VIROLOGY
90,
147-150
Expression
(1978)
of Polyoma Virus in Heterokaryons between Carcinoma Ceils and Differentiated Cells MARTINE
Dbpartement
de Biologic
BOCCARA moliculaire,
Znstitut Accepted
FRANCOISE
AND Pasteur, June
Embryonal
KELLY
25 rue du Dr. Roux,
75015 Paris,
France
I, 1978
Mouse embryonal carcinoma cells are resistant to infection with SV40 and polyoma virus and neither early T nor late V antigens can be detected by indirect immunofluorescence. In order to investigate the position of the block, we have examined the expression of polyoma virus in heterokaryons between the embryonal carcinoma cells PCC3/A/l and two types of susceptible cells: the PCD3-differentiated cells derived from teratocarcinoma which are fully permissive to polyoma virus and BHKZl cells which allow only the expression of early viral genes. The results of these experiments permit to circumscribe the position of the block at a very early step of the lytic cycle, after penetration of the viral particle and before synthesis of the T antigen.
Recent studies by Swartzendruber and Lehman (1) have shown that the pluripotent embryonal carcinoma cells of the mouse teratocarcinoma OTT6050 (2) are resistant to infection by simian virus 40 and polyoma virus, whereas differentiated derivatives are susceptible to both viruses. We have confirmed these results on several established cell lines derived from the same teratocarcinoma, two multipotent embryonal carcinoma lines and three differentiated derivatives lines ( 3). A first step in trying to understand the mechanism of the resistance of embryonal carcinoma cells to viral infection consists in the localization of the block. Recent data by Swartzendruber et al. (4) indicate that the viruses enter the cells, However, the methods used in their studies allow detection of physical particles, but give little information about infectious particles. In the experiments described here, we have examined the expression of polyoma virus in heterokaryons between infected embryonal carcinoma cells and differentiated cells, which allows us to follow the fate of the infectious particles themselves. We have used two teratocarcinomaderived cell lines: an embryonal carcinoma cell line PCC3/A/l which differentiates in vivo and in vitro, and a differentiated cell line PCD3 derived from embryoid bodies
grown in vitro which has a stable fibroblast morphology in culture. Both cell lines have been described elsewhere (5). We have shown earlier that cells from the PCCS/A/l line are resistant to infection by polyoma virus and SV40 when maintained in their undifferentiated state ( 3). In contrast, cells from the PCD3 line are fully permissive for polyoma virus and can be abortively infected and transformed by SV40 (3). We have also used BHK21 cells, from a fibroblastic line derived from baby hamster kidney, which allow only the expression of the early genes of polyoma virus (6) and are a gift of Dr. Montagnier (Institut Pasteur). In the experiments described here, PCC3/A/l cells were fused with either PCD3 cells or BHK21 cells. Cells from one parent were first infected with polyoma virus then fused with cells from the other parent. The fraction of V antigen-positive heterokaryons was determined 48 hr after infection. The cells, grown as described earlier (3), were seeded at a density of 5.10” cells per 60-mm tissue culture dish. They were infected 24 hr later with polyoma virus at a multiplicity of infection of 20 PFU per cell and the virus was adsorbed 1 hr at 37”. In some crosses, infected cells were treated in the interval between viral infection and 147 0042~6822/78/0901-0147$02.00/O Copyright 0 1978 by Academic
AU rights of reproduction
Press, Inc. in any form reserved.
