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Localization of type C retrovirus restriction in embryonal carcinoma cells by cytochalasin B-mediated cellular fractionation
Ann. Virol. (Inst. Pasteur) 1982, 133 E, 107-113
LOCALIZATION OF IN
TYPE
EMBRYONAL
BY
C RETROVIRUS CARCINOMA
CYTOCHALASIN
CELLULAR
RESTRICTION
CELLS
B-MEDIATED
FRACTIONATION
by M. Sall~, J. Tobaly, L. d'Auriol, R. EmanoI1-Ravicovitch and J. P~ri~s U.107 I N S E R M , LOI-CNRS, Ddpartemenl d'Oncologie Expdrimentale, lnslilat de Recherches sur les Maladies du Sang, Hdpilal Saint-Louis, 7574,5 Paris Cedex 10
SUMMARY Murine type C retroviruses do not replicatc in embryonal carcinoma cells (ECC), whilst they do normally in the differentiated derivatives of these cells. I lowever, coculture in the presence of a fusing agent allows the recovery of the input virus up to 5 days after infection. Cytoplasmic and nuclear fractions were obtained at different times after infection by treatment of the infected ECC with cytochalasin B and differential centrifugation. Virus was recovered up to 24 h post-infection by fusion of the cytoplasmic fraction with a permissive cell, whereas beyond 24 h virus was only recovered from the nuclear fraction. These results indicate that the cytoplasmic events of viral replication in the restrictive ECC are seemingly normal and that the block is very probably localized in the nucleus. KEY-WORDS : Retrovirus type C, Carcinogenesis, Restriction, Cytoehalasin B ; Fusion, Permissive cell, Cocultivation, Mouse.
INTRODUCTION ]'he susceptibility of murine teratocarcinoma-derived cell lines to ecotropic mouse type C retrovirus has been shown to be affected by the state of cellular differentiation [4, 8]. Embryonal carcinoma cells (ECC), which are the undifferentiated stem components of teratocarcinomas, Manuscrit re~u le 5 fdvrier 1982, acceptd le 21 avril 1982.
108
M. SALLI~ AND COLL.
are refractory to murine leukaemia virus (MuLV) multiplication. Differentiated teratocarcinoma-derived cells, however, are fully susceptible to MuLV. In a preceeding paper we demonstrated t h a t after infecting ECC with MuLV it was impossible to activate or rescue the input virus, even b y long-term cocultivation with highly susceptible mouse cells [5]. In the mixed cultures, the detection of virus production was never possible. Teich el al. [8] also reported t h a t fusion mediated b y Sendal virus of MuLVinfected ECC to mouse fibroblasts never resulted in detectable activation or rescue of the infecting virus. Taken together, these results suggest t h a t the viral DNA, if synthetised in infected ECC, is either biologically inactive or else placed under a v e r y strong restriction mechanism. However, Gautsch [2] has demonstrated t h a t restriction of MuLV in ECC can be overcome b y fusion with permissive cells during the 12-h period immediately following infection. The present study, which was undertaken in parallel with molecular biology experiments designed to examine the early phase of MuLV infection in ECC [1, 10], confirms the results of Gaustch and indicates t h a t the blocking event v e r y p r o b a b l y occurs after the newly synthetized provirus leaves the cytoplasm to enter the nucleus.
MATERIALS AND METHODS Cells.
PCC4 aza(-) cultures derived from a transplantable teratocarcinoma of the 129/SV mouse [3] were kindly supplied by F. Jacob and E. Jacob (Institut Pasteur, Paris). They are composed of totipotential ECC and give rise to multitissular tumours when injected into syngenic mice either subcutaneously or intraperitoneally. However, they never differentiate when maintained in vitro. The D55 murine cell line derived from BALB/c was the kind gift of R. Bassin (NC I, Bethesda, USA). It is highly permissive to type C murine retrovirus infection because it does not express the FV1 genetic system of restriction. Virus.
Moloney murine leukaemia virus (M-MuLV) was produced b y a chronically infected cell line (3T3 Swiss) inoculated with M-MuLV from the laboratory of G. Todaro (NCI). This cell line releases an NB, ecotropic, XC-positive virus with a titre of about 106 pfu/ml. When used as inoculum on PCC4 cells, the virus was employed at a multiplicity of 1. Viral deleclion.
Two methods were applied to detect and quantify viral production. Virion reverse transcriptase activity was measured in supernatant fluids after pelletization of the virus using synthetic template poly(A)-oligo(dT) and expressed as cpm CB = c y t o c h a l a s i n B. E C C = e m b r y o n a l c a r c i n o m a cell. MEM = (Eagle's) minimal essential medium.
M-MuLV = Moloney murine leukaemia virus. MuLV = murine leukaemia virus. PEG = p o l y e t h y l e n e glycol.
