Yaba virus-specific DNA in the host cell nucleus

59

Biochimica et Biophysica Acta, 478 (1977) 59--67 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 8 9 9 0

YABA VIRUS-SPECIFIC DNA IN THE HOST CELL NUCLEUS

J E R R Y L. T A Y L O R

and H. R O U H A N D E H

*

Laboratory of Molecular and Cancer Virology, Department of Microbiology, Southern Illinois University,Carbondale, III.62901 (U.S.A.) (Received January 21st, 1977) (Revised manuscript received May 9th, 1977)

Summary DNA-DNA hybridization studies show that Yaba virus-specific DNA is present in the host cell nucleus late in the infection cycle. The nuclear DNA appears to exist as a complete genome, not covalently linked to host cell DNA, as demonstrated by sedimentation analyses. The DNA appears to be synthesized in the nucleus, since its level of incorporation of label is ten times the background incorporation detectable in the cytoplasm. Extraction of the nuclei by treatment with SDS and EDTA after precipitation with 1 M NaC1 separates most of cellular DNA from the Yaba virus-specific DNA.

Introduction Poxviruses have been characterized as a family of large, DNA-containing viruses which replicate in the cytoplasm of the host cell. Pennington and Follet [I] studied vaccinia virus replication in cells enucleated by cytochlasin B treatment. They found that complete virus particles are not produced in these cells. Other studies have demonstrated the presence of poxvirus-specific D N A in the host cell nucleus [2--4]. These studies indicate that poxvirus replication depends upon some involvement with host cell nucleus. The purpose of this study is to determine whether Yaba virus D N A is present in the nuclei of infected host cellsand to characterize the nature of this D N A . Materials and Methods Cells. Jinet cells [5], a continuous line of cynomolgus monkey kidney cells, and CV-1 [6], a continuous line of cercopithecus monkey kidney cells, were

* To whom reprint requests should be addressed.

60 used for these studies. Cells were grown in Modified Eagles Medium with Earle's salts, and 5% fetal calf serum. Virus. Yaba virus was propagated at 35°C in roller bottle culture and purified by the m e t h o d of Joklik [7]. Virus titier was estimated from absorbance readings on purified virus with one A~60 unit representing 1.2 • 10 ~° particles or 2.4 • 107 focus forming units [8]. Extraction and purification of DNA. Yaba virus DNA was extracted from purified virions b y the m e t h o d of Oda and Joklik [9]. DNA from cells or cellular fractions was extracted b y the method of Fuginaga et al. [10]. Labeling and fractionation of cells. Cells were infected with Yaba virus at 3 focus forming units/cell. At various times after infection as indicated in each experiment, cultures were pulse-labeled with 0.5 to 1 pCi/ml of [3H]thymidine (57 Ci/mol) for the indicated period o f time. Cells were fractionated by either of t w o procedures, the double detergent treatment of Penman et al. [11], or the citric acid m e t h o d of Knowler et al. [12]. For the double detergent treatment, cells were resuspended in extraction buffer (0.01 M NaC1/0.01 M Tris, pH 7.4/0.02 M MgC12) with 0.5% Nonidet-P40 (NP40). The cells were placed in an ice bath for 15 min, then the nuclei were pelleted b y centrifugation at 250 X g for 10 min. The pellet was resuspended in reticulocyte swelling buffer (RS buffer) (0.01 M NaC1/0.01 M Tris, pH 7.4/0.0015 M MgC12) and 0.15 ml of 10% Tween 80110% NP40 (2 : 1) was added for each ml of RS buffer. The nuclear suspension was vortexed at t o p speed for 4 s and nuclei were again pelleted by centrifugation. The cytoplasmic fractions were combined. Alternately, the citric acid m e t h o d of Knowler et al. [ 12] was used. Cells were resuspended in 10 vols. of 2.5% (w/w) citric acid and disrupted by 20 strokes with a Dounce homogenizer. The nuclei were pelleted and resuspended in 10 vols. of 0.25 M sucrose in 1.5% (w/w) citric acid. The suspension was layered on 2 vols. of 0.88 M sucrose in 1.5% citric acid and the nuclei pelleted by centrifugation at 250 × g for 10 min. Preparations were stained with acridine orange and examined b y fluorescent microscopy. Preparations prepared by these methods showed no cytoplasmic material clinging to the clean nuclear preparations. The nuclear fraction, in some experiments, was further fractionated by treatm e n t with 0.6% SDS and 0.01 M EDTA and precipitation with I M NaC1. [13,14]. DNA-DNA hybridization. The m e t h o d of hybridization was as described by Green et al. [15], using membrane filters. Sedimentation analysis and equilibrium centrifugation of DNA. Yaba virusinfected cells were pulse-labeled for 1 h with 1 pCi/ml [3H]thymidine, immediately after infection and at 48 h after infection. Total nuclear DNA was extracted and treated as described by Jungwirth and Dawid [16]. The DNA was denatured b y boiling for 5 min. CsC1 was added to the DNA to a final density of 1.71 g/cm 3. After incubation for 12 h at 60°C, DNA was centrifuged to equilibrium at 3 5 0 0 0 rev./min for 60 h at 25°C in a Beckman Type 40 rotor. The gradients were fractionated, then precipitated with trichloroacetic acid. The precipitate was filtered o n t o membrane filters and the radioactivity was counted. Sedimentation of 1 M NaCl supernatant nuclear DNA from Yaba virus-infected cells was examined. Infected cells were labeled at 1 gCi/ml [3H]thymi-

