JOURNAL OF VIROLOGY, Jan. 1994, p. 346-351 Vol. 68, No. 10022-538X/94/$04.00+0Copyright © 1994, American Society for Microbiology

A Single Chicken Anemia Virus Protein Induces ApoptosisM. H. M. NOTEBORN,'* D. TODD,2 C. A. J. VERSCHUEREN,' H. W. F. M. DE GAUW,' W. L. CURRAN,2

S. VELDKAMP,3 A. J. DOUGLAS,2 M. S. McNULTY,3 A. J. VAN DER EB,' AND G. KOCH3Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, Leiden University, 2300 RA Leiden,' and Virology

Department, DLO-Central Veterinary Institute, 8200 AJ Lelystad, The Netherlands, and Veterinary Sciences Division,Stormont, Belfast BT4 3SD, Northern Ireland2

Received 19 July 1993/Accepted 19 October 1993

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformedmononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded proteinVP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 formsaggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. Byimmunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro,expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptibleto CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of aC-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.

Chicken anemia virus (CAV) transiently causes severe ane-mia due to destruction of erythroblastoid cells and immuno-deficiency due to depletion of cortical thymocytes in youngchickens (10, 38). Jeurissen et al. (11) have provided evidencethat the observed depletion of the thymocytes occurs viaCAV-induced apoptosis. Apoptosis is considered to be aphysiological process of cell depletion that is part of thehomeostatic regulation of normal tissues (5). CAV in additionto several other viruses, such as human immunodeficiency virustype 1 (2) and parvovirus B19 (19), seems to use apoptosis toexert its cytopathogenic effect.CAV is a small virus with a diameter of about 23 nm and

contains a circular single-stranded DNA of 2.3 kb (27). CAVmultiplies via a circular double-stranded DNA replicativeintermediate, which was recently cloned (18, 21). The clonedCAV genome was proven to be representative for CAVisolates collected worldwide (23, 29). A polycistronic polyade-nylated mRNA (22) which comprises three overlapping openreading frames encoding proteins VP1 (51.6 kDa), VP2 (24.0kDa), and VP3 (13.6 kDa) is transcribed from the CAVgenome.

In the present paper, we report studies on the expression ofthe VP3 protein in CAV-infected cells. VP3 is a putative CAVprotein of 121 amino acids and contains two proline-richstretches and two positively charged regions. So far, there is noevidence that VP3 is present in purified CAV particles (27).However, VP3 seems to be important for the virus life cycle, asfound by VP3-deletion studies (19a). By way of immunolabel-ing experiments with CAV-infected chicken lymphoblastoid Tcells in vitro, we present evidence that VP3 accumulates incondensed DNA. We also show that expression of VP3 aloneis sufficient to induce apoptosis in chicken mononuclear cells.

MATERIALS AND METHODSViruses and cells. The chicken lymphoblastoid T-cell line

MDCC-MSBI, transformed by Marek's disease virus (1), wasinfected with the CAV-Cux-1 isolate, which was originally

* Corresponding author. Mailing address: Laboratory for MolecularCarcinogenesis, Sylvius Laboratory, Leiden University, P.O. Box 9003,2300 RA Leiden, The Netherlands. Phone: (31) 71 276113. Fax: (31)71 276125.

isolated in Germany (34). For DNA transfections, we used thecell line MDCC-MSB1; the myeloid cell line LSCC-HD11transformed by avian myeloblastosis virus (4), which was kindlyprovided by T. Graf; and primary chicken embryo fibroblasts(CEFs).

Immunoblotting. The nuclear protein preparations thatwere used for immunoblotting were produced as follows.MDCC-MSB1 cells that had been infected at a multiplicity ofinfection of about 0.2 median tissue culture infective dose percell were collected by centrifugation at 48 h after infection, anda nucleus-rich pellet was obtained (32). Nuclear material thathad been resuspended in 10 mM Tris HCl-1 mM EDTA (pH8.0), dispersed by thorough sonication, and solubilized with 2%sodium dodecyl sulfate (SDS) was fractionated by gel filtrationthrough a column (45.1 by 5 cm) containing Sephacryl S200.Noninfected MDCC-MSB1 cells were similarly processed forcontrol purposes.