TYPE C MURINE RETROVIRUS RESTBICTION
109
of ~H-TMP incorporated into the final product of the reaction [6]. In addition, in silu XC plaque tests were performed on eocultures as described by Rowe el al. [7].
Cellular enuclealion. Cytochalasine B (CB) cellular enueleation was performed following the method described by Wigler and Weinstein [9]. PCC4 cells, detached with 0.5 mM EDTA, were well homogenised and resuspended at a final concentration of 0.5 to 2 • 107 cells/ml in MEM for suspension culture (without Ca++ or Mg ++) containing 12.5 % Ficoll, 10 y.g/ml CB and 200 ~g/ml each of penicillin and streptomycin. These cell suspensions were carefully applied to a sterile Fieoll density gradient prepared with the following layers: 25, 17, 16, 15, and 12.5 %, and incubated at 37 ~ C for 17 h. All the layers of the gradient were prepared in the same culture medium as described above (with antibiotics and CB). Gradients were centrifuged at 25,000 rpm for 1 h at 31 ~ C in an SW41 rotor (Beckman) prewarmed to 31 ~ C for 1 h in an ~ L350 Beckman ~ ultracentrifuge. After eentrifugation, cytoplasts and karyoplasts were collected separately from the top of the tube with a Pasteur pipette in the 15-17 % and 17-25 % region of the gradient, respectively. P u r i t y of both fractions was monitored by phase-constrast microscopy. Cytoplasts and karyoplasts were diluted in 10 ml of culture medium (DMEM) and centrifuged at 1,000 rpm for 5 min. Each pellet was then fused with entire cells (D55) as described below.
Polyethylene glycol (PEG) /usion technique. Infected and uninfected PCC4 cells or PCC4 cytoplasts and karyoplasts were washed and then centrifuged (1,300 rpm) with an equal number of D55 cells for 5 min at 25 ~ C. One ml of a 25 % P E G solution, prewarmed to 37 ~ C, was added to the mixed cells for exactly 1 min. Afterwards, the P E G solution was diluted with 9 ml of DMEM, and the mixed cultures were centrifuged for (1,300 rpm, 5 min, 25 ~ C), then the medium was removed and the eocultures were resuspended in a new culture medium before seeding in a 250-ml Nunelon flask. The coeultures were then split twice a week. At each passage level a fraction of the cells and their supernatants were submitted to the viral detection tests.
BESULTS T a b l e I p r e s e n t s t h e results of p r e l i m i n a r y e x p e r i m e n t s designed to e x a m i n e t h e effects of P E G - m e d i a t e d fusion on t h e r e c o v e r y of i n p u t virus f r o m P C C 4 - i n f e c t e d cells. T h e v i r u s was easily r e c o v e r e d f r o m 8 h t o 5 d a y s p o s t i n f e c t i o n w h e n t h e s e cells were c o c u l t i v a t e d w i t h D55-suse e p t i b l e cells in t h e p r e s e n c e of t h e fusing agent. A f t e r this t i m e no virus could be isolated f r o m a n y of t h e cocultures. No virus was d e t e c t a b l e in t h e s u p e r n a t a n t s of c o c u l t u r e s p e r f o r m e d in t h e a b s e n c e of P E G . Table II indicatesthat w h e n C B - t r e a t e d PCC4 cell f r a c t i o n s were fused to D55 cells in t h e p r e s e n c e of P E G , virus was r e c o v e r e d f r o m t h e f u s e d c y t o p l a s m i c a n d n u c l e a r f r a c t i o n s o n l y b e t w e e n 8 a n d 24 h a f t e r infection. A t 48- a n d 72-h t i m e points, e v e n t h o u g h t h e c y t o p l a s m i c f r a c t i o n did n o t give rise t o v i r u s p r o d u c t i o n , t h e n u c l e a r f r a c t i o n did, a n d r e c o v e r y of virus was possible. O v e r 72 h, cells were n o t s u b m i t t e d to CB t r e a t m e n t b e c a u s e of t h e i r r e q u i r e m e n t s for cell passage.
110
M.
T A B L E I. after
SALLt~
AND
COLL.
