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Extrachromosomal deer fibromavirus DNA in deer fibromas and virus-transformed mouse cells
VIROLOGY
131,546-550(X983)
SHORT Extrachromosomal
COMMUNICATIONS
Deer Fibromavirus
and Virus-Transformed
DNA in Deer Fibromas Mouse
Cells
DENNIS E. GROFF,**~JOHN P. SUNDBERG,* AND WAYNE D. LANCASTER*? *Department of Obstetrics and Gynecdojry and fDcpartm.cnt of Pathokqy, Gwrgetuwn Univerdy Center, Washington, D. C. 2tWO7, and $Department of Ve’eten’nary Pathobim, College of Veten’mw-g Medicine, University of l&wis, Urbana, Ilkois 61801
Medical
The non-virus-producing fibromatous portions of five deer fibromas were examined for deer fibromavirus (DFV) DNA sequences. Liquid-phase hybridization revealed 100 to 330 copies per cell of the virus genome. Southern blot analysis of undigested deer tumor DNA preparations indicated that most of the DFV DNA was present as monomeric, unintegrated genomes; however, restriction enzyme digestion patterns suggest a small population of resistant DFV sequences. DYV DNA was also present in virus-transformed NIH/W3 mouse cells as multiple, extrachromosomal genomes.
Cutaneous fibromatosis of deer is caused by a papillomavirus. Shope first demonstrated that cell-free filtrates from a naturally occurring deer fibroma induced fibromatosis in experimental deer (1). In 1968, deer fibromavirus (DFV) was observed in keratinizing epidermal cells of the deer fibroma and classified a papillomavirus based on virion architecture, size, and nuclear site of maturation in epithelial cells (2). Recently, DFV was isolated from the infected epithelium of deer fibromas (8). The viral genome is a duplex supercoiled DNA molecule with a mass of about 5.5 X 10’ Da (8.3 kb). DYV DNA has two regions which show homology with bovine papillomavirus types 1 and 2 (RPV-1 and BPV-2) DNAs under stringent hybridization conditions. DFV DNA also shows homology with two discrete regions of human papillomavirus type 1 (HPV-1) DNA under hybridization conditions allowing up to 30% base mismatch; a feature common to all known papillomaviruses (4, ii). Papillomaviruses generally cause benign tumors in the host species. Most papillomaviruses induce proliferations only of the stratified or squamous epithelium (papillomas). Some papillomaviruscs, such as ’ To whom reprint
requests should be addressed.
w424X22/X3 $mHl Copyright Ail righta
BPV-1 and BPV-2, cause fibroblastic as well as epithelial proliferation (fibropapillomas) (for rcvicws see Refs. 6 and 7’). DFV is unique among papillomaviruses in that the neoplastic potential appears to be primarily associated with stimulation of fibroblasts (1). However, the overlying epithelium of the deer fibroma can be variably hypcrplastic (J. P. Sundbcrg, Ph.D. Thesis, University of Connecticut, 1981). Since DFV matures only in the keratinizing cpithelium overlying the fibroma, it was necessary to determine whether the fibroblastic proliferation was caused by DFV infection or was a proliferative reaction by the host to virus-infected epithelium. Therefore, we examined the nonvirus-producing fibromatous portions of deer fibromas for the presence of DFV DNA by liquid-phase and Southern blot hybridizations. The results indicated that multiple topics of cxtrachromosomal DFV DNA were present in naturally occurring tumors. We also detected extrachromosomal viral sequences in D&V-transformed NIH/3T3 mouse fibroblasts. These results suggest that fibrohlastic transformation in deer fibromas is a result of virus infection of those cells. DFV DNA was detected in four fibromas from white-tailed deer (Ortoctile~~ whyi-
0 1YK3 by Academic Press. Inc. of rcproductian in any form rcscrvcd.
