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Synthesis of viral components in hybrids of differentially permissive cells infected with adenovirus Type 12
Copyrighr 8 1972 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental
SYNTHESIS
Cell Research 74 (1972) 423-429
OF VIRAL
DIFFERENTIALLY
COMPONENTS PERMISSIVE
WITH ADENOVIRUS
IN HYBRIDS
OF
CELLS INFECTED TYPE 12
J. WEBER and S. MAK DEpartement de Microbiologic, Centre Hospitalier Universitaire, Universite’ de Sherbrooke, Sherbrooke, Q&bee, and Biology Department, McMaster University, Hamilton, Ont., Canada
SUMMARY Sendai virus induced heterokaryocytes of non-permissive BHK21 cells and permissive HEp2 cells were infected with adenovirus Type 12. Neoantigens, virus structural antigens and viral DNA were detected in the BHK21 nuclei as well as the HEp2 nuclei of heterokaryocytes, thus demonstrating positive control of these viral functions by the permissive cell component in the heterokaryocyte.
A complete understanding of the nature of the regulatory factors involved in the replication of a virus cannot be obtained without also understanding the nature of abortive infection by the same virus. BHK21 cells infected with human adenovirus type 12 (Ad. 12) represents an abortive system which allows only limited expression of viral functions, such as the synthesis of neoantigen and under appropriate conditions neoplastic transformation [3]. The limited response of hamster cells contrasts sharply with the response of HEp2 cells which facilitate the expression of a complete lytic cycle of the virus. In order to determine whether the permissive HEp2 cells contain some factor(s) which facilitate the replication of Ad. 12, HEp2 and BHK21 cells were fused together with inactivated Sendai virus and infected with Ad. 12. In a previous communication [14] it was shown that the normally non-permissive BHK21 cells become permissive as judged 28-
721810
by the formation of viral inclusion bodies (IB) in the hamster nuclei of heterokaryocytes. In this communication evidence is presented which demonstrates the presence of neoantigens, viral structural antigens and viral DNA in the induced BHK21 nuclei ot HEp2/BHK21 heterokaryocytes. MATERIAL
AND METHODS
Cells: Human epithelial (HEp2) and baby hamster kidney (BHK 21) cells were maintained in Eagle minimal essential medium with Hanks salts and 5 “b fetal calf serum. Human oral epidermoid carcinoma (KB) cells were grown in suspension with Joklik-modified minimum essential medium supplemented with 5 “6 horse serum. Viruses: Human adenovirus Type 12 (Ad.12, Huie) was grown in suspension cultures of KB cells purified by CsCl equilibrium centrifugation and titrated by the plaque method [4]. Sendai virus was grown in the allantoic cavity of 8 day old hens’ eggs, as described previously [14]. The infectious allantoic fluids were inactivated with ultraviolet light.
Cell fusion: The techniques of cell fusion, virus infection and autoradiography were described previously [141. Exptl Cell Res 74 (1972)
424
J. Weber & S. Mak
Table 1. Presence of neoantigens in mononucleate and multinucleate cells in Sendai virus fused cultures of HEp2 and BHK21 cells, 48 h after infection with Ad. 12
Cell type
Nuclear type
Nuclei of positive Cells cells containing containing neoantigen ( %) neoantigen ( %)
HEp2= BHK 21= HEp2/HEp2 BHK 21/BHK21 HEp2/BHK 2 1
HEp2 BHK 21 HEp2 BHK 21 HEp2 BHK 21
89 (500)b 30 (500) 91 (76) 35 (84) 57 (82) 57 (82)
89 (5OO)C 30 (500) 97 (301) 95 (324) 95 (183) 89 (120)
a Unfused cells in the same culture with multinucleate cells. b Number of cells examined. ’ Number of nuclei examined.
