To answer these questions, studies employing a broad range of different cell types are currently being performed.
Induction of apoptosis during APV infection correlates with the expression of agnoprotein 1a and agnoprotein 1b. Agnoproteins of the mammalian polyomaviruses SV40, BKV and JCV exhibit no obvious sequence similarities to agnoprotein 1a and agnoprotein 1b of APV. Various functions of SV40 agnoprotein have been discussed, such as effects on virus assembly (Margolskee & Nathans, 1983 ; Carswell & Alwine, 1986
), maturation (Hou-Jong et al., 1987
), regulation of transcription (Alwine, 1982
; Hay & Aloni, 1985
), and release of mature virus (Resnick & Shenk, 1986
). In SV40, agnoprotein expression is not essential for virus replication (Hou-Jong et al., 1987
; Resnick & Shenk, 1986
). In contrast, as shown here, expression of agnoprotein 1a and agnoprotein 1b seems to play an essential role in the life-cycle of APV, because no infectious progeny was produced after pAPVinf.mut transfection. Further experiments will have to be performed to get an insight into the mechanism(s) responsible for blocking APV replication.
At present it may be assumed that agnoproteins of APV and agnoproteins of mammalian polyomaviruses exert different biological functions during virus replication. One of these functions of agnoprotein 1a and agnoprotein 1b is obviously the induction of apoptosis. Induction of apoptosis is known to be a mechanism to destroy cells in the absence of an inflammatory response. It could be speculated that this mechanism also plays a role in pathogenicity of APV, e.g. by allowing efficient virus release from the nucleus and rapid virus spread through the organism.
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Acknowledgments |
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This publication is dedicated to Rudolf Rott on the occasion of his 75th birthday.
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References |
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Received 30 November 1999;
accepted 10 February 2000.