546
SHORT
COMMUNICATIONS
nia.nus) and one from a mule deer (0. hewknus) by liquid-phase hybridization. Tissue was excised from the non-virusproducing fibroblastic layers of the tumors, sectioned on a cryostat, and homogenized in 7 M urea, 0.24 M sodium phosphate (pH 6.8), 1% SDS. Cellular DNA was purified on hydroxyapatite (8). The reassociation of nick-translated =P-labeled DFV DNA in the presence of cellular DNA was assayed on hydroxyapatite. Accelerated rates of reassociation were analyzed as described by Gelb et al. (9). All five deer tumors examined contained DFV DNA ranging from 100 to 330 genome copies per diploid cell (Table 1). DFV sequences in the deer fibromas were further characterized by Southern blot analysis (10). Cellular DNAs were electrophoresed in agarose gels undigested or cleaved with restriction enzymes that recognize one unique site in DFV DNA [BgZII, 0.47 map units (m.u.); EcoRI, 0.0 m.u. (3)],
TABLE
1
QUANTITATION OF DFV DNA SEQUENCES IN TUMORS AND TRAI~SFORMED CELLS
Cellular
DNA
Tumors White-tailed deer 1 2 3 4 Mule deer 5 Transformed cells D584 Control Reconstruction (9 copy)* Calf thymus DNA
DFV genomes/ diploid cell”
330 101 213 150
+ 10 f 9 r 28 I? 4
301
+ 18
3.8 -+ 0.4 9.1 ?z 0.4 0
a Assuming a mass of 5.5 X lo6 Da for DFV DNA and 3.9 X 10” Da for diploid mammalian cellular DNA. *Reconstruction contained 50 pg/ml calf thymus DNA, 6.4 X IO.-’ rg/ml DFV DNA, and 5.0 X lo-’ fig/ ml of nick-translated YzP-labeled DFV probe DNA.
,547
transferred to nitrocellulose filter, and probed with %P-labeled DFV DNA. Analysis of undigested cellular DNAs revealed that the majority of viral DNA sequences corn&rated with unit-length supercoiled (Fo I), open circular (Fo II), or linear (Fo III) DFV DNA extracted from virions (Fig. 1A). Cleavage of cellular DNA with &$I1 converted most of the DFV DNA sequences to unit-length linear molecules (Fig. lB), as did digestion with EcoRI (data not shown). A small fraction of DFV DNA migrating as Fo II molecules was always observed after digestion of cellular DNA with either BglII or EwRI. This may be due to incomplete digestion of the viral sequences or a resistant population of viral DNA molecules in the tumors. Two subtypes of DFV have been identified; subtype a infects mule deer and subtype b infects white-tailed deer (3). Hind111 digestion distinguishes between the DNAs of these two subtypes: subtype a has sites at 0.14, 0.46, and 0.84 mu.; subtype b has sites at 0.14, 0.46, and 0.53 m.u. To determine the subtype of the DFV sequences present in the fibromas, cellular DNAs were digested with Hind111 and analyzed by Southern blot hybridization (Fig. 1C). All four white-tailed deer fibromas showed the subtype b pattern with viral DNA fragments of 5.1,2.7, and 0.5 kb. The muIe deer fibroma contained sequences consistent with the subtype a pattern with viral DNA fragments of 3.1, 2.7, and 2.5 kb. A small amount of high-molecularweight viral DNA was observed in all undigested flbroma DNA preparations (Fig. 1A). Digestion of cellular DNAs with BamHI, which recognizes no site in DFV DNA, produced patterns identical to those observed in undigested tumor DNAs (data not shown). Since digestion with singlecut restriction enzymes yieIded monomeric forms of DFV DNA, it was concluded that the slow-migrating DFV sequences probably represent a complex of intertwined monomeric viral sequences or unintegrated multimeric genomes. Similar forms of viral DNA have been observed in BPV-1 tumor cells (11) and BPV-1 transformed mouse cells (11, ZZ).