Proteins were separated by SDS-polyacrylamide gel electro-phoresis, electroblotted, and subjected to immunodetection asdescribed by Todd et al. (28). Blots were incubated with a1:10,000 dilution of an immunoglobulin fraction (approximate-ly 28.7 mg/ml), which had been produced by precipitation with(NH4)2SO4, of the CAV-specific monoclonal antibody (MAb)3B1 (17) and a 1:1,000 dilution of a peroxidase-labeled goatanti-mouse conjugate.Immunoperoxidase assay and immunofluorescence assay.

MDCC-MSB1 cells, which were synchronized by growing inmedium without leucine and fetal bovine serum, were infectedwith CAV at a multiplicity of infection of about 1 median tissueculture infective dose of virus per cell. The cells were harvestedat 0, 16, 24, 32, and 64 h after infection; spun on coverslips; andfixed with 100% acetone. Immunoperoxidase staining wascarried out with a 100-fold dilution of the MAb CVI-CAV-85.1(85.1) (21) and a 500-fold dilution of a peroxidase-labeledrabbit anti-mouse immunoglobulin G conjugate in phosphate-buffered saline, as described by Jeurissen et al. (9). Immuno-fluorescence assays were carried out on DNA-transfected cellsby using MAb CVI-CAV-85.1 as described by Noteborn et al.(20).Immunoelectron microscopy. MDCC-MSB1 cells were in-

fected with CAV at a multiplicity of infection of about 0.2median tissue culture infective dose of virus per cell. Theinfected and noninfected control cells were harvested 48 h

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APOPTOSIS INDUCED BY A SINGLE CAV PROTEIN 347

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FIG. 1. Immunoblot of nuclear protein fractions from noninfected(lane A) and CAV-infected (lane B) MDCC-MSBl cells. The blot wasinitially allowed to react with MAb 3B1, which is directed against VP3.

after infection. Thin-section electron microscopy and immu-nogold labeling were carried out as described by McNulty et al.(16). The grids were stained with a 10,000-fold dilution of theCAV-specific MAb 3B1 and a 1:50 dilution of 15-nm gold-labeled goat anti-mouse immunoglobulin G conjugate.

Construction of VP3 expression vectors. Plasmid DNAmanipulations were performed essentially according to themethods described by Maniatis et al. (14). The CAV sequences

originated from plasmid pCAV/E, described by Noteborn et al.(21).The sequences encoding VP3 were cloned in the expression

vector pRSV-H20, which contains the Rous sarcoma viruspromoter. From plasmid pEP-VP3 (19a) we isolated a 0.46-kbBamHI-EcoRI DNA fragment with CAV DNA sequencesfrom positions 427 to 868 (21). It contains the coding region ofVP3, a 58-bp 5'-flanking sequence, and a 25-bp 3-flankingsequence. Two synthetic DNA oligomers, 5'-GATCCAACCCGGGTFG-3' and 5'-AATTCAACCCGGGTTG-3', were hy-bridized to form a double-stranded BamHI-EcoRl DNAlinker. The vector pRSV-H20 was linearized with BglII andtreated with calf intestine alkaline phosphatase, after which a

4.3-kb fragment was isolated. The BamHI-EcoRI DNA linkerand the 0.46-kb BamHI-EcoRI DNA fragment were ligatedwith the 4.3-kb BglII DNA fragment. The final construct,pRSV-VP3, contains the VP3 coding region under the controlof the Rous sarcoma virus promoter as shown in Fig. 4 and was

analyzed by restriction enzyme digestions.A truncated VP3 product was made by deleting 11 codons at

the 3' terminus of the VP3 coding sequences and introducinga new stop codon. Plasmid pEP-VP3 was digested with BamHIand HindIlI. The 0.38-kb BamHI-HindIIl DNA fragment was

isolated. Two synthetic DNA oligomers, 5'-AGCTTGATTACCACTACTCCCTGAG-3' and 5'-TCGACTCAGGGAGTAGTGGTAATCA-3', were hybridized to form a double-stranded

64 64D : .

FIG. 2. Indirect immunoperoxidase staining of CAV-infectedMDCC-MSB1 cells with antibody 85.1 directed against VP3. The cellswere fixed and stained 0, 16, 24, 32, 40, 56, and 64 h after infection.

