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Response of somatic cell hybrids to exposure to adenovirus type 2 and 12
Experimental
Cell Research 56 (1969) 319-325
RESPONSE OF SOMATIC CELL HYBRIDS TO EXPOSURE TO ADENOVIRUS TYPE 2 AND 12 J. WEBER and H. F. STICH’ Department of Biology, McMaster University, Hamilton, Ont., Canada
SUMMARY The response of multinucleated homokaryocytes and heterokaryocytes to infection with human adenovirus type 2 and 12 was examined by enumerating the incidence of viral inclusion bodies in their nuclei. Hep-2 cells, which support the replication of adenovirus type 2 and type 12, and BHK-21 cells, in which adenovirus type 12 only induces an abortive replication cycle and adencvirus type 2 replication is restricted to few cells, were used. Cell fusion was induced by UVirradiated Sendai virus. A relatively high incidence of inclusion bodies was found to occur in BHK-21 nuclei of Hep-2/BHK-21 heterokaryocytes. Neither UV-irradiated Sendai virus, nor co-cultivation of Hep-2 and BHK-21 cells appeared to change the capacity of mononucleated Hep-2 or BHK-21 cells to form viral inclusion bodies. It was concluded that the presence of the Hep-2 nucleus or cytoplasm in the heterokaryocytes is essential for the induction of viral inclusion bodies in the non-permissive BHK-21 nuclei.
Induced fusion of various somatic cells has been successfully applied to recover infectious virus from W-40 and Rous sarcoma virus transformed human or mouse cells [7, 9, 10, 13, 141. It is generally assumedthat the addition of a permissive system which favours viral replication to a non-permissive one which contains a complete viral genome will induce the formation of mature infectious virions. In the present paper we report the induction of viral inclusion bodies (IB) in heterokaryocytes of Hep-2 and BHK-21 cells infected with human adenovirus type 12 (Ad. 12). The Hep-2 cells permit a complete viral replication cycle and thus represent the permissive system, whereas the BHK-21 cells support only an abortive replication cycle of Ad. 12 [I, 21. A second series of experiments concerns the enhanced capacity of BHK-21 homokaryocytes and Hep-2/BHK-21 heterokaryocytes to form viral IB following human adenovirus type 2 (Ad. 2) infection. Normally 1 Present address: Cancer Research Centre, University of British Columbia, Vancouver, Canada.
Ad. 2 seems to replicate only in few infected BHK-21 cells as indicated by the low incidence of IB (1). Cell fusion was induced with a relatively small dose of ultraviolet (UV) irradiated Sendai virus which did not induce chromosome damage and micronucleation. MATERIALS AND METHODS Cells A baby hamster kidney (BHK-21) and a human epithelial cell line (Hep-2) were employed in monolayer cultures. Both cell lines were maintained in Eagles minimal essential medium with Hanks salts supplemented with 200 U/ml penicillin and 40 yg/ml streptomycin sulphate and 5 % or 10 % fetal calf serum for the hamster and human line, respectively.
Viruses Ad. 2 and Ad. 12 were grown in suspension cultures of KB cells and titrated by plaquing following the methods described bv Green 13.41.Examination of the virus stocks in the electron m&&cope showed that all preparations were free of adeno-associated virus. Sendai virus was grown in the allantoic cavity of hens’ eggs. The allantoic fluids were collected 48 h post-infection, clarified by centrifugation and exposed to ultraviolet light (UV) for 8 min at a distance of 15 cm. All virus stocks were stored in liquid nitrogen. Exptl Cell Res 56
320
J. Weber & H. F. Stich
Table 1. Frequency of various multinucleated Hep-2 and BHK-21 homokaryocytes and Hep-2/BHK-21 heterokaryocytes following exposure to UV-irradiated Sendai virus Number of nuclei in fused cells 2
3
4
56789
10 11 12 13 14 15 16 17 18 19 20 21
Hep-2 homokaryocytes
61.0 21.8
7.9 4.2 2.2 1.4 0.9 0.3 0
0.2
0.1
0.1
0.1
BHK-21 homokaryocytes
53.1 25.7 13.6 4.4 2.2 1.0
Hep-2/BHK-21 heterokaryocytes
32.0 26.2 12.5 9.5 4.7 2.6 2.6 2.8 2.1 2.1 0.2 1.1 0.4 0.4 0.2 0.2 0.2 0.4
RESULTS
Cell fusion The method used followed, essentially, that discussed by Harris [5]. Prior to fusion the BHK-21 line was labeled for identification with 0.2 ,uc/ml of *H-thymidine (specific activity 6.7 c/mM) for two days. This technique resulted in labeling 97 % of the cells checked at 48 h after fusion. At the time of fusion lOO-fold excess of cold thymidine was added. Four to six million cells in 0.5 ml of each of the parental lines to be fused were mixed together in the presence of 1 ml of UV irradiated Sendai virus containing 256 hemaglutinating units. The mixture was allowed to stand for 15 min at 4”C, then was shaken for an additional 30 min at 37°C. The excess virus was removed and the fused cells were resuspended in growth medium containing 5 % fetal calf serum and seeded in Leighton tubes containing coverslips. The cultures were infected with the adenoviruses 12 h after fusion with 0.5 ml of virus suspension, the titer being adjusted to give an input multiplicity of x 10. Following a 2 h adsorption period the virus was rinsed off twice with phosphate buffered saline and then maintained in normal medium. Autoradiography was performed on aceto-orcein stained coverslips using Kodak NTB-3 emulsion. The preparations were usually exposed for a week.