R e s c u e o f i n p u t v i r u s f r o m M - M u L V - i n f e c t e d P C C 4 cells PEG-mediated f u s i o n w i t h D55-susceptible cells (1). Time after infection 8 12 1 2 4 5 6 8
h h day days days days days days
XC t e s t
Reverse transcriptase a c t i v i t y (2)
+ (a) § + + -~ + 0 (a) 0
1,327 1,631 1,493 1,686 719 973 0 (4) 0
(1) Triplicate c u l t u r e s of f u s e d infected a n d u n i n f e c t e d PCC4/D55 cells were t e s t e d d u r i n g a period of 3 m o n t h s b y reverse t r a n s c r i p t a s e a n d in s i l u XC assays. (2) A m o u n t s given ( • 10 -3 c p m ) a r e t h e m e a n s of m o r e t h a n t h r e e successive passages a f t e r e n z y m a t i c activities r e a c h e d a plateau. (8) Syncitia too n u m e r o u s to be c o u n t e d were c o n s t a n t l y p r e s e n t over t h r e e or m o r e successive p a s s a g e s . (4) N e g a t i v e r e s u l t s were confirmed for b o t h t e s t s d u r i n g a period of 3 m o n t h s ; f u s e d u n i n f e c t e d P C C 4 / D 5 5 cell c u l t u r e s a l w a y s gave n e g a t i v e results.
T A B L E I I. - - R e s c u e o f i n p u t v i r u s f r o m n u c l e a r a n d c y t o p l a s m i c f r a c t i o n s o f M - M u L V - i n f e c t e d PCC4 cells fused to D 5 5 c e l l s .
Time after infection 8 12 24 48 72
h h h h h
Cytoplasmic fraction § (8) T (8) -~ (12) --
Nuclear fraction § • -~ + +
(8) (8) (8) (12) (12)
Triplicate c u l t u r e s of infected PCC4 cytop l a s m i c a n d n u c l e a r f r a c t i o n s f u s e d at each t i m e p o i n t to p e r m i s s i v e D55 ceils; t h e i r d e s c e n d e n t s were t e s t e d d u r i n g a period of 3 m o n t h s b y reverse t r a n s c r i p t a s e a n d in s i l u XC a s s a y s . Cocultures were considered as positive ( + ) w h e n t h e two viral m a r k e r s ( n u m e r o u s s y n c i t i a a n d reverse t r a n s c r i p t a s e a s s a y > 20 • 10 -3 c p m ) were s i m u l t a n e o u s l y positive over m o r e t h a n t h r e e successive passages, and negative (--) when both criteria were s u m u l t a n e o u s l y n e g a t i v e . N u m b e r s w i t h i n p a r e n t h e s e s indicate t h e n u m b e r of d a y s after c o c u l t i v a t i o n elapsed before t h e a s s a y b e c a m e positive.
TYPE C MURINE RETROVIRUS RESTRICTION
111
DISCUSSION Fusion of CB-fractionated M-MuLV-infected PCC4 cells to highly permissive fibroblastic mouse cells has been used in this s t u d y to localize the cellular region where the restriction of ecotropic t y p e C virus replication in the cells takes place. In confirmation with recent results [2], we have demonstrated t h a t fusion with an efficient fusing agent is sufficient to overcome the restriction event. Moreover, the block of viral multiplication probably occurs at the level of the cellular nucleus, after the cytoplasmic events of the virus replication cycle have been completed. These results raise an i m p o r t a n t question: was the virus which was recovered by cocultivation of whole cells and cellular fractions t h e progeny of the input M-MuLV? Several genetic markers (results not shown) such as XC test positivity, species tropism (ecotropism), FVI gene-dependent tropism, and pathogenicity (induction of leukemia) strongly indicated t h a t this was the case since t h e y are characteristics of the infecting M-MuLV and are not those of any t h e known Swiss/NIH (D55) endogenous viruses. Moreover, o ocultivation of uninfected PCC4 cells with D55 cells never led to virus activation. In addition, the fact t h a t no virus could be isolated from the m e d i u m used to wash cells after inoculation, and t h a t direct t r e a t m e n t of virus inocula with P E G destroyed all virus activity, seems to exclude any possible contamination of indicator cells with residual input virus (data not shown). Another argument used to explain the isolation of infectious virus from PCC4-infected cultures is the existence of some differentiated cells among the PCC4 cell population. These permissive cells would have supplied the virus detected after fusion and cocultivation. This possibility, however, is not consistent with our observations of the absolute negativity of several different viral replication markers in PCC4 cultures after infection with M-MuLV such as immunofluorescence, in situ XC tests, infectious centers and particularly direct cocultivation with permissive cells [4, 5]. One interesting observation m a d e during our studies was t h a t virus activity can be induced by fusion and cocultivation of the infected cells up to 5 days after inoculation. This result is not in agreement with the results of Gautsch [2], who was able to detect virus activity from ECC only up to 20 h after infection. This difference could be explained by the fact t h a t we used a m e t h o d involving viral amplification as evidence of the presence of virus; t h a t is our technique is more sensitive t h a n the in situ XC technique employed by Gaustch. In conclusion, the data presented here indicate t h a t the block of viral replication in PCC4 cells is v e r y probably localized in the nucleus. T h e y also confirm previous results indicating t h a t neosynthesis of infectious provirus during early steps of viral replication is accomplished normally, at least in PCC4 cells [9, 10]. These results however do not solve the import a n t problem of the presence or absence of normal proviral integration in PCC4 genomic DNA. This problem is the subject of a subsequent paper.