148
SHORT
COMMUNICATIONS
fusion with a l/1000 dilution of anti-polyoma capsid antiserum (the antiserum was a gift of Dr. Denise Paulin, Institut Pasteur, and its titer determined by hemagglutination test was l/300,000) and neuraminidase (cholera filtrate, Sigma, l/200). The other parent had been previously labeled for 48 hr with 1 @/ml [“Hlthymidine (25 Ci/mM, C.E.A.), then placed in cold medium containing 0.01 mM thymidine for 3 hr. Both parent cells (about 5.10” cells of each type) were harvested, fused according to the procedure described by Watkins and Dulbecco ( 7) using P-propionolactone-inactivated Sendai virus (a gift from Esther Vogt, Institut Pasteur) and seeded on glass coverslips. The cells were maintained in the presence of antipolyoma capsid antiserum and neuraminidase as above. Coverslips were removed 48 hr after infection, fixed and stained for V antigen by indirect immunofluorescence (8) using anti-polyoma capsid sheep antiserum (a gift of Dr. Denise Paulin) as the specific reagent, and fluoresceine-conjugated y-globulins (Institut Pasteur Production). The cell preparations were then processed for autoradiography with L4 Ilford emulsion and developed after 6 days. Cells were examined with a Zeiss microscope equipped with ultraviolet optics for the presence of V antigen positive and/or radioactively labeled nuclei. In the labeling conditions used, grains were found in nearly 100% of the labeled cells. Heterokaryons were therefore identified as cells having several nuclei (2 to 4 on the average), one of them at least containing grains, and the percentage of V antigen positive heterokaryons was scored. One such V antigen positive heterokaryon is shown in Fig. 1. Unfused cells from both parents were also examined. The results are summarized in Table 1. 1. When PCC3/A/l cells were infected and fused 4 hr later with PCD3 cells, 20% of the heterokaryons become V antigenpositive. The contribution of both parent cells is necessary for virus expression, because fewer than 1 in lo4 PCC3/A/l and PCD3 cells and no homokaryons were stained. Inasmuch as none of the PCC3/A/l homokaryons express V antigen, it is unlikely that V antigen expression
in heterokaryons is induced by the process of fusion. Direct infection of the PCD3 cells by free extracellular virus can also be excluded because most single PCD3 cells and PCD3 homokaryons remain V antigen-negative. This result strongly suggests that the virus enters PCCS/A/l cells but is expressed only upon fusion with the permissive cells. The virus appears to be stable within the cell for at least 24 hr inasmuch as delaying fusion between infected PCC3/A/l cells and PCD3 cells by that period of time did not significantly affect the proportion of positive heterokaryons. 2. When PCCB/A/l cells are fused with polyoma-infected PCD3 cells, 60% of the heterokaryons were V antigen-positive, a fraction identical with that observed for unfused PCD3 cells. Clearly, expression of polyoma virus in PCD3 cells is unaffected by fusion with PCC3/A/l cells, which rules out the existence of a diffusible repressor in the embryonal carcinoma cells. This is strengthened by direct infection of the heterokaryons: PCC3/A/l cells and [“HIthymidine-labeled PCD3 cells were fused and infected 24 hr after fusion with polyoma virus. The fraction of V antigen positive cells 48 hr after infection was 45%’ for the heterokaryons, 49% for the PCD3 homokaryons, and 34% for the isolated PCD3 cells. PCC3/A/l homokaryons were all negative. Heterokaryons, therefore, appear to have a susceptibility to viral infection comparable to that of PCD3 homokaryons or isolated PCD3 cells. 3. When PCC3/A/l cells were fused with the nonpermissive BHK21 cells previously infected with polyoma virus, 5% of the heterokaryons were V antigen-positive. None of the unfused BHK21 cells (lo4 cells examined) were stained and BHK21 homokaryons were also negative. It is reasonable to assume that PCCS/A/l cells contain cellular factors which are not present in BHK21 cells and allow late expression of the virus in the heterokaryons, although at a rather low efficiency. In any one heterokaryon, all nuclei were either stained or unstained for V antigen. The fusion experiments reported here strongly indicate that resistance to viral infection is not due to a lack of penetration
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FIG. 1. Heterokaryon between polyoma virus-infected PCC3/A/I cells and cH]thymidine-labeled PCD3 cells. Staining for polyoma V antigen by indirect immunofluorescence was followed by autoradiography. A, dark field ultraviolet illumination. The two nuclei in the heterokaryon are V antigen-positive and one of them shows grains. The irregular outline of the fluorescence in the radioactively labeled nucleus is probably an artifact caused by the grains. None of the isolated PCC3/A/l cells (small) and PCD3 cells (large) shows immunofluorescence. One V antigen negative PCC3/A/I homokaryon is also shown. B, bright field illumination. One isolated PCD3 cell and a PCD3 nucleus in the heterokaryon have incorporated comparable amounts of cH]thymidine. 800x.