Frequency of homokaryocyte and heterokaryocyte formation
Cell fusion, which was induced by UV-irradiated Sendai virus in a mixed population of Hep-2 and BHK-21 cells, resulted in a great variety of multinucleated homo- and heterokaryocytes. The relative frequencies of various multinucleated cells with only Hep-2 nuclei, only BHK-21 nuclei or with Hep-2 and BHK-21 nuclei are shown in table 1. The incidence of homokaryocytes of the two cell lines was similar when small numbers of cells were participating. The fusion of larger numbers of cells appeared to take place only among Hep-2 cells. With the exception of binucleated cells, the incidence of
Table 2. The effect of UV-irradiated
Sendai virus and co-cultivation on the incidence of Ad. 2 and Ad. 12 induced inclusion bodies (IB) in nonfused Hep-2 and BHK-21 cells IB in Hep-2 ( %)
Sendai virus
Pure culture U-W2)
Mixed culture U-W-2/ BHK-21)
Pure culture (BHK-21)
48
t+
35.6 36.1 75.7 71.8
33.0 35.8 69.0 66.8
0.0 0.0 :::
48
+
41.5 43.7
42.0 45.8
0.0 0.0
virus
Sample time (h)
Ad. 2
24
Ad. 12
Exptl Cell Res 56
IB in BHK-21 ( %) Mixed culture Wep-Y BHK-21) 0.0 i-z 2:3 0.0 0.0
Somatic cell hybrids exposed to adenovirus type 2 and 12
321
Table 3. Effect of homologous cell fusion on the incidence of nucleia with Ad. 2 or Ad. 12 induced IB
Virus Ad. 2 Ad. 12
Hep-2 nuclei of
BHK-21 nuclei of
Sample time @I
Mononucleated Multinucleated cells ( %) cells ( X)
Mononucleated Multinucleated cells ( %) cells ( %)
24 48 48
35.8 66.8 45.8
0.4 2.3 0.0
41.0 61.7 64.3
1.8 12.5 0.0
a A minimum of 300 nuclei were examined.
heterokaryocytes corresponded to that of Hep-2 homokaryocytes. The excess of binucleated homokaryocytes of both types over binucleated heterokaryocytes could be attributed to incomplete dispersion of cells prior to fusion and to failure of cytokinesis following nuclear division. Effect of Sendai virus on the incidence of Ad. 2 and Ad. 12 induced inclusion bodies in nonfused mononucleated cells
The effect of Sendai virus on the capacity of Hep-2 cells to form intranuclear IB was examined by comparing Hep-2 coverslip cultures infected with Ad. 2 and Ad. 12 with those exposed to UV-irradiated Sendai virus 12 h prior to infection with the two adenoviruses. The incidence of IB was enumerated in nonfused mononucleated cells. The results, summarized in table 2, show that UV-irradiated Sendai virus has no enhancing or retarding effect on the capacity of mononucleated Hep-2 cells to form IB. A similar experiment, which was performed with BHK-21 cultures, also revealed the inability of UVirradiated Sendai virus to affect the incidence of Ad. 2 or Ad. 12 induced IB in mononucleated cells (table 2). Effect of co-cultivation of Hep-2 and BHK-21 cells on the incidence of Ad. 2 and Ad. 12 inclusion bodies in nonfused mononucleated cells
Aliquots of Hep-2 and BHK-21 cells, which have been previously labeled with 3H-thymidine, were mixed, seeded on coverslips and infected with Ad. 2 or Ad. 12. A second set of cultures
consisting of the mixed Hep-2 and BHK-21 were treated with UV-irradiated Sendai virus 12 h prior to infection with the two adenoviruses. The incidence of IB was determined in the nonfused Hep-2 and BHK-21 cells of the infected mixed cultures. The BHK-21 cells were identified by their labeled nuclei on autoradiographic preparations. The results are presented in table 2. Co-cultivation of the two cell lines had no detectable influence on the incidence of IB in Hep-2 or BHK-21 mononucleated cells. The addition of UV-impaired Sendai virus to the mixed cultures prior to infection with the adenoviruses also did not alter the frequency of IB in mononucleated cells. This latter observation is in agreement with the above described results on non-mixed Hep-2 and BHK-21 cell populations exposed to UV-impaired Sendai virus and infected with Ad. 2 or Ad. 12. Response of Hep-2 or BHK-21 homokaryocytes to infection with Ad. 2 or Ad. 12
The effect of cell fusion on the formation of Ad. 2 and Ad. 12 IB was evaluated by comparing the incidence of IB containing nuclei in mononucleated cells with that in multinucleated cells of cultures treated with UV-impaired Sendai virus and thereupon infected with Ad. 2 or Ad. 12 (table 3). In the 24 h sample the frequency of nuclei in Hep-2 homokaryocytes with Ad. 2 induced IB (figs 1, 3) is 41 %, which comprises a slight increase over that in mononucleated cells (35.8 %). In the 48 h sample the incidence of IB in the nuclei of Hep-2 homokaryocytes is lower than that in the unfused mononucleated cells. This may be due to an earlier lysis of Ad. 2 Exptl Cell Res 56
322 J. Weber& H. F. Stich
Figs 1-4. Homokaryocytes of Hep-2 cells 48 h after infection. Inclusion bodies are present in all nuclei with the excention of those marked by an arrow: such nuclei which fail to form an inclusion body were rare. Figs 1 and 3 show cells infected with Ad: 2; figs 2 and 4 show cells infected with Ad. 12. x 550. Fi,. 5. Homokaryocyte _ _ of BHK-21 cells 48 h after infection with Ad. 2. Inclusion bodies are present in all nuclei.
x-&O.