548
SHORT
COMMUNICATIONS
FIG. 1. Blot. hybridization of 3”P-labe1ed DFV DNA to deer fibroma DNA. DNA (5 pgg).purified by standard procedures from the non-virus-producing portion of deer fibromas, was treated as described below, electrophoresed through 0.8% agarose gels in TPE buffer (36 mMTris, 30 mMsodium phosphate, 1 mMEDTA, pH 7.5) (16) plus ethidium bromide (0.5 fig/ml), transferred by blotting to nitrocellulose filters (/I/), hybridized according to Wahl et al (17) with DFV subtype a/pBR322 recombinant (pD72) DNA lab&d with 9 by nick translation (18), and exposed to X-ray film for 36-48 hr. (A) Undigested tumor cell DNA was electrophoresed at 30 V for 10 hr and depurinatcd before blotting. (B) Cellular DNA was digested with &AI and electrophorescd at 30 V for 10 hr. (C) Ccllular DNA was digested with Hind111 and elcctro-
The mule deer fibroma contained a second form of DFV DNA in addition to unitlength viral DNA. &$I1 digestion produced a distinct 12.4-kb fragment as well as genomic 8.3-kb DNA (Fig. 1B). Hind111 digestion of the mule deer fibroma DNA produced distinct bands of 5.0 and 1.6 kb in addition to the genomic 3.1-, 2.7-, and 2.5-kb bands (Fig. 1C). EcoRI digestion yielded a 4.1-kb fragment in addition to unit-length linear DFV DNA. These data are consistent with the interpretation that a subpopulation of circular viral DNA contains a 4.1-kb insert. It has been reported that deleted papillomavirus DNA can accumulate during natural infection. In one case of equine sarcoid, a tumor induced by BPV-1 or BPV-2 (IS’), a class of BPV-1 molecules deleted by about 9% was detected in addition to full-length RPV-1 DNA (1.4). The altered DFV DNA in the mule deer fibroma suggests that insertions in papillomavirus DNA may also be perpetuated during natural infection. Mouse cells transformed by the linearized 69% transforming fragment of BPV-1 DNA contain circularized BPV DNA which have undergone rearrangements (12). Some of these rearranged BPV molecules contain insertions of nonviral DNA at the site of circularization. Whether the rearranged DFV DNA in the mule deer fibroma contained inserted host DNA was not determined. DFV transforms mouse fibroblasts in cell culture (D. Lowy, personal communication). Infection of subconfluent cultures of NIH/3T3 mouse cells with CsCl-purified DFV (subtype a) virions produced morphologically distinct, multilayered foci on monolayers of normal cells within 14 days of infection. These cells had the charac-
phoresed at 15 V for 12 hr; sample 4 was electrophoresed separately at 30 V for 10 hr. Digestion was verified by monitoring cthidium bromide staining of HindHI-digested A-phage DNA added to each reaction. Lanes marked M are lo-copy reconstructions containing 71 pg DFV DNA and 5 rg calf thymus DNA. The positions of supercoiled (Fo I), open circular (Fo II), and linear (Fo III) DFV DNA are indicated. The positions for sample 4 in (C) are marked separately.
SHORT
teristics of transformed cells in that they grew in 1% fetal calf serum, formed colonies in 0.35% agar, and were tumorigenic in nude mice. The transformation was specifically inhibited by antisera raised against DFV virions. NIHMI’3 mouse fibroblasts transformed by DFV subtype a (D584 cells) contained viral sequences. Analysis of reassociation kinetics of D584 DNA indicated about four copies of DFV DNA per diploid cell (Table 1). Southern blot hybridization showed that Bun&HI, BQIIII, EcoRI, and Hind111 digestion of D584 DNA produced only those patterns expected from free monomeric circular DFV subtype a DNA (Fig. 2). The intensity of the DFV DNA in the undigested sample was less than expected. This was probably due to inefficient transfer of the supercoiled DNA from the gel. The
Foil Folll Fol
-
:#+ w
549
COMMUNICATIONS
lower intensity of the signals from the Hind111 digest was expected because the virus DNA had been cleaved into three fragments. Since the signal from the smallest Hind111 fragment shown in Fig. 2 would be equivalent to the signal produced by one genome copy per cell, this experiment would have detected less than one copy of integrated DFV DNA per cell. It was concluded that DFV DNA was exclusively extrachromosomal (within our limits of sensitivity) in D584 cells. The presence of intact extrachromosomal DFV genomes in the proliferating fibroblasts of deer fibromas and the fact that DFV can transform mouse fibroblasts suggests that the virus is responsible for the neoplastic state of the cells in fibromas. Although this report does not address the question of DFV gene activity in fibromas, histological similarities to the fibroblastic proliferation caused by BPV-1 in cattle, where viral gene transcription has been demonstrated (x), would also suggest a functional role of the DFV genome in fibroblastic transformation. The transcription of DFV DNA in virus-transformed mouse fibroblasts is currently under investigation.