112
M. SALLI~ AND COLL.
RI~SUMI~ LOCALISATION DU PHI~NOMt~NE DE RESTRICTION DE LA MULTIPLICATION DES RI~TROVIRUS DE TYPE C, DANS LES CELLULES DU CARCINOME EMBRYONNAIRE~ PAR FRACTIONNEMENT )t LA CYTOCHALASINE B
Les r~trovirus murins ne se r~pliquent pas dans les cellules indiff~renei6es du eareinome embryonnaire (CCE) de la souris tandis qu'ils le font normalement dans les eellules diff6renci~es de m~me origine. Cependant la eoeulture en presence d'un agent fusionnant permet de r~cup6rer le virus infeetant et eela jusqu'h cinq jours apr~s infection. Les fractions cytoplasmiques et nuel6aires ont 6t~ obtenues, h des temps variables apr~s infection, grfiee h un t r a i t e m e n t des CCE par la eytochalasine B et une eentrifugation diff~rentielle. Le virus n'est r~isol~ dans la fraction cytoplasmique fusionn~e avec des eellules permissives que p e n d a n t les 24 premieres heures apr~s l'infeetion virale, tandis que la fraction nucl6aire p e r m e t de r~cup6rer le virus au-delh de 24 heures. Ces r~sultats m o n t r e n t que les ~tapes cytoplasmiques de la r~plication virale dans les CCE non permissives semblent normales et que le bloeage se situe tr~s probablem e n t au niveau nuel6aire. MOTS-CLt~S : R~trovirus de t y p e C, Carcinogen~se, Restriction, Cytochalasine B ; Fusion, Cellule permissive, Cocultivation, Souris.
REFERENCES [1] D'AuRIOL, L., YANG, W. K., TOBALY, J., CAVALIEBI, P., PgaIgs, J. & EMA-
NOiL-RAVICOVITCH, R., Studies on the restriction of ecotropic murine [2] [3]
[4]
[5]
[6]
retrovirus replication in mouse teratocarcinoma cells. J. gen. Virol., 1981, 55, 117-122. GAUTSCH, J. W., Embryonal carcinoma stein cells lack a function required for virus replication. Nature (Lond.), 1980, 285, 111-112. JAKOB, H., BOON, T., GAILLABD, J., NICOLAS, J. F., & JACOB, F., Teratocarcinome de la souris : isolement, culture et propri6tds de cellules h potentialitds multiples. Ann. Microbiol. (Inst. Pasteur), 1973, 124 B, 269-282. P~RII~S, J., ALVES-CARDOSO, E., CANIVET, M., DEBONS-GuILLEMIN, M., & LASNERET, J., Lack of multiplication of ecotropic murine C type viruses in mouse teratocarcinoma primitive cells. J. nat. Cancer Inst., 1977, 59, 463-465. PI~RII~S, J., DEBONS-GUILLEMIN, M. C., CANIVET, M., EMANOIL-RAvICOVITCH, R., TAVITIAN, A. & BoInOS, M., l~tude de la multiplication des virus murins de type C dans des ligndes cellulaires ddrivdes du tdratocarcinome de la souris. Nouv. Rev. [ranc. Hdmat., 1977, 18, 383-390. Ross, J., SCOLNICK,E. M., AARONSON, S. A. & TODABO, G. J., Separation of murine cellular and murine leukemia virus DNA polymerases. Nature New Biol. (Lond.), 1971, 231, 163-169.
TYPE C MURINE RETROVIRUS RESTRICTION
113
[7] ROWE, W. P., PuGn, W. E. & HARTLEY, J . W . , Plaque assay technique for murine leukemia viruses. Virology, 1970, 42, 1136-1139. [81 TEICH, N. M., WEISS, R . A., MARTIN, G. R. & LOWRY, D. R., Virus infection of inurine teratocarcinoma stem cell lines. Cell, 1977, 12, 973-982. [9] ~VIGLER, M. H. & WEINSTEIN, I. B., A preparative method for obtaining enucleated mammalian cells. Biochem. biophgs. BeN. commun., 1975, 63, 669-674. [10] YANG, W. K., D'AuRIOL, L., YANG, D. M., KIGGANS, J. O., 0u, C. Y., Pt~RII~S, oI & EMANOiL-RAVICOVITCH,R., Restricted infectivity of ecotropic type C retrovirus in mouse teratocarcinoma cells : studies on viral DNA intermediates. J. supramol. Slruct., 1980, 14, 223-232.