Fig. 6. Homokaryocyte of BHK-21 cells 48 h after infection with Ad. 2. Inclusion bodies appear only in the lower two nuclei. This type of incomplete response of the homokaryocyte nuclei was rare. x 500.
infected multinucleated cells and their loss from the cell population. An enhancing effect of cell fusion was also observed in Hep-2 cultures infected with Ad. 12. The incidence of IB in the nuclei of Hep-2 homokaryocytes (figs 2, 4) Exptl
Cell Res 56
increased by about 19 yO over that in mononucleated cells. A pronounced stimulating effect of cell fusion on the incidence of IB was observed in BHK-21 cells infected with Ad. 2: 1.8 yO (24 h sample)
Fig. 7. Heterokaryocyte
48 h after infection with Ad. 2. The unlabeled Hep-2 nucleus and the labeled BHK-21 nucleus both have inclusion bodies. x 500. Fig. 8. Heterokaryocyte 48 h after infection with Ad, 2. Only the unlabeled Hep-2 nucleus (arrow) contains an inclusion body. x 500. Fig. 9. Heterokaryocyte 48 h after infection with Ad. 12. Inclusion bodies appear in all nuclei, including the labeled BHK-21 nucleus. x 500. Fig. IO. Heterokaryocyte 48 h after infection with Ad. 12. Only two Hep-2 nuclei (arrows) contain inclusion bodies. The labeled BHK-21 nuclei do not contain inclusion bodies. x 500.
and 12.5 % (48 h sample) in homokaryocytes (figs 5, 6) as compared to 0.4 % and 2.3 % in mononucleated cells. However infection with Ad. 12 failed to elicit IB in mononucleated or multinucleated BHK-21 cells. Response of Hep-2/BHK-21 heterokaryocytes to infection with Ad. 2 and Ad. 12
Equal numbers of 3H-thymidine labeled BHK-21 and unlabeled Hep-2 cells were mixed, treated with UV-irradiated Sendai virus, seeded on coverslips, infected 12 h later with Ad. 2, and sampled 24 or 48 h post-infection. The various types of multinucleated cells were identified on autoradiographic preparations and the incidences of IB were determined in Hep-2 and BHK-21 nuclei of heterokaryocytes. The results are expressedin terms of cells as well as nuclei, as the effect of fusion between Hep-2 and BHK21-
691803
21 cells on the incidence of IB in BHK-21 nuclei (table 4). As previously mentioned cell fusion increased the capacity of BHK-21 cells to form Ad. 2. A further enhancement of this capacity occurred in the Hep-2/BHK-21 heterokaryocyte (figs 7, 8). If the presence of a Hep-2 nucleus or cytoplasm in the heterokaryocyte has no positive influence on the formation of IB in BHK-21 nuclei, then their frequency should not exceed that found in BHK-21 homokaryocytes. The actually observed incidence of IB containing BHK-21 nuclei was 28.6 % compared to 2.1 % in homokaryocytes, at 24 h, and 36.9 % compared to 10.7 % at 48 h post-infection. This enhancement of IB formation in BHK-21 nuclei by the presence of Hep-21 nuclei and cytoplasm is unaltered when the analysis is based on cells (table 4). An experimental procedure similar to that Exptl Cell Res 56
324
J. Weber & H. F. Stich
Table 4. Effect of heterologous cell fusion on the incidence of Ad. 2 or Ad. 12 induced IB in BHK-21 nuclei Homokaryocytes
Virus Ad. 2 Ad. 12
Heterokaryocytes
Sample time 0-d
Cells with IB containing BHK-21 nuclei ( %)
IB containing BHK-21 nuclei ( X)
Cells with IB containing BHK-21 nuclei ( %)
IB containing BHK-21 nuclei ( X)
24 48 48
1.8 (378)’ 12.5 (136) 0 (138)
2.1 (921)b 10.7 (364) 0 (781)
30.9 (295) 65.4 (165) 35.5 (247)
28.6 (413) 36.9 (390) 33.2 (421)
a Total number of multinucleated cells examined. ’ Total number of BHK-21 nuclei in the multinucleated cells examined.