- 8.3kb - 3.1 kb - 2.7kb 7 2.5kb
ACKNOWLEDGMENTS The authors thank A. Bennett Jenson for helpful discussions and Douglas R. Lowy for D584 DNA. This work was supported by Public Health Service Grants CA32663 and CA32638 from the National Cancer Institute. REFERENCES
FIG. 2. Blot hybridization of =P-labeled DFV DNA to cellular DNA from DFV-transformed NIH/3T3 mouse fibroblasts (D584 cells). Cellular DNA (15 pg) was digested with the indicated restriction enzyme, electrophoresed through a 0.8% agarose gel at 30 V for 10 hr, transferred to nitrocellulose, hybridized to =P-labeled pD72 DNA, and exposed for 8 days at -70’ with intensifying screens. The positions of supercoiled (Fo I), open circular (Fo II), and linear (Fo III) DFV DNA are indicated at the left; the positions of DFV DNA restriction enzyme digestion products are on the right.
I. SHOPE,
R. E., MANGOLD, R., MCNAMARA, L. G., K. R., J. EXJI. Med. 108.797-802
and DUMBELL, (1958). R TAJIMA,
M., GORDON, D. E., and OLSON, C.. Amer.
J. Vet Res 29, 11851194 (1958). 3. LANCASTER,
W. D., and SUNDUERG,
123,212-216
J. P., Vi?-&gg
(1982).
4 LAW, M.-F., LAXASTER, W. D., and HOWLEY, P. M., .I Vird 32, 199-207 (1979). 5. HOWLEY, P. M., LAW, M.-F., HELLMAN, C., ENGEI., L., Ar.o~so, M. C., TSREAL, M. A., Lowu, D. R., and LANNCA~~ER, W. D., In “Viruses in Naturally
550
6.
7. 8. 9. 10.
11.
SHORT
COMMUNICATIONS
Occurring Cancer” (M. Essex, G. Todaro, and H. zur Hausen, eds.), pp. 233-247. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980. HO~LEY, P. M., In “Viruses Associated with Human Cancer” (L. Phillips, ed.), pp. 253-306. Dekker, New York, 1982. LANCASTER, W. D., and OLSON, C., MimobioL Rev. 46.191-207 (1982). LANCASTER, W. D., OLSON, C., and MF,INKE, W., J Virvl. 17, 824831 (1976). GELB, L. D., KOHNE, D. E., and MAKTIX, M. A., J. Mol. Biol 57.129-145 (1971). SOIJTHERN, E. M., J. MoL BioL 98,503-517 (1975). LANCASTER, W. D., Virology 108.25-255 (1981).
1% LAW, M.-F., LOWY, D. R., DVORET~SKY, I., and HOWLEY, P. M., Pm. Natl Arid. Sci. USA 78, 2727-2731 (1981). 1s. LANCASTEK, W. D., TIN&EN, G. H., and OLSON, c., Jnteruirologu 11. m-233 (1979). 14 iiMTMAh7rl, E., MLXLER, H., and SAUER, G., J. Vim1 35, 962-964 (1980). 15. AMTMANN, E., and SAUEK, G., J. ViroL 43, 59-66 (1982). 11%LOENING, U. E., Bidwwt. .I. 113, 131-138 (1969). 17’. WAHL, G. M., STERN, M., and STARK, G. R., PIWC. NatL Acud Sci. USA 76.3683-3687 (1979). 18. RIGBY, P. W., DIECKMANN, M., RIIOUES, C., and BERG, P.. J. MoL Bid 113, 237-251 (1977).