HindIII-SalI DNA linker. Plasmid pRSV-H20 was digestedwith BglII and Sall and treated with calf intestine alkalinephosphatase, and a 4.3-kb fragment was isolated. The HindIll-Sall DNA linker and the 0.38-kb BamHI-HindIII DNA frag-ment were ligated with the 4.3-kb BglII-SalI DNA fragment.The final construct, pRSV-tr, was analyzed by restrictionenzyme digestions and by sequencing of the newly introducedbases (see Fig. 4).DNA transfection and analysis of cellular DNA. Plasmid

DNA was purified by centrifugation in a CsCl gradient and bycolumn chromatography in Sephacryl S500 (Pharmacia). ForDNA transfections of MDCC-MSB1 and LSCC-HD11 cells,the DEAE-dextran method of Luthman and Magnusson (13),in which the chloroquine treatment was replaced with a

dimethyl sulfoxide boost, was used. CEFs were transfectedwith DNA according to the calcium phosphate precipitationmethod of Graham and Van der Eb (8). DNA extracted fromDNA-transfected MDCC-MSB1 cells was analyzed as de-scribed by Jeurissen et al. (11).

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348 NOTEBORN ET AL.

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FIG. 3. Immunogold labeling of electron-dense nuclear inclusionsin CAV-infected MDCC-MSB1 cells. Infected cells harvested 48 hafter infection were embedded in Lowicryl and allowed to react withMAb 3B1, which is directed against VP3. Magnification, x 26,000.

RESULTS

Expression of VP3 during CAV infection of cultured cells.We have analyzed the expression of VP3 during a CAVinfection of chicken lymphoblastoid T cells. The CAV-infectedcells were analyzed by immunoblotting and an immunoperoxi-dase assay using CAV-specific MAbs 3B1 (17) and 85.1 (21).Both MAbs are specific for CAV VP3, since these specificallybind to recombinant VP3 synthesized with a baculovirussystem (19a). Immunoblotting experiments showed that frac-tions of the nuclear extracts of CAV-infected cells that elutedclose to the void volume of the Sephacryl S200 columncontained a 16-kDa protein that specifically reacted with MAb3B1 (Fig. 1). Under the immunodetection conditions used,faint nonspecific staining of other proteins in the profilesderived from infected and noninfected cells was also observed.Expression of sequences encoding VP3 in a baculovirus systemyields a protein with an identical molecular mass (19a).

Immunoperoxidase staining of CAV-infected MDCC-MSB1cells with MAb 85.1 was carried out at several time points afterinfection (Fig. 2). As early as 24 h after infection, VP3 waspresent in the nucleus. The distribution of VP3 was very faintand finely granular. Later after infection, the granules in-creased in size, and gradually aggregates appeared. The aggre-gates were especially prominent around 60 h after infection. Atthis stage of infection, the CAV-specific cytopathogenic effectwas clearly visible.The aggregates containing VP3 resemble the electron-dense

structures described by Jeurissen et al. (11) that occur duringthe apoptotic process of CAV-infected cells. We have shownby immunogold electron microscopy that MAb 3B1 bindsstrongly to many, but not all, of the electron-dense apoptotic

1-M N A L Q E D T P P G P S T VF R P P T S S R P L E T P H CR E I R I G I A G I T I T L SL C G C A N A R A P T L R S AT A D N S E S T G F K N V P DL R T D Q P K P P S K K R S CD P S E Y R V S E L K E S L IT T T P S R P R T A K R R I RL-1 21

FIG. 4. (A) Schematic representation of the expression vectorspRSV-VP3 and pRSV-tr. Both vectors are constructed as described inMaterials and Methods. The CAV sequence encoding VP3 is drawn asa filled box, that for VP3-tr is drawn as a striped box, the Rous sarcomavirus promoter region is drawn as a dotted box, and the simian virus 40sequences, containing a poly(A) addition site, is drawn as an open box.(B) Amino acid sequence of VP3. The 11 amino acids which weredeleted from the C terminus of the truncated form of VP3 areunderlined. The proline residues are in italics, and the lysine andarginine residues are in boldface type.

structures in the nucleus of CAV-infected cells (Fig. 3).Apparently, VP3 accumulates in these apoptotic structures.VP3 induces apoptosis in cultured avian mononuclear cells.