described above was used to examine the response of heterokaryocytes to infection with Ad. 12. The results are expressedas in the above experiment (table 4). Since Ad. 12 does not appear to induce IB in mononucleated or multinucleated BHK-21 homokaryocytes, their formation in BHK-21 nuclei of heterokaryocytes (35.5 % when the analysis is based on cells, 33.2 % when based on nuclei) must be due to the effect of Hep-2 nuclei or cytoplasm in these hybrid cells (figs 9, 10). To examine the effect of the number of parental nuclei in the heterokaryocytes on the formation of IB, the data was expressed as shown in table 5. The incidence of nuclei with IB of either parental type appears to vary independently of the number present in the heterokaryocyte. The increasing variation in the percentage of positive nuclei among the hetero-
karyocytes with increasing number of nuclei can be explained by the fewer number of cells within each class. DISCUSSION A high incidence of viral inclusion bodies was found to occur in hamster nuclei of multinucleated heterokaryocytes of Hep-2 and BHK21 cells infected with Ad. 12, which appears neither to replicate nor to induce IB in cultured BHK-21 cell populations. A pronounced enhancement of IB was also observed in heterokaryocytes infected with Ad. 2, which normally produces IB in only few BHK-21 cells. These results indicate that the formation of the intranuclear IB is under positive control by the Hep-2 nucleus or cytoplasm within the hybrid cell. The increased capacity of multinucleated homokaryocytes of Hep-2 cells and of BHK-21
Table 5. The effect of the number of nuclei in Ad. 12 infected heterokaryon cells on the frequency of IB in Hep-2 and BHK-21 nuclei Number of nuclei in heterokaryocytes
Hep-2 nuclei with IB ( %) BHK-21 nuclei with IB (a) Total number of nuclei analyzed Exptl Cell Res 56
4
2
3
100
96.8 97
5
6
7
8
9
10
11
12 13
88.9 95.0 92.8 79.5 74.4 94.9 90.0 -
85.7 80.8 70.4 77.8 72.2 92.8 88.9 35.7 77.8 50.0 28
57
60
65
54
42
48
54 100
33
-
14 15
16
17
18 19
70.0 100 84.6 84.6 57.1 100 100 66.7 100 50.0 33.3 66.7 100 52
28 30
16
17
0.0
18 38
Somatic cell hybrids exposed to adenovirus type 2 and 12
homokaryocytes to form IB following Ad. 2 infection seems to represent a phenomenon separate from that described above. Since the fused cells represent a larger target for the virus, it is likely that more particles penetrate these, than mononucleated cells. The presence of a single infectious particle may be sufficient to ensure a protein-composed IB for each nucleus of the heterokaryocyte by virtue of the shared cytoplasm. In this connection it is of interest to note the lack of IB in multinucleated BHK-21 homokaryocytes infected with Ad. 12 present in Ad. 12 infected heterokaryocytes. The inability of this adenovirus to replicate in BHK-21 cells is not affected by cell fusion. This contrasts with the capacity of Ad. 12 to induce IB in the heterokaryons, indicating that the presence of Hep-2 nucleus or cytoplasma rather than the process of cell fusion is responsible for the induction of Ad. 12 IB in the heterokaryocytes. At present one can only speculate about the mechanism responsible for the induction of viral IB in the normally non-permissive BHK-21 nuclei. One of the difficulties is our meager knowledge concerning virus formation in BHK21 nuclei [l]. Previous studies in lytic systems indicate that IB contain virus structural antigens [6], are the site of viral DNA synthesis [8] and that the components form mature virions [6]. Current studies employing immunofluorescence indicate that the IB in the BHK-21 nuclei indeed contain viral antigens. Titration of adenovirus by the number of cells forming IB indicates that results by this method over a reasonable range correspond very closely to results obtained by the tube titration method (unpublished observations). Therefore the presence of IB in these nuclei may indicate a viral replication cycle, however the possibility that the observed accumulation of viral products does not lead to the assembly of viruses cannot be completely discarded. The present study also does not show
325
whether transcription of the Ad. 2 or Ad. 12 genome in the BHK-21 nuclei of the heterokaryocytes is induced. The various components of the IB could be synthesized in the cytoplasm under control of the viral genome present in Hep-2 nuclei and thereupon transferred into the BHK-21 nuclei of hybrid cells. Since the synthesis of adenovirus coat proteins seemsto take place in the cytoplasm and a movement of these proteins into the nucleus appears to occur [ 11, 121 this possibility cannot be ruled out. The application of electron microscopy combined with autoradiography in the analysis of the hybrid cells may provide an answer to these unsolved problems. The nature of the induced IB in the BHK-21 nuclei is currently being investigated. This work was supported by a grant from the National Research Council of Canada.
REFERENCES 1. Cooper, J E K, Stich, H F & Yohn, D S, Virology 33 (1967) 533. 2. Doerfler, W, Proc natl acad sci US 60 (1968) 636. 3. Green, M & Kimes, R, Virology 31 (1967) 562. 4. Green, M & Pina, M, Ibid 20 (1963) 199. 5. Harris, J, Watkins, J F, Ford, C E & Schoefl, G I, J cell sci 1 (1966) 1. 6. Kalnins, V I, Stich, H F, Gregory, C & Yohn, D S, Cancer res 27 (1967) 1874. I. Koprowski, H, Jensen, F C & Steplewski, Z, Proc natl acad sci US 58 (1967) 127. 8. Martinez-Palomo, A & Granboulan, N J, Virology 1 (1967) 1010. 9. Svoboda, J, Hlozanek, T & Machala, 0, J gen virol 2 (1968) 461. 10. Takemoto, K, Todaro, G & Habel, K, Virology 35 (1968) 1. 11. Thomas, D C & Green, M, Proc natl acad sci US 56 (1966) 243. 12. Velicer, L F & Ginsberg, J S, Bact proc (1968) 167. 13. Watkins, J F & Dulbecco, R, Proc natl acad sci US 58 (1967) 1396. 14. Yamaguchi, N, Takeuchi, N & Yamamoto, T, Japan j exptl med 37 (1967) 83. Received November 5, 1968 Revised version received February 7, 1969
Exptl Cel! Res 56
Cell Research 56 (1969) 319-325
RESPONSE OF SOMATIC CELL HYBRIDS TO EXPOSURE TO ADENOVIRUS TYPE 2 AND 12 J. WEBER and H. F. STICH’ Department of Biology, McMaster University, Hamilton, Ont., Canada
SUMMARY The response of multinucleated homokaryocytes and heterokaryocytes to infection with human adenovirus type 2 and 12 was examined by enumerating the incidence of viral inclusion bodies in their nuclei. Hep-2 cells, which support the replication of adenovirus type 2 and type 12, and BHK-21 cells, in which adenovirus type 12 only induces an abortive replication cycle and adencvirus type 2 replication is restricted to few cells, were used. Cell fusion was induced by UVirradiated Sendai virus. A relatively high incidence of inclusion bodies was found to occur in BHK-21 nuclei of Hep-2/BHK-21 heterokaryocytes. Neither UV-irradiated Sendai virus, nor co-cultivation of Hep-2 and BHK-21 cells appeared to change the capacity of mononucleated Hep-2 or BHK-21 cells to form viral inclusion bodies. It was concluded that the presence of the Hep-2 nucleus or cytoplasm in the heterokaryocytes is essential for the induction of viral inclusion bodies in the non-permissive BHK-21 nuclei.