150
SHORT
COMMUNICATIONS TABLE
EXPRESSION Parent cell Infecwd” PCCS/A/I’ PCC3/A/I’ PCD3 BHK
Noninfected6 PCD.3 PCD3 PCC3/A/l PCC3/A/
1
OF POLYOMA Time of fusion after infection” (hr)
1
VIRUS
IN HETEROKARYONS
% of V antigenpositive heterokaryons 48 hr after infection”
4 24 4 4
20 30 60 5
‘% of V antigen-positive Infected
parent
cro.01
90,
147-150
Expression
(1978)
of Polyoma Virus in Heterokaryons between Carcinoma Ceils and Differentiated Cells MARTINE
Dbpartement
de Biologic
BOCCARA moliculaire,
Znstitut Accepted
FRANCOISE
AND Pasteur, June
Embryonal
KELLY
25 rue du Dr. Roux,
75015 Paris,
France
I, 1978
Mouse embryonal carcinoma cells are resistant to infection with SV40 and polyoma virus and neither early T nor late V antigens can be detected by indirect immunofluorescence. In order to investigate the position of the block, we have examined the expression of polyoma virus in heterokaryons between the embryonal carcinoma cells PCC3/A/l and two types of susceptible cells: the PCD3-differentiated cells derived from teratocarcinoma which are fully permissive to polyoma virus and BHKZl cells which allow only the expression of early viral genes. The results of these experiments permit to circumscribe the position of the block at a very early step of the lytic cycle, after penetration of the viral particle and before synthesis of the T antigen.
Recent studies by Swartzendruber and Lehman (1) have shown that the pluripotent embryonal carcinoma cells of the mouse teratocarcinoma OTT6050 (2) are resistant to infection by simian virus 40 and polyoma virus, whereas differentiated derivatives are susceptible to both viruses. We have confirmed these results on several established cell lines derived from the same teratocarcinoma, two multipotent embryonal carcinoma lines and three differentiated derivatives lines ( 3). A first step in trying to understand the mechanism of the resistance of embryonal carcinoma cells to viral infection consists in the localization of the block. Recent data by Swartzendruber et al. (4) indicate that the viruses enter the cells, However, the methods used in their studies allow detection of physical particles, but give little information about infectious particles. In the experiments described here, we have examined the expression of polyoma virus in heterokaryons between infected embryonal carcinoma cells and differentiated cells, which allows us to follow the fate of the infectious particles themselves. We have used two teratocarcinomaderived cell lines: an embryonal carcinoma cell line PCC3/A/l which differentiates in vivo and in vitro, and a differentiated cell line PCD3 derived from embryoid bodies
grown in vitro which has a stable fibroblast morphology in culture. Both cell lines have been described elsewhere (5). We have shown earlier that cells from the PCCS/A/l line are resistant to infection by polyoma virus and SV40 when maintained in their undifferentiated state ( 3). In contrast, cells from the PCD3 line are fully permissive for polyoma virus and can be abortively infected and transformed by SV40 (3). We have also used BHK21 cells, from a fibroblastic line derived from baby hamster kidney, which allow only the expression of the early genes of polyoma virus (6) and are a gift of Dr. Montagnier (Institut Pasteur). In the experiments described here, PCC3/A/l cells were fused with either PCD3 cells or BHK21 cells. Cells from one parent were first infected with polyoma virus then fused with cells from the other parent. The fraction of V antigen-positive heterokaryons was determined 48 hr after infection. The cells, grown as described earlier (3), were seeded at a density of 5.10” cells per 60-mm tissue culture dish. They were infected 24 hr later with polyoma virus at a multiplicity of infection of 20 PFU per cell and the virus was adsorbed 1 hr at 37”. In some crosses, infected cells were treated in the interval between viral infection and 147 0042~6822/78/0901-0147$02.00/O Copyright 0 1978 by Academic
AU rights of reproduction
Press, Inc. in any form reserved.