To establish whether VP3 alone is able to induce apoptosis, theexpression vector pRSV-VP3 was constructed (Fig. 4). Thisvector contains CAV DNA sequences encoding only VP3.After transfection with DNA of pRSV-VP3, VP3 was tran-siently expressed in cultured chicken lymphoblastoid T cells.The cells were harvested after 48 h, acetone fixed, and stainedwith MAb 85.1. In addition, the cells were treated withpropidium iodide (PI), which is known to stain intact nucleistrongly but apoptotic nuclei relatively weakly (26). CAV-infected cells containing apoptotic inclusion structures, whichwere comparable to the structures observed at 64 h afterinfection (Fig. 2), were weakly and irregularly stained by PI(data not shown). We observed 60% of the transfected cellswith nuclei exhibiting a very finely granular distribution ofVP3. The DNA of cells with this type of VP3 staining wasalways clearly stained by PI. In contrast, 40% of VP3-positivecells contained aggregates of VP3, and their DNA invariablystained weakly and irregularly with PI. Three days aftertransfection, almost all VP3-positive cells contain VP3 aggre-gates and negatively PI-stained DNA (Fig. 5). In contrast,expression of a C-terminally truncated VP3 (Fig. 4) yieldedalmost no apoptosis in MDCC-MSB1 cells. Three days aftertransfection, 80 to 90% of the cells expressing truncated VP3

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APOPTOSIS INDUCED BY A SINGLE CAV PROTEIN 349

A B m 1 2 3 4

C D

E F

FIG. 5. Indirect immunofluorescence of MDCC-MSB1 cells trans-fected with plasmid pRSV-VP3 (A to D) or pRSV-tr (E and F). Thecells were harvested 48 h (A and B) or 72 h (C to F) after transfection,fixed, and stained either with MAb 85.1 directed against VP3 (A, C,and E), or with PI (B, D, and F).

still have normally PI-stained nuclei (Fig. 5). At this time point,the cellular DNA of MDCC-MSB1 cells expressing VP3showed the apoptosis-specific nucleosomal laddering. Thisladdering was proven to be much less in DNA from cellstransfected with plasmid pRSV-tr or absent in DNA fromMDCC-MSB1 cells transfected with plasmid pRSV-CAT,which expressed the chloramphenicol transferase gene (Fig. 6).We conclude that expression of VP3 alone can induce theapoptotic process that is observed during CAV infection andresults in cell death.To test whether VP3 can also induce apoptosis in other

chicken cell types, it was expressed in the myeloid cell lineLSCC-HD11 and in primary CEFs. LSCC-HD11 is susceptibleto CAV, whereas CEFs are not. Transfection of DNA encod-ing VP3 in LSCC-HD1 1 cells yielded the formation of VP3aggregates and weak and irregular staining of the cellular DNAby PI. CEFs expressing VP3 revealed only granular staining ofVP3 and strong staining with PI (Fig. 7). This suggests thatVP3 can induce apoptosis in LSCC-HD11 cells but not inCEFs, or at least to a much lesser extent.

slot-

FIG. 6. DNA fragmentation in MDCC-MSBI cells undergoingVP3-mediated cell death. DNAs from CAV-infected MDCC-MSBIcells which were harvested 72 h after infection (lane 1) and fromMDCC-MSB1 cells transfected with DNA of plasmids pRSV-VP3(lane 2), pRSV-tr (lane 3), and pRSV-CAT (lane 4) were analyzed ona 1% agarose-ethidium bromide gel. Lane m, DNA markers (lambda-HindIll fragments; Promega). About 10% of the MDCC-MSBI cellsexpressed VP3 or VP3-tr protein, as determined by immunofluores-cence (data not shown).

DISCUSSION

CAV induces cell death of chicken lymphoblastoid T cellsvia apoptosis (11). By use of immunoassays and DNA analysis,we have established that the putative open reading frame forVP3 of the CAV genome (18, 21) is indeed expressed inCAV-infected cells. Early after infection, VP3 is colocalizedwith cellular DNA. Later after infection, VP3 is bound tonuclear aggregates that are known to be apoptotic bodies (5,1 1). Expression of only VP3 in MDCC-MSB1 cells mimics theprocess of CAV-induced apoptosis. In general, apoptosis is aprocess leading to cell death that occurs rapidly (36). Themajority of CAV-infected MDCC-MSB1 cells become apop-totic within 64 h after infection. In vitro expression of VP3 intransfected cells causes CAV-like apoptosis within 3 days.These data suggest that VP3 alone can trigger the apoptoticpathway in CAV-infected cells.How does the expression of VP3 cause apoptosis? In

general, apoptosis can be induced by a variety of viral andnonviral external stimuli, which can converge on a commonintracellular pathway, resulting in the activation of an endonu-clease (15). The amino acid sequence of VP3 does not showany distinct homology with the sequences of the receptors thatmediate apoptosis (6, 24, 25, 35) or of c-myc and p53 oncogeneproducts (7, 37), known to induce apoptosis. So, it is unlikelythat VP3 mimics the activity of one of these cellular proteins.Further experiments have to be carried out to prove whetherVP3 causes apoptosis by inhibition of the antiapoptosis activityof the oncogene product Bcl-2 (33). We also cannot excludethe possibility that VP3 induces apoptosis by binding to one of