Induced fusion of various somatic cells has been successfully applied to recover infectious virus from W-40 and Rous sarcoma virus transformed human or mouse cells [7, 9, 10, 13, 141. It is generally assumedthat the addition of a permissive system which favours viral replication to a non-permissive one which contains a complete viral genome will induce the formation of mature infectious virions. In the present paper we report the induction of viral inclusion bodies (IB) in heterokaryocytes of Hep-2 and BHK-21 cells infected with human adenovirus type 12 (Ad. 12). The Hep-2 cells permit a complete viral replication cycle and thus represent the permissive system, whereas the BHK-21 cells support only an abortive replication cycle of Ad. 12 [I, 21. A second series of experiments concerns the enhanced capacity of BHK-21 homokaryocytes and Hep-2/BHK-21 heterokaryocytes to form viral IB following human adenovirus type 2 (Ad. 2) infection. Normally 1 Present address: Cancer Research Centre, University of British Columbia, Vancouver, Canada.
Ad. 2 seems to replicate only in few infected BHK-21 cells as indicated by the low incidence of IB (1). Cell fusion was induced with a relatively small dose of ultraviolet (UV) irradiated Sendai virus which did not induce chromosome damage and micronucleation. MATERIALS AND METHODS Cells A baby hamster kidney (BHK-21) and a human epithelial cell line (Hep-2) were employed in monolayer cultures. Both cell lines were maintained in Eagles minimal essential medium with Hanks salts supplemented with 200 U/ml penicillin and 40 yg/ml streptomycin sulphate and 5 % or 10 % fetal calf serum for the hamster and human line, respectively.
Viruses Ad. 2 and Ad. 12 were grown in suspension cultures of KB cells and titrated by plaquing following the methods described bv Green 13.41.Examination of the virus stocks in the electron m&&cope showed that all preparations were free of adeno-associated virus. Sendai virus was grown in the allantoic cavity of hens’ eggs. The allantoic fluids were collected 48 h post-infection, clarified by centrifugation and exposed to ultraviolet light (UV) for 8 min at a distance of 15 cm. All virus stocks were stored in liquid nitrogen. Exptl Cell Res 56
320
J. Weber & H. F. Stich
Table 1. Frequency of various multinucleated Hep-2 and BHK-21 homokaryocytes and Hep-2/BHK-21 heterokaryocytes following exposure to UV-irradiated Sendai virus Number of nuclei in fused cells 2
3
4
56789
10 11 12 13 14 15 16 17 18 19 20 21
Hep-2 homokaryocytes
61.0 21.8
7.9 4.2 2.2 1.4 0.9 0.3 0
0.2
0.1
0.1
0.1
BHK-21 homokaryocytes
53.1 25.7 13.6 4.4 2.2 1.0
Hep-2/BHK-21 heterokaryocytes
32.0 26.2 12.5 9.5 4.7 2.6 2.6 2.8 2.1 2.1 0.2 1.1 0.4 0.4 0.2 0.2 0.2 0.4
RESULTS
Cell fusion The method used followed, essentially, that discussed by Harris [5]. Prior to fusion the BHK-21 line was labeled for identification with 0.2 ,uc/ml of *H-thymidine (specific activity 6.7 c/mM) for two days. This technique resulted in labeling 97 % of the cells checked at 48 h after fusion. At the time of fusion lOO-fold excess of cold thymidine was added. Four to six million cells in 0.5 ml of each of the parental lines to be fused were mixed together in the presence of 1 ml of UV irradiated Sendai virus containing 256 hemaglutinating units. The mixture was allowed to stand for 15 min at 4”C, then was shaken for an additional 30 min at 37°C. The excess virus was removed and the fused cells were resuspended in growth medium containing 5 % fetal calf serum and seeded in Leighton tubes containing coverslips. The cultures were infected with the adenoviruses 12 h after fusion with 0.5 ml of virus suspension, the titer being adjusted to give an input multiplicity of x 10. Following a 2 h adsorption period the virus was rinsed off twice with phosphate buffered saline and then maintained in normal medium. Autoradiography was performed on aceto-orcein stained coverslips using Kodak NTB-3 emulsion. The preparations were usually exposed for a week.