61

dine from 50 to 70 h after infection. DNA was extracted and adjusted to a density of 1.73 g/cm 3 with CsC1. Gradients were centrifuged at 35000 rev./min for 6 6 h at 20°C in an SW50.1 rotor. Fractions were collected and incorporation of label was assayed as above. Purified Yaba virus DNA, cellular DNA, and 1 M NaC1 supernatant DNA of Yaba virus-infected cell nuclei were centrifuged in 5--20% (w/w) alkaline sucrose gradients (in 0.1 M NaOH/0.9 M NaC1/0.001 M EDTA) at 39000 rev./ min for 1.5 h at 10°C in an SW50.1 rotor. Fractions were collected as described above and examined for DNA content by the method of Burton [17]. Samples were treated with trichloroacetic acid and the precipitate was collected on cellulose nitrate filters and analysed in a liquid scintillation counter for incorporated radioactivity. Results

Hybridization o f nuclear DNA from Yaba virus-infected cells with Yaba virus DNA Hybridization experiments were performed to determine homology between Yaba virus DNA and whole nuclear DNA from Yaba virus-infected cells. The nuclear DNA was labeled with [3H]thymidine from 36 to 48 h after infection and purified (specific activity 8490 cpm/pg). When this DNA was hybridized to purified Yaba virus DNA, results indicate (Fig. 1) that a maximum of 1.1% of the infected nuclear DNA is homologous with Yaba virus DNA. Hybridization between labeled Yaba virus DNA and uninfected cell DNA did not occur at levels above those of nonspecific binding, indicating the absence of homology between Yaba virus DNA and normal cellular DNA. Examination o f Yaba virus-specific nuclear DNA Studies were conducted to determine whether or not the virus-specific DNA in the nucleus was covalently linked to cell DNA. Cultures were pulse-labeled with [3H]thymidine for 1 h immediately after infection or late in infection (48 h after infection). DNA was extracted from nuclei isolated by the citric

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F i g . 1 . H y b r i d i z a t i o n o f Y a b a v i r u s - i n f e c t e d cell n u c l e a r D N A w i t h Y a b a v i r u s D N A . I n f e c t e d eells w e r e l a b e l e d w i t h 1 p C i / m l [ 3 H ] t h y m i d l n e f r o m 3 6 t o 4 8 h a f t e r i n f e c t i o n . Ceils w e r e f r a c t i o n a t e d b y t r e a t m e n t w i t h 0 . 5 % N P 4 0 a n d a c o m b i n a t i o n o f N P 4 0 and T w e e n 8 0 . D N A w a s e x t r a c t e d f r o m n u c l e i and 5 ~g samples (~eciflc aet/vity 8490 epm/pg) were hybridized to inereas/nl amounts of Yaba virus DNA o n m e m b r a n e filters.