Ann. Virol. [lnsL PasL), 133 E, n ~ I, 1982.
8
LOCALIZATION OF IN
TYPE
EMBRYONAL
BY
C RETROVIRUS CARCINOMA
CYTOCHALASIN
CELLULAR
RESTRICTION
CELLS
B-MEDIATED
FRACTIONATION
by M. Sall~, J. Tobaly, L. d'Auriol, R. EmanoI1-Ravicovitch and J. P~ri~s U.107 I N S E R M , LOI-CNRS, Ddpartemenl d'Oncologie Expdrimentale, lnslilat de Recherches sur les Maladies du Sang, Hdpilal Saint-Louis, 7574,5 Paris Cedex 10
SUMMARY Murine type C retroviruses do not replicatc in embryonal carcinoma cells (ECC), whilst they do normally in the differentiated derivatives of these cells. I lowever, coculture in the presence of a fusing agent allows the recovery of the input virus up to 5 days after infection. Cytoplasmic and nuclear fractions were obtained at different times after infection by treatment of the infected ECC with cytochalasin B and differential centrifugation. Virus was recovered up to 24 h post-infection by fusion of the cytoplasmic fraction with a permissive cell, whereas beyond 24 h virus was only recovered from the nuclear fraction. These results indicate that the cytoplasmic events of viral replication in the restrictive ECC are seemingly normal and that the block is very probably localized in the nucleus. KEY-WORDS : Retrovirus type C, Carcinogenesis, Restriction, Cytoehalasin B ; Fusion, Permissive cell, Cocultivation, Mouse.
INTRODUCTION ]'he susceptibility of murine teratocarcinoma-derived cell lines to ecotropic mouse type C retrovirus has been shown to be affected by the state of cellular differentiation [4, 8]. Embryonal carcinoma cells (ECC), which are the undifferentiated stem components of teratocarcinomas, Manuscrit re~u le 5 fdvrier 1982, acceptd le 21 avril 1982.
108
M. SALLI~ AND COLL.
are refractory to murine leukaemia virus (MuLV) multiplication. Differentiated teratocarcinoma-derived cells, however, are fully susceptible to MuLV. In a preceeding paper we demonstrated t h a t after infecting ECC with MuLV it was impossible to activate or rescue the input virus, even b y long-term cocultivation with highly susceptible mouse cells [5]. In the mixed cultures, the detection of virus production was never possible. Teich el al. [8] also reported t h a t fusion mediated b y Sendal virus of MuLVinfected ECC to mouse fibroblasts never resulted in detectable activation or rescue of the infecting virus. Taken together, these results suggest t h a t the viral DNA, if synthetised in infected ECC, is either biologically inactive or else placed under a v e r y strong restriction mechanism. However, Gautsch [2] has demonstrated t h a t restriction of MuLV in ECC can be overcome b y fusion with permissive cells during the 12-h period immediately following infection. The present study, which was undertaken in parallel with molecular biology experiments designed to examine the early phase of MuLV infection in ECC [1, 10], confirms the results of Gaustch and indicates t h a t the blocking event v e r y p r o b a b l y occurs after the newly synthetized provirus leaves the cytoplasm to enter the nucleus.
MATERIALS AND METHODS Cells.
PCC4 aza(-) cultures derived from a transplantable teratocarcinoma of the 129/SV mouse [3] were kindly supplied by F. Jacob and E. Jacob (Institut Pasteur, Paris). They are composed of totipotential ECC and give rise to multitissular tumours when injected into syngenic mice either subcutaneously or intraperitoneally. However, they never differentiate when maintained in vitro. The D55 murine cell line derived from BALB/c was the kind gift of R. Bassin (NC I, Bethesda, USA). It is highly permissive to type C murine retrovirus infection because it does not express the FV1 genetic system of restriction. Virus.
Moloney murine leukaemia virus (M-MuLV) was produced b y a chronically infected cell line (3T3 Swiss) inoculated with M-MuLV from the laboratory of G. Todaro (NCI). This cell line releases an NB, ecotropic, XC-positive virus with a titre of about 106 pfu/ml. When used as inoculum on PCC4 cells, the virus was employed at a multiplicity of 1. Viral deleclion.
Two methods were applied to detect and quantify viral production. Virion reverse transcriptase activity was measured in supernatant fluids after pelletization of the virus using synthetic template poly(A)-oligo(dT) and expressed as cpm CB = c y t o c h a l a s i n B. E C C = e m b r y o n a l c a r c i n o m a cell. MEM = (Eagle's) minimal essential medium.
M-MuLV = Moloney murine leukaemia virus. MuLV = murine leukaemia virus. PEG = p o l y e t h y l e n e glycol.