Frequency of homokaryocyte and heterokaryocyte formation
Cell fusion, which was induced by UV-irradiated Sendai virus in a mixed population of Hep-2 and BHK-21 cells, resulted in a great variety of multinucleated homo- and heterokaryocytes. The relative frequencies of various multinucleated cells with only Hep-2 nuclei, only BHK-21 nuclei or with Hep-2 and BHK-21 nuclei are shown in table 1. The incidence of homokaryocytes of the two cell lines was similar when small numbers of cells were participating. The fusion of larger numbers of cells appeared to take place only among Hep-2 cells. With the exception of binucleated cells, the incidence of
Table 2. The effect of UV-irradiated
Sendai virus and co-cultivation on the incidence of Ad. 2 and Ad. 12 induced inclusion bodies (IB) in nonfused Hep-2 and BHK-21 cells IB in Hep-2 ( %)
Sendai virus
Pure culture U-W2)
Mixed culture U-W-2/ BHK-21)
Pure culture (BHK-21)
48
t+
35.6 36.1 75.7 71.8
33.0 35.8 69.0 66.8
0.0 0.0 :::
48
+
41.5 43.7
42.0 45.8
0.0 0.0
virus
Sample time (h)
Ad. 2
24
Ad. 12
Exptl Cell Res 56
IB in BHK-21 ( %) Mixed culture Wep-Y BHK-21) 0.0 i-z 2:3 0.0 0.0
Somatic cell hybrids exposed to adenovirus type 2 and 12
321
Table 3. Effect of homologous cell fusion on the incidence of nucleia with Ad. 2 or Ad. 12 induced IB
Virus Ad. 2 Ad. 12
Hep-2 nuclei of
BHK-21 nuclei of
Sample time @I
Mononucleated Multinucleated cells ( %) cells ( X)
Mononucleated Multinucleated cells ( %) cells ( %)
24 48 48
35.8 66.8 45.8
0.4 2.3 0.0
41.0 61.7 64.3
1.8 12.5 0.0
a A minimum of 300 nuclei were examined.
heterokaryocytes corresponded to that of Hep-2 homokaryocytes. The excess of binucleated homokaryocytes of both types over binucleated heterokaryocytes could be attributed to incomplete dispersion of cells prior to fusion and to failure of cytokinesis following nuclear division. Effect of Sendai virus on the incidence of Ad. 2 and Ad. 12 induced inclusion bodies in nonfused mononucleated cells
The effect of Sendai virus on the capacity of Hep-2 cells to form intranuclear IB was examined by comparing Hep-2 coverslip cultures infected with Ad. 2 and Ad. 12 with those exposed to UV-irradiated Sendai virus 12 h prior to infection with the two adenoviruses. The incidence of IB was enumerated in nonfused mononucleated cells. The results, summarized in table 2, show that UV-irradiated Sendai virus has no enhancing or retarding effect on the capacity of mononucleated Hep-2 cells to form IB. A similar experiment, which was performed with BHK-21 cultures, also revealed the inability of UVirradiated Sendai virus to affect the incidence of Ad. 2 or Ad. 12 induced IB in mononucleated cells (table 2). Effect of co-cultivation of Hep-2 and BHK-21 cells on the incidence of Ad. 2 and Ad. 12 inclusion bodies in nonfused mononucleated cells
Aliquots of Hep-2 and BHK-21 cells, which have been previously labeled with 3H-thymidine, were mixed, seeded on coverslips and infected with Ad. 2 or Ad. 12. A second set of cultures
consisting of the mixed Hep-2 and BHK-21 were treated with UV-irradiated Sendai virus 12 h prior to infection with the two adenoviruses. The incidence of IB was determined in the nonfused Hep-2 and BHK-21 cells of the infected mixed cultures. The BHK-21 cells were identified by their labeled nuclei on autoradiographic preparations. The results are presented in table 2. Co-cultivation of the two cell lines had no detectable influence on the incidence of IB in Hep-2 or BHK-21 mononucleated cells. The addition of UV-impaired Sendai virus to the mixed cultures prior to infection with the adenoviruses also did not alter the frequency of IB in mononucleated cells. This latter observation is in agreement with the above described results on non-mixed Hep-2 and BHK-21 cell populations exposed to UV-impaired Sendai virus and infected with Ad. 2 or Ad. 12. Response of Hep-2 or BHK-21 homokaryocytes to infection with Ad. 2 or Ad. 12
The effect of cell fusion on the formation of Ad. 2 and Ad. 12 IB was evaluated by comparing the incidence of IB containing nuclei in mononucleated cells with that in multinucleated cells of cultures treated with UV-impaired Sendai virus and thereupon infected with Ad. 2 or Ad. 12 (table 3). In the 24 h sample the frequency of nuclei in Hep-2 homokaryocytes with Ad. 2 induced IB (figs 1, 3) is 41 %, which comprises a slight increase over that in mononucleated cells (35.8 %). In the 48 h sample the incidence of IB in the nuclei of Hep-2 homokaryocytes is lower than that in the unfused mononucleated cells. This may be due to an earlier lysis of Ad. 2 Exptl Cell Res 56
322 J. Weber& H. F. Stich
Figs 1-4. Homokaryocytes of Hep-2 cells 48 h after infection. Inclusion bodies are present in all nuclei with the excention of those marked by an arrow: such nuclei which fail to form an inclusion body were rare. Figs 1 and 3 show cells infected with Ad: 2; figs 2 and 4 show cells infected with Ad. 12. x 550. Fi,. 5. Homokaryocyte _ _ of BHK-21 cells 48 h after infection with Ad. 2. Inclusion bodies are present in all nuclei.
x-&O.