62

acid m e t h o d of Knowler et al. [ 12]. This DNA was treated by the method oI' Jungwirth and Dawid [16] to separate cellular and viral DNA by density. These workers demonstrated that vaccinia virus DNA renatures much more rapidly than cellular DNA following heat denaturation probably because of crosslinkages in the virus DNA. As shown in Fig. 2, only one species of DNA was synthesized immediately after infection. This DNA sedimented at the density of denatured cellular DNA. At 48 h after infection, t w o species of DNA were evident: the predominant one sedimented at the density of native Yaba virus DNA, the second as denatured

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Fig. 2. CsC1 s e d i m e n t a t i o n o f Y a b a v i r u s - i n f e c t e d cell n u c l e a r D N A a f t e r d e n a t u r a t i o n a n d r e n a t u r a t i o n . I n f e c t e d cells w e r e p u l s e - l a b e l e d a t 0 o r 4 0 h a f t e r i n f e c t i o n f o r 1 h w i t h 1 / ~ C i / m l [ 3 H ] t h y m i d i n e . T h e n u c l e i w e r e i s o l a t e d b y t r e a t m e n t w i t h 2.5% citric a c i d . D N A w a s e x t r a c t e d a n d d e n a t u r e d b y h e a t i n g at 1 0 0 ~ C f o r 5 rain. T h e s u s P e n s i d n w a s m a d e 1.71 g / c m 3 w i t h CsCI a n d i n c u b a t e d a t 6 0 ° C f o r 12 h a n d t h e n c e n t r i f u g e d a t 3 5 0 0 0 r e v . / m i n f o r 6 0 h in a B e c k m a n T y p e 4 0 r o t o r . F r a c t i o n s w e r e c o l l e c t e d a n d c o u n t e d f o r t r i c h l o r o a c e t i c p r e c i p i t a b l e r a d i o a c t i v i t y . U p p e r p a n e l , 0 h a f t e r i n f e c t i o n ; l o w e r p a n e l , 48 h after infection.

63 cellular DNA. These results demonstrate that Yaba virus DNA is n o t covalently linked to cellular DNA in the host nucleus. Although cellular DNA synthesis was reduced b y Yaba virus infection, i t was evident from this experiment and the previous one that cellular DNA synthesis continues even late in Yaba virus infection.

Selective extraction o f Yaba virus DNA from the host cell nucleus An a t t e m p t was made to extract selectively the nuclear Yaba virus DNA. An adaptation of a procedure used b y Randall and Gafford [14] for fowlpox DNA and Hirt [13] for p o l y o m a DNA was used. Both of these procedures involve precipitation of very high molecular weight molecules with 1 M NaC1. Cellular DNA is precipitated by this procedure while virus DNA remains in the supernatant and can be easily separated and extracted. This procedure was used to fractionate DNA from Yaba virus infected cell nuclei which had been isolated by double-detergent treatment. The DNA which did n o t precipitate with 1 M NaCI was examined for the presence of Yaba virus-specific sequences. This material will be termed low molecular weight nuclear DNA. Time-course synthesis o f low molecular weight nuclear DNA from Yaba virusinfected cells Studies were designed to determine when Yaba virus-specific DNA appears in the nucleus. Cells were infected with Yaba virus and pulse-labeled for 1 h at various times after infection to determine the amount of radioactive thymidine incorporated in the low molecular weight nuclea~ DNA. The results indicated that isolation b y this procedure is n o t consistent, however at 40 h after infection, the synthesis of DNA in this fraction increased and is always higher than uninfected cells up to 110 h after infection (Fig. 3). Sedimentation analysis o f low molecular weight nuclear DNA Further investigations were made into the physical nature of this nuclear DNA fraction. Cells infected with Yaba virus were labeled from 50 to 70 h after infection. The nuclei were obtained by double-detergent treatment and were then treated with 1 M NaCI. The resulting supernatant fraction was layered onto alkaline sucrose gradients and centrifuged. The results are shown in Fig. 4. Purified Yaba virus DNA and uninfected cellular DNA were sedi. mented as controls. The low molecular weight fraction from Yaba virus infected cell nuclei sedimented as a single band. This band was at the same location as Yaba virus DNA. No other labeled material was detected. Cellular DNA sedimented in several bands, probably as a result of breakage during processing. When each fraction of the low molecular weight nuclear fraction gradient was examined for DNA content by the Burton test, other bands in addition to the labeled one were present. These bands probably represented a small proportion of cellular DNA which was present in this low molecular weight fraction, b u t was n o t being synthesized at the time the samples were taken. The fact that there was not a significant amount of cellular DNA labeled in the supernatant fraction indicates that cell DNA was probably n o t being synthesized and subsequently broken down to any great extent during the time of labeling, nor was cellular DNA synthesized as fragments.