131,546-550(X983)
SHORT Extrachromosomal
COMMUNICATIONS
Deer Fibromavirus
and Virus-Transformed
DNA in Deer Fibromas Mouse
Cells
DENNIS E. GROFF,**~JOHN P. SUNDBERG,* AND WAYNE D. LANCASTER*? *Department of Obstetrics and Gynecdojry and fDcpartm.cnt of Pathokqy, Gwrgetuwn Univerdy Center, Washington, D. C. 2tWO7, and $Department of Ve’eten’nary Pathobim, College of Veten’mw-g Medicine, University of l&wis, Urbana, Ilkois 61801
Medical
The non-virus-producing fibromatous portions of five deer fibromas were examined for deer fibromavirus (DFV) DNA sequences. Liquid-phase hybridization revealed 100 to 330 copies per cell of the virus genome. Southern blot analysis of undigested deer tumor DNA preparations indicated that most of the DFV DNA was present as monomeric, unintegrated genomes; however, restriction enzyme digestion patterns suggest a small population of resistant DFV sequences. DYV DNA was also present in virus-transformed NIH/W3 mouse cells as multiple, extrachromosomal genomes.
Cutaneous fibromatosis of deer is caused by a papillomavirus. Shope first demonstrated that cell-free filtrates from a naturally occurring deer fibroma induced fibromatosis in experimental deer (1). In 1968, deer fibromavirus (DFV) was observed in keratinizing epidermal cells of the deer fibroma and classified a papillomavirus based on virion architecture, size, and nuclear site of maturation in epithelial cells (2). Recently, DFV was isolated from the infected epithelium of deer fibromas (8). The viral genome is a duplex supercoiled DNA molecule with a mass of about 5.5 X 10’ Da (8.3 kb). DYV DNA has two regions which show homology with bovine papillomavirus types 1 and 2 (RPV-1 and BPV-2) DNAs under stringent hybridization conditions. DFV DNA also shows homology with two discrete regions of human papillomavirus type 1 (HPV-1) DNA under hybridization conditions allowing up to 30% base mismatch; a feature common to all known papillomaviruses (4, ii). Papillomaviruses generally cause benign tumors in the host species. Most papillomaviruses induce proliferations only of the stratified or squamous epithelium (papillomas). Some papillomaviruscs, such as ’ To whom reprint
requests should be addressed.
w424X22/X3 $mHl Copyright Ail righta
BPV-1 and BPV-2, cause fibroblastic as well as epithelial proliferation (fibropapillomas) (for rcvicws see Refs. 6 and 7’). DFV is unique among papillomaviruses in that the neoplastic potential appears to be primarily associated with stimulation of fibroblasts (1). However, the overlying epithelium of the deer fibroma can be variably hypcrplastic (J. P. Sundbcrg, Ph.D. Thesis, University of Connecticut, 1981). Since DFV matures only in the keratinizing cpithelium overlying the fibroma, it was necessary to determine whether the fibroblastic proliferation was caused by DFV infection or was a proliferative reaction by the host to virus-infected epithelium. Therefore, we examined the nonvirus-producing fibromatous portions of deer fibromas for the presence of DFV DNA by liquid-phase and Southern blot hybridizations. The results indicated that multiple topics of cxtrachromosomal DFV DNA were present in naturally occurring tumors. We also detected extrachromosomal viral sequences in D&V-transformed NIH/3T3 mouse fibroblasts. These results suggest that fibrohlastic transformation in deer fibromas is a result of virus infection of those cells. DFV DNA was detected in four fibromas from white-tailed deer (Ortoctile~~ whyi-
0 1YK3 by Academic Press. Inc. of rcproductian in any form rcscrvcd.