Fig. 6. Homokaryocyte of BHK-21 cells 48 h after infection with Ad. 2. Inclusion bodies appear only in the lower two nuclei. This type of incomplete response of the homokaryocyte nuclei was rare. x 500.
infected multinucleated cells and their loss from the cell population. An enhancing effect of cell fusion was also observed in Hep-2 cultures infected with Ad. 12. The incidence of IB in the nuclei of Hep-2 homokaryocytes (figs 2, 4) Exptl
Cell Res 56
increased by about 19 yO over that in mononucleated cells. A pronounced stimulating effect of cell fusion on the incidence of IB was observed in BHK-21 cells infected with Ad. 2: 1.8 yO (24 h sample)
Fig. 7. Heterokaryocyte
48 h after infection with Ad. 2. The unlabeled Hep-2 nucleus and the labeled BHK-21 nucleus both have inclusion bodies. x 500. Fig. 8. Heterokaryocyte 48 h after infection with Ad, 2. Only the unlabeled Hep-2 nucleus (arrow) contains an inclusion body. x 500. Fig. 9. Heterokaryocyte 48 h after infection with Ad. 12. Inclusion bodies appear in all nuclei, including the labeled BHK-21 nucleus. x 500. Fig. IO. Heterokaryocyte 48 h after infection with Ad. 12. Only two Hep-2 nuclei (arrows) contain inclusion bodies. The labeled BHK-21 nuclei do not contain inclusion bodies. x 500.
and 12.5 % (48 h sample) in homokaryocytes (figs 5, 6) as compared to 0.4 % and 2.3 % in mononucleated cells. However infection with Ad. 12 failed to elicit IB in mononucleated or multinucleated BHK-21 cells. Response of Hep-2/BHK-21 heterokaryocytes to infection with Ad. 2 and Ad. 12
Equal numbers of 3H-thymidine labeled BHK-21 and unlabeled Hep-2 cells were mixed, treated with UV-irradiated Sendai virus, seeded on coverslips, infected 12 h later with Ad. 2, and sampled 24 or 48 h post-infection. The various types of multinucleated cells were identified on autoradiographic preparations and the incidences of IB were determined in Hep-2 and BHK-21 nuclei of heterokaryocytes. The results are expressedin terms of cells as well as nuclei, as the effect of fusion between Hep-2 and BHK21-
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21 cells on the incidence of IB in BHK-21 nuclei (table 4). As previously mentioned cell fusion increased the capacity of BHK-21 cells to form Ad. 2. A further enhancement of this capacity occurred in the Hep-2/BHK-21 heterokaryocyte (figs 7, 8). If the presence of a Hep-2 nucleus or cytoplasm in the heterokaryocyte has no positive influence on the formation of IB in BHK-21 nuclei, then their frequency should not exceed that found in BHK-21 homokaryocytes. The actually observed incidence of IB containing BHK-21 nuclei was 28.6 % compared to 2.1 % in homokaryocytes, at 24 h, and 36.9 % compared to 10.7 % at 48 h post-infection. This enhancement of IB formation in BHK-21 nuclei by the presence of Hep-21 nuclei and cytoplasm is unaltered when the analysis is based on cells (table 4). An experimental procedure similar to that Exptl Cell Res 56
324
J. Weber & H. F. Stich
Table 4. Effect of heterologous cell fusion on the incidence of Ad. 2 or Ad. 12 induced IB in BHK-21 nuclei Homokaryocytes
Virus Ad. 2 Ad. 12
Heterokaryocytes
Sample time 0-d
Cells with IB containing BHK-21 nuclei ( %)
IB containing BHK-21 nuclei ( X)
Cells with IB containing BHK-21 nuclei ( %)
IB containing BHK-21 nuclei ( X)
24 48 48
1.8 (378)’ 12.5 (136) 0 (138)
2.1 (921)b 10.7 (364) 0 (781)
30.9 (295) 65.4 (165) 35.5 (247)
28.6 (413) 36.9 (390) 33.2 (421)
a Total number of multinucleated cells examined. ’ Total number of BHK-21 nuclei in the multinucleated cells examined.