TYPE C MURINE RETROVIRUS RESTBICTION
109
of ~H-TMP incorporated into the final product of the reaction [6]. In addition, in silu XC plaque tests were performed on eocultures as described by Rowe el al. [7].
Cellular enuclealion. Cytochalasine B (CB) cellular enueleation was performed following the method described by Wigler and Weinstein [9]. PCC4 cells, detached with 0.5 mM EDTA, were well homogenised and resuspended at a final concentration of 0.5 to 2 • 107 cells/ml in MEM for suspension culture (without Ca++ or Mg ++) containing 12.5 % Ficoll, 10 y.g/ml CB and 200 ~g/ml each of penicillin and streptomycin. These cell suspensions were carefully applied to a sterile Fieoll density gradient prepared with the following layers: 25, 17, 16, 15, and 12.5 %, and incubated at 37 ~ C for 17 h. All the layers of the gradient were prepared in the same culture medium as described above (with antibiotics and CB). Gradients were centrifuged at 25,000 rpm for 1 h at 31 ~ C in an SW41 rotor (Beckman) prewarmed to 31 ~ C for 1 h in an ~ L350 Beckman ~ ultracentrifuge. After eentrifugation, cytoplasts and karyoplasts were collected separately from the top of the tube with a Pasteur pipette in the 15-17 % and 17-25 % region of the gradient, respectively. P u r i t y of both fractions was monitored by phase-constrast microscopy. Cytoplasts and karyoplasts were diluted in 10 ml of culture medium (DMEM) and centrifuged at 1,000 rpm for 5 min. Each pellet was then fused with entire cells (D55) as described below.
Polyethylene glycol (PEG) /usion technique. Infected and uninfected PCC4 cells or PCC4 cytoplasts and karyoplasts were washed and then centrifuged (1,300 rpm) with an equal number of D55 cells for 5 min at 25 ~ C. One ml of a 25 % P E G solution, prewarmed to 37 ~ C, was added to the mixed cells for exactly 1 min. Afterwards, the P E G solution was diluted with 9 ml of DMEM, and the mixed cultures were centrifuged for (1,300 rpm, 5 min, 25 ~ C), then the medium was removed and the eocultures were resuspended in a new culture medium before seeding in a 250-ml Nunelon flask. The coeultures were then split twice a week. At each passage level a fraction of the cells and their supernatants were submitted to the viral detection tests.
BESULTS T a b l e I p r e s e n t s t h e results of p r e l i m i n a r y e x p e r i m e n t s designed to e x a m i n e t h e effects of P E G - m e d i a t e d fusion on t h e r e c o v e r y of i n p u t virus f r o m P C C 4 - i n f e c t e d cells. T h e v i r u s was easily r e c o v e r e d f r o m 8 h t o 5 d a y s p o s t i n f e c t i o n w h e n t h e s e cells were c o c u l t i v a t e d w i t h D55-suse e p t i b l e cells in t h e p r e s e n c e of t h e fusing agent. A f t e r this t i m e no virus could be isolated f r o m a n y of t h e cocultures. No virus was d e t e c t a b l e in t h e s u p e r n a t a n t s of c o c u l t u r e s p e r f o r m e d in t h e a b s e n c e of P E G . Table II indicatesthat w h e n C B - t r e a t e d PCC4 cell f r a c t i o n s were fused to D55 cells in t h e p r e s e n c e of P E G , virus was r e c o v e r e d f r o m t h e f u s e d c y t o p l a s m i c a n d n u c l e a r f r a c t i o n s o n l y b e t w e e n 8 a n d 24 h a f t e r infection. A t 48- a n d 72-h t i m e points, e v e n t h o u g h t h e c y t o p l a s m i c f r a c t i o n did n o t give rise t o v i r u s p r o d u c t i o n , t h e n u c l e a r f r a c t i o n did, a n d r e c o v e r y of virus was possible. O v e r 72 h, cells were n o t s u b m i t t e d to CB t r e a t m e n t b e c a u s e of t h e i r r e q u i r e m e n t s for cell passage.
110
M.
T A B L E I. after
SALLt~
AND
COLL.