546
SHORT
COMMUNICATIONS
nia.nus) and one from a mule deer (0. hewknus) by liquid-phase hybridization. Tissue was excised from the non-virusproducing fibroblastic layers of the tumors, sectioned on a cryostat, and homogenized in 7 M urea, 0.24 M sodium phosphate (pH 6.8), 1% SDS. Cellular DNA was purified on hydroxyapatite (8). The reassociation of nick-translated =P-labeled DFV DNA in the presence of cellular DNA was assayed on hydroxyapatite. Accelerated rates of reassociation were analyzed as described by Gelb et al. (9). All five deer tumors examined contained DFV DNA ranging from 100 to 330 genome copies per diploid cell (Table 1). DFV sequences in the deer fibromas were further characterized by Southern blot analysis (10). Cellular DNAs were electrophoresed in agarose gels undigested or cleaved with restriction enzymes that recognize one unique site in DFV DNA [BgZII, 0.47 map units (m.u.); EcoRI, 0.0 m.u. (3)],
TABLE
1
QUANTITATION OF DFV DNA SEQUENCES IN TUMORS AND TRAI~SFORMED CELLS
Cellular
DNA
Tumors White-tailed deer 1 2 3 4 Mule deer 5 Transformed cells D584 Control Reconstruction (9 copy)* Calf thymus DNA
DFV genomes/ diploid cell”
330 101 213 150
+ 10 f 9 r 28 I? 4
301
+ 18
3.8 -+ 0.4 9.1 ?z 0.4 0
a Assuming a mass of 5.5 X lo6 Da for DFV DNA and 3.9 X 10” Da for diploid mammalian cellular DNA. *Reconstruction contained 50 pg/ml calf thymus DNA, 6.4 X IO.-’ rg/ml DFV DNA, and 5.0 X lo-’ fig/ ml of nick-translated YzP-labeled DFV probe DNA.
,547
transferred to nitrocellulose filter, and probed with %P-labeled DFV DNA. Analysis of undigested cellular DNAs revealed that the majority of viral DNA sequences corn&rated with unit-length supercoiled (Fo I), open circular (Fo II), or linear (Fo III) DFV DNA extracted from virions (Fig. 1A). Cleavage of cellular DNA with &$I1 converted most of the DFV DNA sequences to unit-length linear molecules (Fig. lB), as did digestion with EcoRI (data not shown). A small fraction of DFV DNA migrating as Fo II molecules was always observed after digestion of cellular DNA with either BglII or EwRI. This may be due to incomplete digestion of the viral sequences or a resistant population of viral DNA molecules in the tumors. Two subtypes of DFV have been identified; subtype a infects mule deer and subtype b infects white-tailed deer (3). Hind111 digestion distinguishes between the DNAs of these two subtypes: subtype a has sites at 0.14, 0.46, and 0.84 mu.; subtype b has sites at 0.14, 0.46, and 0.53 m.u. To determine the subtype of the DFV sequences present in the fibromas, cellular DNAs were digested with Hind111 and analyzed by Southern blot hybridization (Fig. 1C). All four white-tailed deer fibromas showed the subtype b pattern with viral DNA fragments of 5.1,2.7, and 0.5 kb. The muIe deer fibroma contained sequences consistent with the subtype a pattern with viral DNA fragments of 3.1, 2.7, and 2.5 kb. A small amount of high-molecularweight viral DNA was observed in all undigested flbroma DNA preparations (Fig. 1A). Digestion of cellular DNAs with BamHI, which recognizes no site in DFV DNA, produced patterns identical to those observed in undigested tumor DNAs (data not shown). Since digestion with singlecut restriction enzymes yieIded monomeric forms of DFV DNA, it was concluded that the slow-migrating DFV sequences probably represent a complex of intertwined monomeric viral sequences or unintegrated multimeric genomes. Similar forms of viral DNA have been observed in BPV-1 tumor cells (11) and BPV-1 transformed mouse cells (11, ZZ).