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F i g . 3. T i m e c o u r s e o f l o w m o l e c u l a r w e i g h t n u c l e a r D N A s y n t h e s i s i n Y a b a v i r u s - i n f e c t e d cell n u c l e i . I n f e c t e d a n d u n i n t e c t e d cells w e r e p u l s e - l ~ b c l e d f o r I h w i t h 0 . 5 # C i / m l [ 3 H ] t h y m i d i n e a t v a r i o u s t i m e s a f t e r i n f e c t i o n . T h e cells w e r e t h e n f r a c t i o n a t e d b y d o u b l e d e t e r g e n t t r e a t m e n t w i t h N P 4 0 a n d T w c e n 8 0 and the nuclei fractionated by precipitation with 1 M NaCI. Thc supernatant fraction was analyzed to d e t e r m i n e a e i d - p r e c i p i t a b l e r a d i o a c t i v i t y , e . u n i n f e c t e d cells; o, i n f e c t e d cells. F i g . 4. AlkalLne s u c r o s e g r a d i e n t s e d i m e n t a t i o n o f l o w m o l e c u l a r w e i g h t n u c l e a r D N A . Cells w e r e i n f e c t e d w i t h Y a b a v i r u s a n d l a b e l e d w i t h 0 . 5 /=Ci/ml [ 3 H ] t h y m i d i n e f r o m 5 0 t o 7 0 h a f t e r i n f e c t i o n . T h e y w e r e f r s c t i o n a t e d u s i n g N P 4 0 - T w e e n 8 0 t r e a t m e n t a n d t h e n u c l e i e x t r a c t e d u s i n g S D S 1 M NaC1. T h e s u p e r n a t a n t D N A w a s c e n t r i f u g e d o n a 5--20% ( w / w ) s u c r o s e g r a d i e n t in 0 . 1 M N a O H / 0 . 9 M N a C I / 0 . O 0 1 M E D T A a t 3 9 0 0 0 r e v . / m i n f o r 1 . 5 h in a n S W S 0 . 1 r o t o r . F r a c t i o n s w e r e c o l l e c t e d a n d a n a l y s e d f o r a c i d precipitable radioactivity. Upper panel, uninfected cellular DNA; middle panel, low molecular weight n u c l e a r D N A f r o m Y a b a v i r u s - i n f e c t e d cells; l o w e r p a n e l , Y a b a v i r u s D N A ,

The supematant fraction was also examined by CsC1 gradient sedimentation. Again the supernatant fraction came to equilibrium at the same density as Yaba virus D N A (Fig. 5).

65

3H-Labeled uninfected cell DNA and 3H-labeled Yaba virus DNA were hybridized to the low mol. wt. nuclear DNA from Yaba virus-infected cells. Hybridization was detected between the low molecular weight nuclear material and

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Fig. 6. S a t u r a t i o n h y b r i d i z a t i o n of Y a b a virus D N A or cellular D N A to l o w m o l e c u l a r w e i g h t n u c l e a r D N A f r o m Y a b a v i r u s i n f e c t e d cells. I n f e c t e d cells w e r e f r a c t i o n a t e d a t 70 h a f t e r i n f e c t i o n b y t r e a t m e n t w i t h N P 4 0 a n d t h e n SDS 1 M NaC1 t r e a t m e n t o f t h e n u c l e i . T h e s u p e r n a t a n t D N A at 3.5 p g / s a m p l e was h y b r i d i z e d to i n c r e a s i n g a m o u n t s o f 3 H - l a b e l e d Y a b a v i r u s D N A (specific a c t i v i t y 7 9 8 1 c p m / p g ) (@) or 3 H - l a b e l e d cellular D N A (specific a c t i v i t y 581 cpm/~tg) (o) o n m e m b r a n e filters, S i m i l a r l y , u n l a b e l e d uni n f e c t e d cellular D N A was h y b r i d i z e d w i t h 3 H - l a b e l e d Y a b a virus D N A (4),