R e s c u e o f i n p u t v i r u s f r o m M - M u L V - i n f e c t e d P C C 4 cells PEG-mediated f u s i o n w i t h D55-susceptible cells (1). Time after infection 8 12 1 2 4 5 6 8
h h day days days days days days
XC t e s t
Reverse transcriptase a c t i v i t y (2)
+ (a) § + + -~ + 0 (a) 0
1,327 1,631 1,493 1,686 719 973 0 (4) 0
(1) Triplicate c u l t u r e s of f u s e d infected a n d u n i n f e c t e d PCC4/D55 cells were t e s t e d d u r i n g a period of 3 m o n t h s b y reverse t r a n s c r i p t a s e a n d in s i l u XC assays. (2) A m o u n t s given ( • 10 -3 c p m ) a r e t h e m e a n s of m o r e t h a n t h r e e successive passages a f t e r e n z y m a t i c activities r e a c h e d a plateau. (8) Syncitia too n u m e r o u s to be c o u n t e d were c o n s t a n t l y p r e s e n t over t h r e e or m o r e successive p a s s a g e s . (4) N e g a t i v e r e s u l t s were confirmed for b o t h t e s t s d u r i n g a period of 3 m o n t h s ; f u s e d u n i n f e c t e d P C C 4 / D 5 5 cell c u l t u r e s a l w a y s gave n e g a t i v e results.
T A B L E I I. - - R e s c u e o f i n p u t v i r u s f r o m n u c l e a r a n d c y t o p l a s m i c f r a c t i o n s o f M - M u L V - i n f e c t e d PCC4 cells fused to D 5 5 c e l l s .
Time after infection 8 12 24 48 72
h h h h h
Cytoplasmic fraction § (8) T (8) -~ (12) --
Nuclear fraction § • -~ + +
(8) (8) (8) (12) (12)
Triplicate c u l t u r e s of infected PCC4 cytop l a s m i c a n d n u c l e a r f r a c t i o n s f u s e d at each t i m e p o i n t to p e r m i s s i v e D55 ceils; t h e i r d e s c e n d e n t s were t e s t e d d u r i n g a period of 3 m o n t h s b y reverse t r a n s c r i p t a s e a n d in s i l u XC a s s a y s . Cocultures were considered as positive ( + ) w h e n t h e two viral m a r k e r s ( n u m e r o u s s y n c i t i a a n d reverse t r a n s c r i p t a s e a s s a y > 20 • 10 -3 c p m ) were s i m u l t a n e o u s l y positive over m o r e t h a n t h r e e successive passages, and negative (--) when both criteria were s u m u l t a n e o u s l y n e g a t i v e . N u m b e r s w i t h i n p a r e n t h e s e s indicate t h e n u m b e r of d a y s after c o c u l t i v a t i o n elapsed before t h e a s s a y b e c a m e positive.
TYPE C MURINE RETROVIRUS RESTRICTION
111
DISCUSSION Fusion of CB-fractionated M-MuLV-infected PCC4 cells to highly permissive fibroblastic mouse cells has been used in this s t u d y to localize the cellular region where the restriction of ecotropic t y p e C virus replication in the cells takes place. In confirmation with recent results [2], we have demonstrated t h a t fusion with an efficient fusing agent is sufficient to overcome the restriction event. Moreover, the block of viral multiplication probably occurs at the level of the cellular nucleus, after the cytoplasmic events of the virus replication cycle have been completed. These results raise an i m p o r t a n t question: was the virus which was recovered by cocultivation of whole cells and cellular fractions t h e progeny of the input M-MuLV? Several genetic markers (results not shown) such as XC test positivity, species tropism (ecotropism), FVI gene-dependent tropism, and pathogenicity (induction of leukemia) strongly indicated t h a t this was the case since t h e y are characteristics of the infecting M-MuLV and are not those of any t h e known Swiss/NIH (D55) endogenous viruses. Moreover, o ocultivation of uninfected PCC4 cells with D55 cells never led to virus activation. In addition, the fact t h a t no virus could be isolated from the m e d i u m used to wash cells after inoculation, and t h a t direct t r e a t m e n t of virus inocula with P E G destroyed all virus activity, seems to exclude any possible contamination of indicator cells with residual input virus (data not shown). Another argument used to explain the isolation of infectious virus from PCC4-infected cultures is the existence of some differentiated cells among the PCC4 cell population. These permissive cells would have supplied the virus detected after fusion and cocultivation. This possibility, however, is not consistent with our observations of the absolute negativity of several different viral replication markers in PCC4 cultures after infection with M-MuLV such as immunofluorescence, in situ XC tests, infectious centers and particularly direct cocultivation with permissive cells [4, 5]. One interesting observation m a d e during our studies was t h a t virus activity can be induced by fusion and cocultivation of the infected cells up to 5 days after inoculation. This result is not in agreement with the results of Gautsch [2], who was able to detect virus activity from ECC only up to 20 h after infection. This difference could be explained by the fact t h a t we used a m e t h o d involving viral amplification as evidence of the presence of virus; t h a t is our technique is more sensitive t h a n the in situ XC technique employed by Gaustch. In conclusion, the data presented here indicate t h a t the block of viral replication in PCC4 cells is v e r y probably localized in the nucleus. T h e y also confirm previous results indicating t h a t neosynthesis of infectious provirus during early steps of viral replication is accomplished normally, at least in PCC4 cells [9, 10]. These results however do not solve the import a n t problem of the presence or absence of normal proviral integration in PCC4 genomic DNA. This problem is the subject of a subsequent paper.