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350 NOTEBORN ETAL.J.Vto.

A B

E F

FIG. 7. Indirect immunofluorescence of LSCC-HDI I cells and

CEFs which were transfected with plasmid pRSV-VP3, which encodes

VP3. The LSCC-HDII cells (A to D) were harvested 48 h after

transfection, and the CEFs (E and F) were harvested 72 h after

transfection. The cells were fixed and stained with MAb 85.1 directed

against VP3 (A, C, and E) and with PI (B3, D, and F).

the membrane-bound receptors. However, this seems to be

unlikely, as VP3 is located in the nucleus. VP3 could induce

apoptosis by regulating genes involved in apoptosis either

directly, via the induction of endonucleases, or indirectly, via

the induction of, e.g., the oncogene c-myc or p53 or repressionof the putative oncogene bcl-2.

The results of the immunoassays revealed that VP3 is strictlylocated within the cellular chromatin structures. The basic

character of VP3 and the C-terminal region in particular mightbe the reason that VP3 has binding properties to the cellular

DNA. The results obtained with the VP3-tr mutant showed

that deletion of the C-terminal basic region significantly re-

duced the apoptotic activity of VP3. Does VP3 act as an

endonuclease? Findings with CEFs argue against this idea, and

computer analyses did not reveal any sequence homology of

VP3 with one of the known endonucleases.

The small size of VP3 and its rather basic character may

allow it to interact with histone and/or nonhistone proteinswithin the chromatin structure. Thus, the presence of VP3 in

the chromatin structure may result in a breakdown of the

supercoiling organization. Eventually, these modifications of

the chromatin structure may lead to fragmentation and con-

densation of the cellular DNA. A disturbance of the supercoil-ing organization by VP3 might be caused by its high prolinecontent. Induction of apoptosis by disruption of the superchro-matin structure was described for apoptotic rat thymocytes(31) and rat ventral prostate cells after castration (12). Analternative explanation might be that VP3 interacts directlywith the histone protein HI, which binds to the intranucleo-somal DNA. For instance, binding of VP3 to this regioninstead of histone HI might enable endonucleases to digest theintranucleosomal DNA. Arends et al. (3) have observed thatthe oligonucleosomes become depleted of histone HI duringthe apoptotic process.Under our experimental conditions, the expression of VP3

in CEFs and cells of the human epithelia] cell line HeLa (datanot shown) did not result in the observed disruption of thecellular DNA. However, neither cell line is susceptible toCAy, in contrast to MDCC-MSBi and HDll cells. It mightwell be that VP3 can cause degradation of cellular DNA onlyin cells that are susceptible to CAy. An alternative explanationmight be that VP3 induces a cascade of events, resulting insingle-strand modification, i.e., the generation of alkaline-sensitive sites in the cellular DNA. Apparently, in lymphocytesand monocytes these modifications lead to double-strandedcleavage, whereas HeLa cells and CEFs remain arrested at thestage of single-strand modification. This would account for theobservation that lymphocytes and monocytes fragment theirDNA within hours after treatment with VP3. This explanationwas offered by Tomei (30) for the glucocorticoid-inducedapoptosis observed in lymphocytes but not in fibroblasts andHeLa cells.We conclude that a single CAV protein can induce apoptosis

in chicken lymphoblastoid T and myeloid cells. Experimentsthat will unravel the mechanism by which VP3 induces celldeath are under way.

ACKNOWLEDGMENTS

We thank Hans van Ormondt for critically reading the manuscript,Alt Zantema for helpful discussions, and Frans van Bussel for hisexcellent drawing work.

This research was made possible partially with research grants fromThe Netherlands Ministry of Economy Affairs and Aesculaap By,Boxtel, The Netherlands.