both cell DNA and Yaba virus DNA. Saturation of Yaba virus DNA and cell DNA with this fraction was approximately 2% {Fig. 6). This represented a relative increase over the level of Yaba virus DNA detected in whole nuclear DNA preparations. Therefore, this extraction procedure separated Yaba virus DNA from much of the cell DNA. Discussion One of the c o m m o n characteristics of DNA oncogenic viruses is the presence of viral DNA in the nucleus. The virus families containing t u m o r inducing viruses, including Herpes, papova, retro-, and adenoviruses, all replicate in the host cell nucleus. In addition, integration of the viral genome into the host cell genome has been demonstrated in many of these viruses. Yaba virus induces benign histiocytomas in monkeys and man [18--21]. It has been unusual because it is a DNA virus which causes t u m o r formation and y e t is cytoplasmic in its replication. The reported results indicate that there is nuclear involvement in the infected cell. This supports the work of LaColla and Weissbach [2], Bolden et al. [3], and Gafford and Randall [4], who also detected virus-specific DNA in the nuclei of poxvirus-infected cells. The virus DNA detected here exists as a complete genome, not covalently linked to the host cell DNA. The procedures used do n o t eliminate the possibility that virus DNA may also be linked to cell DNA. The presence of this DNA may permit better correlation with the other DNA-oncogenic virus systems.

67 Acknowledgements This investigation was supported by Public Health Service research grant CA10724 from the National Cancer Institute. We also wish to thank Dr. Donald C. Graves for his review of the manuscript. References 1 2 3 4

Pennington. T~I. and FoUett, E.A.C. (1974) J. ViroL 13. 488--493 LaCoHa, P. and Weissbach, A. (1975) J. Virol, 15, 305--315 Bolden, A., Aucker, J. and Weissbach, A. (1975) J. Virol. 16, 1584--1592 Gafford, L. and Randall, C.C. (1975) Abstracts, Annual Meeting of the American Society for Microbiology $314. p. 266. 5 Tsuchiya, Y., Tagaya, I. and Tsuruhara, T. (1969) Jap. J. Microbiol. 13,103--117 6 Jensen, R.C., Girardi, A~I., Gilden, R.V. and Koprowski, H. (1964) Proc. Natl. Acad. Sci. U.S. 52, 53--54 7 Joklik, W.K, (1962) Biochim. Biophys. Acta 61,290--301 8 Yuasa, T. and Tagaya, I. (1971) Jap. J. Med. Sci. Biol. 24, 357--364 9 0 d a , K. and Joklik, W.K. (1967) J. Mol. Biol. 27,395--419 10 Fujinaga, K., Rankin, A., Yamazaki, H., Sekikawa, K , Bragdon, J. and Green, M. (1973) Virology 56. 484---495 11 Penman, S., Greenburg, H. and Williams, M. (1969) in Fundamental Techniques in Virology (Habel, K. and Salzman, N.P., eds.), pp. 49--58, Academic Press, New York 12 Knowler, J.T., Moses, H.L. and Spelsberg, T.C. (1973) J. Cell Biol. 59,685--695 13 Hirt, B. (1967) J. Mol. Biol. 26,365--369 14 Randall, C.C. and Gafford0 L.G. (1969) Am. J, Pathol. 40, 51--59 15 Green, M., Fujinaga, K. and Pina, M. (1969) in Fundamental Tehcniques in Vizology, (Habel, K. and Salzman, N.P., eds.), pp. 467--484, Academic Press, New York 16 Jungwtxth, C, and Dawid, I.B. (1967) Arch Ges, Virusforsh. 20, 464)468 17 Burton, K. (1968) in Methods in Enzymology (Grossman° L. and Moldave, K., eds.), pp. 163--166, Academic Press, New York 18 Niven, J.S.F., Armstrong, J.A.° Andrews, C.H., Pereira, H.G. ~tnd Valentine, R.C. (1961) J. Pathol. BacterloL 81, 1 --14 19 Beazcroft, W.C.G. and Jamieson, M.F. (1958) Nature 182,195--196 20 Metzgaz, R.S., Grace, Jr., J.T. and Sproul, E.E. (1962) Ann. Acad. Sci. 101,192--202 21 Grace, Jr., J.T. and Mirand, E.A. (1963) Ann. N.Y. Acad. of Sci. 108, 1123--1128