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Enucleation of cells made simple and rescue of SV40 by enucleated cells made even simpler
VIROLOGY
61, 2277229 (1973)
Short Enucleation
of Cells Made
Communications Simple
Cells Made Fusion of SV40-transformed nonjwrmissiw cells with African Green Monkey Cells (AG;\II<), which are permissive for SV40, is the most efficient method for rescue of SV40 gcnome (1,s). Which fact’or in the permissive cells is involved in the excision and replication of SV40 genome in the nonpermissivc r~ucleus in the heterokaryocyte is still unknown (3, 4). It’ may be of either nuclear or cytoplasmic origin, or both. In order to investigate this problem, wc dccidcd to cnuclratr CV-1 cells of AGRIK origin (5) and fuse the enucleated ~11s with mKSBUlO0 mouse crlls transformed by sv40 (6). 4 X IO6 CV-1 wlls were grown direct’ly on the inner surface of nitrocellulow ultracentrifuge tubes fitting an SW 25.1 rotor. The walls of the tubes were treated with concentrated sulfuric acid for 4 min prior to seeding (7), followed by thorough washing with 700/b cathanol and by distilled HzO. The tubes wew then sterilized by exposure to UV light. The cells wcrc placed in t,hc tubes in 5 ml of Basal Eagle’s I\Iedium (BRIE) containing lO0]0 fatal calf serum (FCS), 2 n2M glutaminch, and antibiotics. The tubes \vcrc stoppcrrd wit)h a rubber stopper and placed horizont,ally in a roller apparatus at 37°C for 48-72 hr in order to obtain uniform attachment of the cells to the imwr surface of the tubes. At thcl cbndof this period, the tubw wwc rcmowd from the roller apparatus and filled wit,h BJIE with 10% 1’CS containing %I0 pg/ml of cytochalasin B (8) (from Imperial Chemical Industries). The cultures were left in this solution for 145 hr at 37°C. Sftcr this, th(l medium was wplaced by a medium un1 This work was supported in part by the ?Jat.ional Institutes of Health Gr’allt CA0 1534 and COliIC (:rarlt CA 10815.
Copyright ~11 rights
@ 1973 lay Academic Press, Inc. of rrproduction in any f
and Rescue of SV40 by Enucleated Even Simpler’ taiuing 2.5 pg/ml of cytochalasin B. The tubes, placed in SW 25.1 buckets sterilized by UV, were centrifuged at’ 23,000 rpm for 4.5 min at 25°C in an L-2 Beckman ultracentrifuge. Examination of cells att,ached to the surface of the tube aftw ccntrifugation rcvraled tht: presence of elongated thin st,ructures, apparently without’ nuclei. The medium \vas removed, the pellet discnrdcd and the tube mashed two or three times with BI\IE wit’h 10% FCS. The tubes were then refilled with BME with 10% PCS, stopprred, and incubated in a vertical position for ‘2 hr at 37°C. .4t that time, wc observed that the elongated, thin structures seen immediatcly after ccntrifugation regained tho shape of the original (~~11,but, without nuclei. The medium was then rcmovcd and the cells detached from the walls of t,he tubes by treatment with 0.05% vcrsenc in PBS (Cal+and Xlg2+-free). The rrlls were spun c3owI at 180~ for 5 min and resuspended in 1\IER4 without serum at pH S.0 (9) at the (wccntration of 9 X 10” wlls per ml. The prrccntagc of cinucleatcd CV-1 ~~~11s was checked by the follo\ving methods: 1. Counting thcl cells on the wall of the wntrifuge tubes by cutting out strips of the nitrowllulosc tubes and examination of the strips undw light microscopy. 2. Counting of the cells on smears made by spwading tho vcrsenizcd wlls on glass slidw. 3. Plating the vcrsenizc>d cells on coverslips in petri dishrs in th(b presence of B1IE with 10% of wrum and incubating for 10 hr at 37°C. Th(x wlls ww fixed in ice-cold ethanol: awtic acid (3: 1) and stained in buffrwd Girmsa wt~pH 5.75.