548
SHORT
COMMUNICATIONS
FIG. 1. Blot. hybridization of 3”P-labe1ed DFV DNA to deer fibroma DNA. DNA (5 pgg).purified by standard procedures from the non-virus-producing portion of deer fibromas, was treated as described below, electrophoresed through 0.8% agarose gels in TPE buffer (36 mMTris, 30 mMsodium phosphate, 1 mMEDTA, pH 7.5) (16) plus ethidium bromide (0.5 fig/ml), transferred by blotting to nitrocellulose filters (/I/), hybridized according to Wahl et al (17) with DFV subtype a/pBR322 recombinant (pD72) DNA lab&d with 9 by nick translation (18), and exposed to X-ray film for 36-48 hr. (A) Undigested tumor cell DNA was electrophoresed at 30 V for 10 hr and depurinatcd before blotting. (B) Cellular DNA was digested with &AI and electrophorescd at 30 V for 10 hr. (C) Ccllular DNA was digested with Hind111 and elcctro-
The mule deer fibroma contained a second form of DFV DNA in addition to unitlength viral DNA. &$I1 digestion produced a distinct 12.4-kb fragment as well as genomic 8.3-kb DNA (Fig. 1B). Hind111 digestion of the mule deer fibroma DNA produced distinct bands of 5.0 and 1.6 kb in addition to the genomic 3.1-, 2.7-, and 2.5-kb bands (Fig. 1C). EcoRI digestion yielded a 4.1-kb fragment in addition to unit-length linear DFV DNA. These data are consistent with the interpretation that a subpopulation of circular viral DNA contains a 4.1-kb insert. It has been reported that deleted papillomavirus DNA can accumulate during natural infection. In one case of equine sarcoid, a tumor induced by BPV-1 or BPV-2 (IS’), a class of BPV-1 molecules deleted by about 9% was detected in addition to full-length RPV-1 DNA (1.4). The altered DFV DNA in the mule deer fibroma suggests that insertions in papillomavirus DNA may also be perpetuated during natural infection. Mouse cells transformed by the linearized 69% transforming fragment of BPV-1 DNA contain circularized BPV DNA which have undergone rearrangements (12). Some of these rearranged BPV molecules contain insertions of nonviral DNA at the site of circularization. Whether the rearranged DFV DNA in the mule deer fibroma contained inserted host DNA was not determined. DFV transforms mouse fibroblasts in cell culture (D. Lowy, personal communication). Infection of subconfluent cultures of NIH/3T3 mouse cells with CsCl-purified DFV (subtype a) virions produced morphologically distinct, multilayered foci on monolayers of normal cells within 14 days of infection. These cells had the charac-
phoresed at 15 V for 12 hr; sample 4 was electrophoresed separately at 30 V for 10 hr. Digestion was verified by monitoring cthidium bromide staining of HindHI-digested A-phage DNA added to each reaction. Lanes marked M are lo-copy reconstructions containing 71 pg DFV DNA and 5 rg calf thymus DNA. The positions of supercoiled (Fo I), open circular (Fo II), and linear (Fo III) DFV DNA are indicated. The positions for sample 4 in (C) are marked separately.
SHORT
teristics of transformed cells in that they grew in 1% fetal calf serum, formed colonies in 0.35% agar, and were tumorigenic in nude mice. The transformation was specifically inhibited by antisera raised against DFV virions. NIHMI’3 mouse fibroblasts transformed by DFV subtype a (D584 cells) contained viral sequences. Analysis of reassociation kinetics of D584 DNA indicated about four copies of DFV DNA per diploid cell (Table 1). Southern blot hybridization showed that Bun&HI, BQIIII, EcoRI, and Hind111 digestion of D584 DNA produced only those patterns expected from free monomeric circular DFV subtype a DNA (Fig. 2). The intensity of the DFV DNA in the undigested sample was less than expected. This was probably due to inefficient transfer of the supercoiled DNA from the gel. The
Foil Folll Fol
-
:#+ w
549
COMMUNICATIONS
lower intensity of the signals from the Hind111 digest was expected because the virus DNA had been cleaved into three fragments. Since the signal from the smallest Hind111 fragment shown in Fig. 2 would be equivalent to the signal produced by one genome copy per cell, this experiment would have detected less than one copy of integrated DFV DNA per cell. It was concluded that DFV DNA was exclusively extrachromosomal (within our limits of sensitivity) in D584 cells. The presence of intact extrachromosomal DFV genomes in the proliferating fibroblasts of deer fibromas and the fact that DFV can transform mouse fibroblasts suggests that the virus is responsible for the neoplastic state of the cells in fibromas. Although this report does not address the question of DFV gene activity in fibromas, histological similarities to the fibroblastic proliferation caused by BPV-1 in cattle, where viral gene transcription has been demonstrated (x), would also suggest a functional role of the DFV genome in fibroblastic transformation. The transcription of DFV DNA in virus-transformed mouse fibroblasts is currently under investigation.