REFERENCES1. Akiyama, Y., and S. Kato. 1974. Two cell lines from lymphomas of

Mareks disease. Biken J. 17:105-117.2. Ameisen, J. C., and A. Capron. 1991. Cell dysfunction and

depletion in AIDS: the programmed cell death hypothesis. Immu-nol. Today 12:102-105.

3. Arends, M. J., R. G. Morris, and A. H. Wyllie. 1990. Apoptosis: therole of the endonucleases. Am. J. Pathol. 136:593-608.

4. Beug, H., A. Von Kirkbach, G. Doiderlein, J. F. Conscience, and T.Graf. 1979. Chicken hematopoietic cells transformed by sevenstrains of defective avian leukemia viruses display three differentphenotypes of differentiation. Cell 18:375-390.

5. Cohen, J. J. 1993. Apoptosis. Immunol. Today 14:126-130.6. Ellis, R. E., J. Yuan, and H. R. Horvitz. 1991. Mechanisms and

functions of cell death. Annu. Rev. Cell Biol. 7:663-698.7. Evan, G. I., A. H. Wyllie, C. S. Gilbert, T. D. Littlewood, H. Land,

M. Brooks, C. M. Waters, L. Z. Penn, and D. C. Hancock. 1992.Induction of apoptosis in fibroblasts by c-myc protein. Cell 69:119-128.

8. Graham, F. L., and A. J. Van der Eb. 1973. A new technique forthe assay of infectivity of human adenovirus 5 DNA. Virology52:456-467.

9. Jeurissen, S. H. M., E. M. Janse, S. Ekino, P. Nieuwenhuis, G.Koch, and G. F. de Boer. 1988. Monoclonal antibodies as probesfor defining cellular subsets in the bone marrow, thymus, bursa of

J. VIROL.

on February 7, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

APOPTOSIS INDUCED BY A SINGLE CAV PROTEIN 351

Fabricius, and spleen of the chicken. Vet. Immunol. Immuno-pathol. 19:225-238.

10. Jeurissen, S. H. M., J. M. A. Pol, and G. F. de Boer. 1989.Transient depletion of cortical thymocytes induced by chickenanaemia agent. Thymus 14:115-123.

11. Jeurissen, S. H. M., F. Wagenaar, J. M. A. Pol, A. J. van der Eb,and M. H. M. Noteborn. 1992. Chicken anemia virus causesapoptosis of thymocytes after in vivo infection and of cell linesafter in vitro infection. J. Virol. 66:7383-7388.

12. Kyprianou, N., and J. T. Isaacs. 1988. Activation of programmedcell death in the rat ventral prostate after castration. Endocrinol-ogy 122:552-662.

13. Luthman, H., and G. Magnusson. 1983. High efficiency polyomaDNA transfection of chloroquine treated cells. Nucleic Acids Res.11:1295-1308.

14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

15. Martz, E., and D. M. Howell. 1989. CTL: virus control cells firstand cytolytic cells second? Immunol. Today 10:79-86.

16. McNulty, M. S., T. J. Connor, F. McNeilly, M. F. McLoughlin, andK. S. Kirkpatrick. 1990. Preliminary characterization of isolates ofchicken anemia agent from the United Kingdom. Avian Pathol.19:67-73.

17. McNulty, M. S., W. L. Curran, D. Todd, and D. P. Mackie. 1990.Chicken anemia agent: an electron microscopic study. Avian Dis.34:736-743.

18. Meehan, B. M., D. Todd, J. L. Creelan, J. A. P. Earle, E. M. Hoey,and M. S. McNulty. 1992. Characterization of viral DNAs fromcells infected with chicken anemia agent: sequence analysis of thecloned replicative form and transfection capabilities of clonedgenome fragments. Arch. Virol. 124:301-309.

19. Morey, A. L., D. J. P. Ferguson, and K. A. Fleming. 1993.Ultrastructural features of fetal erythroid precursors infected withparvovirus B19 in vitro: evidence of cell death by apoptosis. J.Pathol. 169:213-220.

19a.Noteborn, M. H. M. Unpublished results.20. Noteborn, M. H. M., G. F. de Boer, A. Kant, G. Koch, J. L. Bos, A.

Zantema, and A. J. van der Eb. 1990. Expression of avianleukemia virus env-gp85 in Spodoptera frugiperda cells by use ofa baculovirus expression vector. J. Gen. Virol. 71:2641-2648.