228
SHORT
COMMUNICATIONS
Incorporation of amino acids in protein in the enucleatcd cells was checked by exposure of the versenized cells on coverslips t,o tritiated lcucine (sp act 5 Curies per mmole) 10 &$‘ml for 18 hr at 37°C in BXIE with 10% fetal calf serum. Aft,cr this the cells were fixed in ice-cold acetic acid:ethanol (1:3), washed in 5% trichloric acetic acid for 15 min at 4”C, and processed for autoradiography according to standard procedure (10). The monolayers of mIiSBUlO0 cells were trypsinized, the cells resuspended in medium, and pelleted down after centrifugation at 18Og for 5 min. They were then resuspended at a concentration of 9 X 106/ml in serumfree MEM at pH 8.0. One half milliliter of enucleat)ed CV-1 cells containing, urhss otherwise stated, 4.5 X lo6 cells and 0.5 ml of mIiSBUlO0 containing 4.5 X 10” cells were mixed together and fused in the presence of 4000 hemagglutinating units of Beta propiolactone-inactivated Sendai virus. The cell mixtures were kept on ice for 20 min, followed by incubation in a water bath at 37°C for 30 min. The cells were mechanically shaken during incubation. After that, the cells were centrifuged at 18Og for 5 min, resuspended in BME containing 10% fetal calf serum, and plated in serial IO-fold dilution 011 glass GO-mm petri dishes con-
RELEASE
OF INFECTIOUS
taining an almost confluent monolayer of CV-1 cells. Each dilution of cells was plated on 10 petri dishes and the dishes incubated for 10 hr at 37°C in an atmosphere of 4y0 CO2 . The medium was removed, and the monolayers were washed with BME and overlaid with 5 ml of 1% solution of agar in lacto-albumin hydrolyzate (11). An additional 5 ml of overlay was added after 7 days of incubation at 37°C. Ten days later, 2 ml of overlay medium containing 1: 10000 neutral red was added and number of plaques recorded 1 day later. For control purposes, CV-1 cells containing nuclei were fused with mKSBUlO0 cells and the fused cells treated as above. As a second control CV-1 cells were mixed with mKSBU100 cells without Sendai virus being present and the cells plated on CV-1 cell monolayers as described above. Repeated determination of the ratio of enucleated to nucleated CV-1 cells after cytochalasin B treatment indicated that more than 98% of the cells counted did not contain nuclei. The results of autoradiography after incorporation of [3H]leucine into enucleated CV-1 cells indicated that more than 90% of the cells showed silver grains over the ctyoplasm within 20 hr after cnucleation. Fusion of enucleated CV-1 cells with mKSBUlO0 cells resulted in the rcscuc of
TABLE 1 SV40 BY FUSED CELLS. EXPERIMENTS 1, 2, AND 3 IN THE PRESENCE HAU OF INACTIVATED SENDAI VIRUS
OF 4000
Cells fused and ratio (in parentheses)
Dilution of the cells plated per petri disha
Average number of infectious centers per petri dish
1
mKSBUlO0 + enucleated CV-lb (4.5 x 106) (4.5 x 106)
2
mKSBUlOO + nucleated CV-1 (4.5 x 106) (4.5 x 106)
3
mKSBUl60 + nucleated CV-1 (4.5 x 106) (1.8 x 106)
1:20 1:206 1:2000 1:20 1:200 1:2000 1:20 1:200 1:2000 1:20
15.0 6.5 1.4 63.0 11.0 1.4 4.7 1.4 0.4 0.7
Fusion experiment
Control
mKSBUlO0 + nucleated CV-lc (4.5 x 106) (4.5 x 106)
a Ten petri dishes containing CV-1 monolayers b Enucleated CV-1 cells 98.8yn. c Cells mixed in the absence of Sendai virus.
plated
for each dilution
of ceils.
SHORT
229
COMMUNICATIONS
infectious SV40 (Table 1). When the same number of nucleated CV-1 cells were fused with mKSBUlO0 the rescue of the virus was approximately two to four times more efficient than in the case of enucleated CV-1 cells. In Expt 3, the number of nucleated CV-1 cells participating in the fusion process was reduced to 47, of those used in Expt 2, in order to compare t,he results with those obtained in Expt 1, where less than 2% of CV-1 cells retained t’heir nuclei after cytochalasin B treatment. The results of Expt 3 indicate that rescue of SV40 in this case was less efficient than that observed after the use of the enucleated CV-1 cells preparation (Expt 1). In the absence of Sendai virus, mixtures of mKSBUlO0 and nucleated CV-1 cells produced less than 1 infectious center per pet,ri dish. The result,s of these experiments indicate that enucleated CV-1 cells are capable of rescuing SV40 genome and that the rescue is not due to the small number of nucleated CV-1 cells present in the preparation. Thus, the cytoplasm of CV-1 cells must contain a factor(s) which permits the SV40 genome present in the nuclei of mKSBU100 cells to be excised and to replicate. Otherwise, the small number of nucleated CV-1 cells would have t’o produce a factor which may have contributed indirectly to the rescue of the virus.