- 8.3kb - 3.1 kb - 2.7kb 7 2.5kb
ACKNOWLEDGMENTS The authors thank A. Bennett Jenson for helpful discussions and Douglas R. Lowy for D584 DNA. This work was supported by Public Health Service Grants CA32663 and CA32638 from the National Cancer Institute. REFERENCES
FIG. 2. Blot hybridization of =P-labeled DFV DNA to cellular DNA from DFV-transformed NIH/3T3 mouse fibroblasts (D584 cells). Cellular DNA (15 pg) was digested with the indicated restriction enzyme, electrophoresed through a 0.8% agarose gel at 30 V for 10 hr, transferred to nitrocellulose, hybridized to =P-labeled pD72 DNA, and exposed for 8 days at -70’ with intensifying screens. The positions of supercoiled (Fo I), open circular (Fo II), and linear (Fo III) DFV DNA are indicated at the left; the positions of DFV DNA restriction enzyme digestion products are on the right.
I. SHOPE,
R. E., MANGOLD, R., MCNAMARA, L. G., K. R., J. EXJI. Med. 108.797-802
and DUMBELL, (1958). R TAJIMA,
M., GORDON, D. E., and OLSON, C.. Amer.
J. Vet Res 29, 11851194 (1958). 3. LANCASTER,
W. D., and SUNDUERG,
123,212-216
J. P., Vi?-&gg
(1982).
4 LAW, M.-F., LAXASTER, W. D., and HOWLEY, P. M., .I Vird 32, 199-207 (1979). 5. HOWLEY, P. M., LAW, M.-F., HELLMAN, C., ENGEI., L., Ar.o~so, M. C., TSREAL, M. A., Lowu, D. R., and LANNCA~~ER, W. D., In “Viruses in Naturally
550
6.
7. 8. 9. 10.
11.
SHORT
COMMUNICATIONS
Occurring Cancer” (M. Essex, G. Todaro, and H. zur Hausen, eds.), pp. 233-247. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980. HO~LEY, P. M., In “Viruses Associated with Human Cancer” (L. Phillips, ed.), pp. 253-306. Dekker, New York, 1982. LANCASTER, W. D., and OLSON, C., MimobioL Rev. 46.191-207 (1982). LANCASTER, W. D., OLSON, C., and MF,INKE, W., J Virvl. 17, 824831 (1976). GELB, L. D., KOHNE, D. E., and MAKTIX, M. A., J. Mol. Biol 57.129-145 (1971). SOIJTHERN, E. M., J. MoL BioL 98,503-517 (1975). LANCASTER, W. D., Virology 108.25-255 (1981).
1% LAW, M.-F., LOWY, D. R., DVORET~SKY, I., and HOWLEY, P. M., Pm. Natl Arid. Sci. USA 78, 2727-2731 (1981). 1s. LANCASTEK, W. D., TIN&EN, G. H., and OLSON, c., Jnteruirologu 11. m-233 (1979). 14 iiMTMAh7rl, E., MLXLER, H., and SAUER, G., J. Vim1 35, 962-964 (1980). 15. AMTMANN, E., and SAUEK, G., J. ViroL 43, 59-66 (1982). 11%LOENING, U. E., Bidwwt. .I. 113, 131-138 (1969). 17’. WAHL, G. M., STERN, M., and STARK, G. R., PIWC. NatL Acud Sci. USA 76.3683-3687 (1979). 18. RIGBY, P. W., DIECKMANN, M., RIIOUES, C., and BERG, P.. J. MoL Bid 113, 237-251 (1977).