21. Noteborn, M. H. M., G. F. de Boer, D. J. Van Roozelaar, C.Karreman, 0. Kranenburg, J. G. Vos, S. H. M. Jeurissen, R. C.Hoeben, A. Zantema, G. Koch, H. van Ormondt, and A. J. van derEb. 1991. Characterization of cloned chicken anemia virus DNAthat contains all elements for the infectious replication cycle. J.Virol. 65:3131-3139.

22. Noteborn, M. H. M., 0. Kranenburg, A. Zantema, G. Koch, G. F.de Boer, and A. J. van der Eb. 1992. Transcription of the chickenanemia virus (CAV) genome and synthesis of its 52-kDa protein.Gene 118:267-271.

23. Noteborn, M. H. M., C. A. J. Verschueren, D. J. van Roozelaar, S.Veldkamp, A. J. van der Eb, and G. F. de Boer. 1992. Detection of

chicken anemia virus by DNA hybridization and polymerase chainreaction. Avian Pathol. 21:107-118.

24. Pinching, A. J., and K. E. Nye. 1990. Defective signal transduc-tion-a common pathway for cellular dysfunction in HIV infec-tion? Immunol. Today 11:256-259.

25. Smith, C. A., G. T. Williams, R. Kingston, E. J. Jenkinson, andJ. J. T. Owen. 1989. Antibodies to CD3/TCR complex inducedeath by apoptosis in immature T cells in thymic culture. Nature(London) 337:181-183.

26. Telford, W. G., L. E. King, and P. J. Fraker. 1992. Comparativeevaluation of several DNA binding dyes in the detection ofapoptosis-associated chromatin degradation by flow cytometry.Cytometry 13:137-143.

27. Todd, D., J. L. Creelan, D. P. Mackie, F. Rixon, and M. S.McNulty. 1990. Purification and biochemical characterisation ofchicken anaemia agent. J. Gen. Virol. 71:819-823.

28. Todd, D., J. Hull, and J. McNair. 1987. Antigenically importantproteins of Aujeszky's disease (pseudorabies) virus identified byimmunoblotting. Arch. Virol. 96:215-224.

29. Todd, D., K. A. Mawhinney, and M. S. McNulty. 1992. Detectionand differentiation of chicken anemia virus isolates by using thepolymerase chain reaction. J. Clin. Microbiol. 30:1661-1666.

30. Tomei, L. D. 1991. Apoptosis: a program for death or survival, p.279. In L. D. Tomei and F. 0. Cope (ed.), Apoptosis: themolecular basis of cell death. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

31. Umansky, S. R., B. A. Korol, and P. A. Nelipovich. 1981. In vivoDNA degradation in thymocytes of gamma-irradiated or hydro-cortisone-treated rats. Biochim. Biophys. Acta 655:9-17.

32. Van Bleck, G. M., and S. G. Natheson. 1990. Isolation of anendogenously processed immunodominant viral peptide from theclass I H-2K molecule. Nature (London) 348:213-216.

33. Vaux, D. L., H. L. Aguila, and I. L. Weissmann. 1992. Bcl-2prevents death of deprived cells but fails to prevent apoptosis intargets of cell-mediated killing. Int. Immunol. 4:821-824.

34. Von Bulow, V., B. Fuchs, B. Viclitz, and H. Landgraf. 1983.Fruhsterblichkeitssyndrom bei Kuken nach Doppelinfektion mitdem Virus des Marekschen Krankheit (MDV) und einen Anamie-Erreger (CAA). J. Vet. Med. B 30:742-750.

35. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A.Jenkins, and S. Nagata. 1992. Lymphoproliferative disorder inmice explained by defects in Fas antigen that mediates apoptosis.Nature (London) 356:314-317.

36. Wyllie, A. H., J. F. R. Kerr, and A. R. Currie. 1980. Cell death: thesignificance of apoptosis. Int. Rev. Cytol. 68:251-306.

37. Yonish-Rouach, E., D. Resnitzky, J. Lottem, L. Sachs, A. Kimchi,and M. Oren. 1991. Wild-type p53 induces apoptosis of myeloidleukaemic cells that is inhibited by interleukin-6. Nature (London)352:345-347.

38. Yuasa, N., T. Taniguchi, and I. Yoshida. 1979. Isolation and someproperties of an agent inducing anaemia in chicks. Avian Dis.23:366-385.

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