REFERENCES
8. 3.
4. 5.
10.
11.
KOPRO~SKI, H., JENSEN, F.,and STEPLE~SKI, Z., Proc. Nat. Acad. Sci. USA 58, 127-133 (1967). WATKINS, J., and DULBECCO, R., Proc. Sat. Acad. Sci. USA 58,1396-1403 (19G ). WEVER, G., KIT, S., and UUBBS, D., J. J'irol. 5, 578-585 (1970). STEPLEWSKI, Z., KNOWLES, B., and KoprowSKI, H., Proc. Sat. Acad. Sci. USA 3, 769776 (1968). JENSEN, F., GIRARDI, A., GILDEN, R., and KOPROWSKI, H., Proc. Nat. Acad. Sci. USA 52, 53-59 (1964). DUHBS, D., KIT,S.,DETORRES, R.,and ANKEN, M., J. Viral. 1,968-979 (1967). PRESCOTT, D., MYERSON, D., and WALLACE, J., Exp. Cell Res. 71, 480-485 (1972). CARTER, S., A’ature (London) 213, 261-264 (1967). CROCS, C., KOPROWSKI, H., and EAGLE, H., Proc. #at. Acad. Sci. USA 64, 1953-1956 (1972. ROGERS, A., “Techniques of Autoradiography,” 1st ed. Amer. Elsevier, New York (1967). SWETLY, P., BARBANTI-BRODANO, G., KNOWLES, B.,and KOPROWSKI, H.,J. Viral. 4, 343-355 (1969). CARLO M.CROCE HILARY KOPROWSKI
The Wistar
Institute
of
Anatomy and Biology Philadelphia, Pennsylvania 19104 Accepted October lb’, 1972
61, 2277229 (1973)
Short Enucleation
of Cells Made
Communications Simple
Cells Made Fusion of SV40-transformed nonjwrmissiw cells with African Green Monkey Cells (AG;\II<), which are permissive for SV40, is the most efficient method for rescue of SV40 gcnome (1,s). Which fact’or in the permissive cells is involved in the excision and replication of SV40 genome in the nonpermissivc r~ucleus in the heterokaryocyte is still unknown (3, 4). It’ may be of either nuclear or cytoplasmic origin, or both. In order to investigate this problem, wc dccidcd to cnuclratr CV-1 cells of AGRIK origin (5) and fuse the enucleated ~11s with mKSBUlO0 mouse crlls transformed by sv40 (6). 4 X IO6 CV-1 wlls were grown direct’ly on the inner surface of nitrocellulow ultracentrifuge tubes fitting an SW 25.1 rotor. The walls of the tubes were treated with concentrated sulfuric acid for 4 min prior to seeding (7), followed by thorough washing with 700/b cathanol and by distilled HzO. The tubes wew then sterilized by exposure to UV light. The cells wcrc placed in t,hc tubes in 5 ml of Basal Eagle’s I\Iedium (BRIE) containing lO0]0 fatal calf serum (FCS), 2 n2M glutaminch, and antibiotics. The tubes \vcrc stoppcrrd wit)h a rubber stopper and placed horizont,ally in a roller apparatus at 37°C for 48-72 hr in order to obtain uniform attachment of the cells to the imwr surface of the tubes. At thcl cbndof this period, the tubw wwc rcmowd from the roller apparatus and filled wit,h BJIE with 10% 1’CS containing %I0 pg/ml of cytochalasin B (8) (from Imperial Chemical Industries). The cultures were left in this solution for 145 hr at 37°C. Sftcr this, th(l medium was wplaced by a medium un1 This work was supported in part by the ?Jat.ional Institutes of Health Gr’allt CA0 1534 and COliIC (:rarlt CA 10815.