described above was used to examine the response of heterokaryocytes to infection with Ad. 12. The results are expressedas in the above experiment (table 4). Since Ad. 12 does not appear to induce IB in mononucleated or multinucleated BHK-21 homokaryocytes, their formation in BHK-21 nuclei of heterokaryocytes (35.5 % when the analysis is based on cells, 33.2 % when based on nuclei) must be due to the effect of Hep-2 nuclei or cytoplasm in these hybrid cells (figs 9, 10). To examine the effect of the number of parental nuclei in the heterokaryocytes on the formation of IB, the data was expressed as shown in table 5. The incidence of nuclei with IB of either parental type appears to vary independently of the number present in the heterokaryocyte. The increasing variation in the percentage of positive nuclei among the hetero-
karyocytes with increasing number of nuclei can be explained by the fewer number of cells within each class. DISCUSSION A high incidence of viral inclusion bodies was found to occur in hamster nuclei of multinucleated heterokaryocytes of Hep-2 and BHK21 cells infected with Ad. 12, which appears neither to replicate nor to induce IB in cultured BHK-21 cell populations. A pronounced enhancement of IB was also observed in heterokaryocytes infected with Ad. 2, which normally produces IB in only few BHK-21 cells. These results indicate that the formation of the intranuclear IB is under positive control by the Hep-2 nucleus or cytoplasm within the hybrid cell. The increased capacity of multinucleated homokaryocytes of Hep-2 cells and of BHK-21
Table 5. The effect of the number of nuclei in Ad. 12 infected heterokaryon cells on the frequency of IB in Hep-2 and BHK-21 nuclei Number of nuclei in heterokaryocytes
Hep-2 nuclei with IB ( %) BHK-21 nuclei with IB (a) Total number of nuclei analyzed Exptl Cell Res 56
4
2
3
100
96.8 97
5
6
7
8
9
10
11
12 13
88.9 95.0 92.8 79.5 74.4 94.9 90.0 -
85.7 80.8 70.4 77.8 72.2 92.8 88.9 35.7 77.8 50.0 28
57
60
65
54
42
48
54 100
33
-
14 15
16
17
18 19
70.0 100 84.6 84.6 57.1 100 100 66.7 100 50.0 33.3 66.7 100 52
28 30
16
17
0.0
18 38
Somatic cell hybrids exposed to adenovirus type 2 and 12
homokaryocytes to form IB following Ad. 2 infection seems to represent a phenomenon separate from that described above. Since the fused cells represent a larger target for the virus, it is likely that more particles penetrate these, than mononucleated cells. The presence of a single infectious particle may be sufficient to ensure a protein-composed IB for each nucleus of the heterokaryocyte by virtue of the shared cytoplasm. In this connection it is of interest to note the lack of IB in multinucleated BHK-21 homokaryocytes infected with Ad. 12 present in Ad. 12 infected heterokaryocytes. The inability of this adenovirus to replicate in BHK-21 cells is not affected by cell fusion. This contrasts with the capacity of Ad. 12 to induce IB in the heterokaryons, indicating that the presence of Hep-2 nucleus or cytoplasma rather than the process of cell fusion is responsible for the induction of Ad. 12 IB in the heterokaryocytes. At present one can only speculate about the mechanism responsible for the induction of viral IB in the normally non-permissive BHK-21 nuclei. One of the difficulties is our meager knowledge concerning virus formation in BHK21 nuclei [l]. Previous studies in lytic systems indicate that IB contain virus structural antigens [6], are the site of viral DNA synthesis [8] and that the components form mature virions [6]. Current studies employing immunofluorescence indicate that the IB in the BHK-21 nuclei indeed contain viral antigens. Titration of adenovirus by the number of cells forming IB indicates that results by this method over a reasonable range correspond very closely to results obtained by the tube titration method (unpublished observations). Therefore the presence of IB in these nuclei may indicate a viral replication cycle, however the possibility that the observed accumulation of viral products does not lead to the assembly of viruses cannot be completely discarded. The present study also does not show
325
whether transcription of the Ad. 2 or Ad. 12 genome in the BHK-21 nuclei of the heterokaryocytes is induced. The various components of the IB could be synthesized in the cytoplasm under control of the viral genome present in Hep-2 nuclei and thereupon transferred into the BHK-21 nuclei of hybrid cells. Since the synthesis of adenovirus coat proteins seemsto take place in the cytoplasm and a movement of these proteins into the nucleus appears to occur [ 11, 121 this possibility cannot be ruled out. The application of electron microscopy combined with autoradiography in the analysis of the hybrid cells may provide an answer to these unsolved problems. The nature of the induced IB in the BHK-21 nuclei is currently being investigated. This work was supported by a grant from the National Research Council of Canada.
REFERENCES 1. Cooper, J E K, Stich, H F & Yohn, D S, Virology 33 (1967) 533. 2. Doerfler, W, Proc natl acad sci US 60 (1968) 636. 3. Green, M & Kimes, R, Virology 31 (1967) 562. 4. Green, M & Pina, M, Ibid 20 (1963) 199. 5. Harris, J, Watkins, J F, Ford, C E & Schoefl, G I, J cell sci 1 (1966) 1. 6. Kalnins, V I, Stich, H F, Gregory, C & Yohn, D S, Cancer res 27 (1967) 1874. I. Koprowski, H, Jensen, F C & Steplewski, Z, Proc natl acad sci US 58 (1967) 127. 8. Martinez-Palomo, A & Granboulan, N J, Virology 1 (1967) 1010. 9. Svoboda, J, Hlozanek, T & Machala, 0, J gen virol 2 (1968) 461. 10. Takemoto, K, Todaro, G & Habel, K, Virology 35 (1968) 1. 11. Thomas, D C & Green, M, Proc natl acad sci US 56 (1966) 243. 12. Velicer, L F & Ginsberg, J S, Bact proc (1968) 167. 13. Watkins, J F & Dulbecco, R, Proc natl acad sci US 58 (1967) 1396. 14. Yamaguchi, N, Takeuchi, N & Yamamoto, T, Japan j exptl med 37 (1967) 83. Received November 5, 1968 Revised version received February 7, 1969
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