112
M. SALLI~ AND COLL.
RI~SUMI~ LOCALISATION DU PHI~NOMt~NE DE RESTRICTION DE LA MULTIPLICATION DES RI~TROVIRUS DE TYPE C, DANS LES CELLULES DU CARCINOME EMBRYONNAIRE~ PAR FRACTIONNEMENT )t LA CYTOCHALASINE B
Les r~trovirus murins ne se r~pliquent pas dans les cellules indiff~renei6es du eareinome embryonnaire (CCE) de la souris tandis qu'ils le font normalement dans les eellules diff6renci~es de m~me origine. Cependant la eoeulture en presence d'un agent fusionnant permet de r~cup6rer le virus infeetant et eela jusqu'h cinq jours apr~s infection. Les fractions cytoplasmiques et nuel6aires ont 6t~ obtenues, h des temps variables apr~s infection, grfiee h un t r a i t e m e n t des CCE par la eytochalasine B et une eentrifugation diff~rentielle. Le virus n'est r~isol~ dans la fraction cytoplasmique fusionn~e avec des eellules permissives que p e n d a n t les 24 premieres heures apr~s l'infeetion virale, tandis que la fraction nucl6aire p e r m e t de r~cup6rer le virus au-delh de 24 heures. Ces r~sultats m o n t r e n t que les ~tapes cytoplasmiques de la r~plication virale dans les CCE non permissives semblent normales et que le bloeage se situe tr~s probablem e n t au niveau nuel6aire. MOTS-CLt~S : R~trovirus de t y p e C, Carcinogen~se, Restriction, Cytochalasine B ; Fusion, Cellule permissive, Cocultivation, Souris.
REFERENCES [1] D'AuRIOL, L., YANG, W. K., TOBALY, J., CAVALIEBI, P., PgaIgs, J. & EMA-
NOiL-RAVICOVITCH, R., Studies on the restriction of ecotropic murine [2] [3]
[4]
[5]
[6]
retrovirus replication in mouse teratocarcinoma cells. J. gen. Virol., 1981, 55, 117-122. GAUTSCH, J. W., Embryonal carcinoma stein cells lack a function required for virus replication. Nature (Lond.), 1980, 285, 111-112. JAKOB, H., BOON, T., GAILLABD, J., NICOLAS, J. F., & JACOB, F., Teratocarcinome de la souris : isolement, culture et propri6tds de cellules h potentialitds multiples. Ann. Microbiol. (Inst. Pasteur), 1973, 124 B, 269-282. P~RII~S, J., ALVES-CARDOSO, E., CANIVET, M., DEBONS-GuILLEMIN, M., & LASNERET, J., Lack of multiplication of ecotropic murine C type viruses in mouse teratocarcinoma primitive cells. J. nat. Cancer Inst., 1977, 59, 463-465. PI~RII~S, J., DEBONS-GUILLEMIN, M. C., CANIVET, M., EMANOIL-RAvICOVITCH, R., TAVITIAN, A. & BoInOS, M., l~tude de la multiplication des virus murins de type C dans des ligndes cellulaires ddrivdes du tdratocarcinome de la souris. Nouv. Rev. [ranc. Hdmat., 1977, 18, 383-390. Ross, J., SCOLNICK,E. M., AARONSON, S. A. & TODABO, G. J., Separation of murine cellular and murine leukemia virus DNA polymerases. Nature New Biol. (Lond.), 1971, 231, 163-169.
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[7] ROWE, W. P., PuGn, W. E. & HARTLEY, J . W . , Plaque assay technique for murine leukemia viruses. Virology, 1970, 42, 1136-1139. [81 TEICH, N. M., WEISS, R . A., MARTIN, G. R. & LOWRY, D. R., Virus infection of inurine teratocarcinoma stem cell lines. Cell, 1977, 12, 973-982. [9] ~VIGLER, M. H. & WEINSTEIN, I. B., A preparative method for obtaining enucleated mammalian cells. Biochem. biophgs. BeN. commun., 1975, 63, 669-674. [10] YANG, W. K., D'AuRIOL, L., YANG, D. M., KIGGANS, J. O., 0u, C. Y., Pt~RII~S, oI & EMANOiL-RAVICOVITCH,R., Restricted infectivity of ecotropic type C retrovirus in mouse teratocarcinoma cells : studies on viral DNA intermediates. J. supramol. Slruct., 1980, 14, 223-232.
Ann. Virol. [lnsL PasL), 133 E, n ~ I, 1982.
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