Copyright ~11 rights
@ 1973 lay Academic Press, Inc. of rrproduction in any f
and Rescue of SV40 by Enucleated Even Simpler’ taiuing 2.5 pg/ml of cytochalasin B. The tubes, placed in SW 25.1 buckets sterilized by UV, were centrifuged at’ 23,000 rpm for 4.5 min at 25°C in an L-2 Beckman ultracentrifuge. Examination of cells att,ached to the surface of the tube aftw ccntrifugation rcvraled tht: presence of elongated thin st,ructures, apparently without’ nuclei. The medium \vas removed, the pellet discnrdcd and the tube mashed two or three times with BI\IE wit’h 10% FCS. The tubes were then refilled with BME with 10% PCS, stopprred, and incubated in a vertical position for ‘2 hr at 37°C. .4t that time, wc observed that the elongated, thin structures seen immediatcly after ccntrifugation regained tho shape of the original (~~11,but, without nuclei. The medium was then rcmovcd and the cells detached from the walls of t,he tubes by treatment with 0.05% vcrsenc in PBS (Cal+and Xlg2+-free). The rrlls were spun c3owI at 180~ for 5 min and resuspended in 1\IER4 without serum at pH S.0 (9) at the (wccntration of 9 X 10” wlls per ml. The prrccntagc of cinucleatcd CV-1 ~~~11s was checked by the follo\ving methods: 1. Counting thcl cells on the wall of the wntrifuge tubes by cutting out strips of the nitrowllulosc tubes and examination of the strips undw light microscopy. 2. Counting of the cells on smears made by spwading tho vcrsenizcd wlls on glass slidw. 3. Plating the vcrsenizc>d cells on coverslips in petri dishrs in th(b presence of B1IE with 10% of wrum and incubating for 10 hr at 37°C. Th(x wlls ww fixed in ice-cold ethanol: awtic acid (3: 1) and stained in buffrwd Girmsa wt~pH 5.75.
228
SHORT
COMMUNICATIONS
Incorporation of amino acids in protein in the enucleatcd cells was checked by exposure of the versenized cells on coverslips t,o tritiated lcucine (sp act 5 Curies per mmole) 10 &$‘ml for 18 hr at 37°C in BXIE with 10% fetal calf serum. Aft,cr this the cells were fixed in ice-cold acetic acid:ethanol (1:3), washed in 5% trichloric acetic acid for 15 min at 4”C, and processed for autoradiography according to standard procedure (10). The monolayers of mIiSBUlO0 cells were trypsinized, the cells resuspended in medium, and pelleted down after centrifugation at 18Og for 5 min. They were then resuspended at a concentration of 9 X 106/ml in serumfree MEM at pH 8.0. One half milliliter of enucleat)ed CV-1 cells containing, urhss otherwise stated, 4.5 X lo6 cells and 0.5 ml of mIiSBUlO0 containing 4.5 X 10” cells were mixed together and fused in the presence of 4000 hemagglutinating units of Beta propiolactone-inactivated Sendai virus. The cell mixtures were kept on ice for 20 min, followed by incubation in a water bath at 37°C for 30 min. The cells were mechanically shaken during incubation. After that, the cells were centrifuged at 18Og for 5 min, resuspended in BME containing 10% fetal calf serum, and plated in serial IO-fold dilution 011 glass GO-mm petri dishes con-
RELEASE
OF INFECTIOUS
taining an almost confluent monolayer of CV-1 cells. Each dilution of cells was plated on 10 petri dishes and the dishes incubated for 10 hr at 37°C in an atmosphere of 4y0 CO2 . The medium was removed, and the monolayers were washed with BME and overlaid with 5 ml of 1% solution of agar in lacto-albumin hydrolyzate (11). An additional 5 ml of overlay was added after 7 days of incubation at 37°C. Ten days later, 2 ml of overlay medium containing 1: 10000 neutral red was added and number of plaques recorded 1 day later. For control purposes, CV-1 cells containing nuclei were fused with mKSBUlO0 cells and the fused cells treated as above. As a second control CV-1 cells were mixed