148
SHORT
COMMUNICATIONS
fusion with a l/1000 dilution of anti-polyoma capsid antiserum (the antiserum was a gift of Dr. Denise Paulin, Institut Pasteur, and its titer determined by hemagglutination test was l/300,000) and neuraminidase (cholera filtrate, Sigma, l/200). The other parent had been previously labeled for 48 hr with 1 @/ml [“Hlthymidine (25 Ci/mM, C.E.A.), then placed in cold medium containing 0.01 mM thymidine for 3 hr. Both parent cells (about 5.10” cells of each type) were harvested, fused according to the procedure described by Watkins and Dulbecco ( 7) using P-propionolactone-inactivated Sendai virus (a gift from Esther Vogt, Institut Pasteur) and seeded on glass coverslips. The cells were maintained in the presence of antipolyoma capsid antiserum and neuraminidase as above. Coverslips were removed 48 hr after infection, fixed and stained for V antigen by indirect immunofluorescence (8) using anti-polyoma capsid sheep antiserum (a gift of Dr. Denise Paulin) as the specific reagent, and fluoresceine-conjugated y-globulins (Institut Pasteur Production). The cell preparations were then processed for autoradiography with L4 Ilford emulsion and developed after 6 days. Cells were examined with a Zeiss microscope equipped with ultraviolet optics for the presence of V antigen positive and/or radioactively labeled nuclei. In the labeling conditions used, grains were found in nearly 100% of the labeled cells. Heterokaryons were therefore identified as cells having several nuclei (2 to 4 on the average), one of them at least containing grains, and the percentage of V antigen positive heterokaryons was scored. One such V antigen positive heterokaryon is shown in Fig. 1. Unfused cells from both parents were also examined. The results are summarized in Table 1. 1. When PCC3/A/l cells were infected and fused 4 hr later with PCD3 cells, 20% of the heterokaryons become V antigenpositive. The contribution of both parent cells is necessary for virus expression, because fewer than 1 in lo4 PCC3/A/l and PCD3 cells and no homokaryons were stained. Inasmuch as none of the PCC3/A/l homokaryons express V antigen, it is unlikely that V antigen expression
in heterokaryons is induced by the process of fusion. Direct infection of the PCD3 cells by free extracellular virus can also be excluded because most single PCD3 cells and PCD3 homokaryons remain V antigen-negative. This result strongly suggests that the virus enters PCCS/A/l cells but is expressed only upon fusion with the permissive cells. The virus appears to be stable within the cell for at least 24 hr inasmuch as delaying fusion between infected PCC3/A/l cells and PCD3 cells by that period of time did not significantly affect the proportion of positive heterokaryons. 2. When PCCB/A/l cells are fused with polyoma-infected PCD3 cells, 60% of the heterokaryons were V antigen-positive, a fraction identical with that observed for unfused PCD3 cells. Clearly, expression of polyoma virus in PCD3 cells is unaffected by fusion with PCC3/A/l cells, which rules out the existence of a diffusible repressor in the embryonal carcinoma cells. This is strengthened by direct infection of the heterokaryons: PCC3/A/l cells and [“HIthymidine-labeled PCD3 cells were fused and infected 24 hr after fusion with polyoma virus. The fraction of V antigen positive cells 48 hr after infection was 45%’ for the heterokaryons, 49% for the PCD3 homokaryons, and 34% for the isolated PCD3 cells. PCC3/A/l homokaryons were all negative. Heterokaryons, therefore, appear to have a susceptibility to viral infection comparable to that of PCD3 homokaryons or isolated PCD3 cells. 3. When PCC3/A/l cells were fused with the nonpermissive BHK21 cells previously infected with polyoma virus, 5% of the heterokaryons were V antigen-positive. None of the unfused BHK21 cells (lo4 cells examined) were stained and BHK21 homokaryons were also negative. It is reasonable to assume that PCCS/A/l cells contain cellular factors which are not present in BHK21 cells and allow late expression of the virus in the heterokaryons, although at a rather low efficiency. In any one heterokaryon, all nuclei were either stained or unstained for V antigen. The fusion experiments reported here strongly indicate that resistance to viral infection is not due to a lack of penetration
.- .- -
.
.
- .-
.- - _
- .
-
- -
-__
-
----
._ --.
-
---.--
FIG. 1. Heterokaryon between polyoma virus-infected PCC3/A/I cells and cH]thymidine-labeled PCD3 cells. Staining for polyoma V antigen by indirect immunofluorescence was followed by autoradiography. A, dark field ultraviolet illumination. The two nuclei in the heterokaryon are V antigen-positive and one of them shows grains. The irregular outline of the fluorescence in the radioactively labeled nucleus is probably an artifact caused by the grains. None of the isolated PCC3/A/l cells (small) and PCD3 cells (large) shows immunofluorescence. One V antigen negative PCC3/A/I homokaryon is also shown. B, bright field illumination. One isolated PCD3 cell and a PCD3 nucleus in the heterokaryon have incorporated comparable amounts of cH]thymidine. 800x.
150
SHORT
COMMUNICATIONS TABLE
EXPRESSION Parent cell Infecwd” PCCS/A/I’ PCC3/A/I’ PCD3 BHK
Noninfected6 PCD.3 PCD3 PCC3/A/l PCC3/A/
1
OF POLYOMA Time of fusion after infection” (hr)
1
VIRUS
IN HETEROKARYONS
% of V antigenpositive heterokaryons 48 hr after infection”
4 24 4 4
20 30 60 5
‘% of V antigen-positive Infected
parent
cro.01