with mKSBU100 cells without Sendai virus being present and the cells plated on CV-1 cell monolayers as described above. Repeated determination of the ratio of enucleated to nucleated CV-1 cells after cytochalasin B treatment indicated that more than 98% of the cells counted did not contain nuclei. The results of autoradiography after incorporation of [3H]leucine into enucleated CV-1 cells indicated that more than 90% of the cells showed silver grains over the ctyoplasm within 20 hr after cnucleation. Fusion of enucleated CV-1 cells with mKSBUlO0 cells resulted in the rcscuc of
TABLE 1 SV40 BY FUSED CELLS. EXPERIMENTS 1, 2, AND 3 IN THE PRESENCE HAU OF INACTIVATED SENDAI VIRUS
OF 4000
Cells fused and ratio (in parentheses)
Dilution of the cells plated per petri disha
Average number of infectious centers per petri dish
1
mKSBUlO0 + enucleated CV-lb (4.5 x 106) (4.5 x 106)
2
mKSBUlOO + nucleated CV-1 (4.5 x 106) (4.5 x 106)
3
mKSBUl60 + nucleated CV-1 (4.5 x 106) (1.8 x 106)
1:20 1:206 1:2000 1:20 1:200 1:2000 1:20 1:200 1:2000 1:20
15.0 6.5 1.4 63.0 11.0 1.4 4.7 1.4 0.4 0.7
Fusion experiment
Control
mKSBUlO0 + nucleated CV-lc (4.5 x 106) (4.5 x 106)
a Ten petri dishes containing CV-1 monolayers b Enucleated CV-1 cells 98.8yn. c Cells mixed in the absence of Sendai virus.
plated
for each dilution
of ceils.
SHORT
229
COMMUNICATIONS
infectious SV40 (Table 1). When the same number of nucleated CV-1 cells were fused with mKSBUlO0 the rescue of the virus was approximately two to four times more efficient than in the case of enucleated CV-1 cells. In Expt 3, the number of nucleated CV-1 cells participating in the fusion process was reduced to 47, of those used in Expt 2, in order to compare t,he results with those obtained in Expt 1, where less than 2% of CV-1 cells retained t’heir nuclei after cytochalasin B treatment. The results of Expt 3 indicate that rescue of SV40 in this case was less efficient than that observed after the use of the enucleated CV-1 cells preparation (Expt 1). In the absence of Sendai virus, mixtures of mKSBUlO0 and nucleated CV-1 cells produced less than 1 infectious center per pet,ri dish. The result,s of these experiments indicate that enucleated CV-1 cells are capable of rescuing SV40 genome and that the rescue is not due to the small number of nucleated CV-1 cells present in the preparation. Thus, the cytoplasm of CV-1 cells must contain a factor(s) which permits the SV40 genome present in the nuclei of mKSBU100 cells to be excised and to replicate. Otherwise, the small number of nucleated CV-1 cells would have t’o produce a factor which may have contributed indirectly to the rescue of the virus.
REFERENCES
8. 3.
4. 5.
10.
11.
KOPRO~SKI, H., JENSEN, F.,and STEPLE~SKI, Z., Proc. Nat. Acad. Sci. USA 58, 127-133 (1967). WATKINS, J., and DULBECCO, R., Proc. Sat. Acad. Sci. USA 58,1396-1403 (19G ). WEVER, G., KIT, S., and UUBBS, D., J. J'irol. 5, 578-585 (1970). STEPLEWSKI, Z., KNOWLES, B., and KoprowSKI, H., Proc. Sat. Acad. Sci. USA 3, 769776 (1968). JENSEN, F., GIRARDI, A., GILDEN, R., and KOPROWSKI, H., Proc. Nat. Acad. Sci. USA 52, 53-59 (1964). DUHBS, D., KIT,S.,DETORRES, R.,and ANKEN, M., J. Viral. 1,968-979 (1967). PRESCOTT, D., MYERSON, D., and WALLACE, J., Exp. Cell Res. 71, 480-485 (1972). CARTER, S., A’ature (London) 213, 261-264 (1967). CROCS, C., KOPROWSKI, H., and EAGLE, H., Proc. #at. Acad. Sci. USA 64, 1953-1956 (1972. ROGERS, A., “Techniques of Autoradiography,” 1st ed. Amer. Elsevier, New York (1967). SWETLY, P., BARBANTI-BRODANO, G., KNOWLES, B.,and KOPROWSKI, H.,J. Viral. 4, 343-355 (1969). CARLO M.CROCE HILARY KOPROWSKI
The Wistar
Institute
of
Anatomy and Biology Philadelphia, Pennsylvania 19104 Accepted October lb’, 1972