Zmmunofluorescence: Air dried cells were fixed for
were drawn, noting their position on the stage micrometer and indicating the presence or absence of neoantigens in the nuclei. The coverslips were subsequently rinsed, and autoradiographs prepared. The neoantigen positive cells were reIocated and the nuclei identified as HEp2 or BHK21 by RESULTS AND DISCUSSION the presence of silver grains. Neoantigens The results are shown in table 1. Almost Both the HEp2 and BHK21 cells infected without exception either all or none of the with Ad. 12 normally contain intranuclear nuclei of HEp2 or BHK21 homokaryocytes neoantigens. In the following experiments contained neoantigen. Among the positive the effect of the presence of several homo- homokaryocytes, only 2-5 %, in tota1, of logous or heterologous nuclei within the the nuclei appeared devoid of neoantigen. Cell fusion enhances the incidence of same cytoplasm on the synthesis of neoantigens and their appearance in the nuclei neoantigens in the nuclei of HEp2 homoof the different types of multinucleated cells karyocytes (from 89 % in monokaryocytes was examined. to 97 %), but especially in the nuclei of Aliquots of BHK21 cells which had been BHK21 homokaryocytes (from 30 % to previously labelled with 3H-thymidine for 95 %). The nuclei of the heterokaryocytes subsequent identification, and HEp2 cells responded similarly, with 95 % of HEp2 nuclei and 89 % of BKH21 nuclei (compared were mixed and fused with UV-inactivated Sendai virus. The cells were seededon cover- with 89 % and 30 % in the monokaryocytes, slips and 12 h later infected with 1 PFU/cell respectively) of positive cells containing of Ad. 12. Forty-eight hours thereafter the neoantigens. Since the multiplicity of infeccells were stained with fluorescein-labelled tion was 1 PFU/cell, the majority of multiantiserum against the neoantigens, and the nucleate cells would contain only one incidence of nuclei with fluorescence was ‘infected’ nucleus. The marked increase in scored. Randomly selected microscope fields the incidence of nuclei of multinucleate
20 min in carbon tetrachloride and stained for neoantigen by the direct method using fluorescein conjugated gamma globulin from Ad.12 tumorbearing Syrian hamsters. Virus structural antigens (V-ag) were stained by the indirect method using commercial antisera.
Synthesis of adenoviral components in cell hybrids
,125
Table 2. Percentages of V-antigen and IB positive nuclei of mononucleate and multinucleated cells containing at least one V-antigen or IB positive nucleus 48 h after infection with Ad. 12.
Cell type
Nuclear type
V-antigen only ( X)
HEp2 BHK 21 HEp2/HEp2 BHK 21/BHK 21 HEpZ/BHK 21 HEp2/BHK 21
HEp2 BHK 21 HEp2 BHK 21 HEp2 BHK 21
1.3 0 7.3 0 3.1 4.4
IB only (o/o)
V-antigen and IB ( %>
No. of nuclei examined
0.7 0 0.6 0 0 0
41.4 0 89.4 0 95.4 91.1
140 151 116 100 66 47
a Presence of viral antigens (V-antigen) and inclusion bodies (IB) were determined on the same cells.
cells which are positive for neoantigen suggests that the neoantigens may be transported into nuclei devoid of infectious viral DNA. A similar observation has previously been made in SV40 infected multinucleated cells [lo]. Enhancement is observable largely in the BHK21 nuclei, as the HEp2 nuclei already exhibit near maximum levels of neoantigen positive nuclei.
prepared for autoradiography. The microscope fields previously drawn were relocated, the BHK21 nuclei were identified by the silver grains, and the presence of IB in the different types of nuclei was scored. Figs 1 and 2 show the fluorescence and the autoradiography on the same positive heterokaryocyte. The results (table 2) are expressedin terms of the percentage of nuclei of infected cells Viral antigens which contained at least one nucleus positive In a previous communication it was already for either V-ag or IB, or both. Since the shown that viral inclusion bodies (IB) are same cells were scored for the expression of induced in the BHK21 nuclei of Ad. 12 both types of cytologically assayable viral infected heterokaryocytes [14]. In order to functions the degree of correlation would determine if such IB also contain viral incidentally indicate the specificity of the structural antigens (V-ag) the following IB assay. As expected none of the BHK21 experiments were performed. cells or homokaryocytes had either V-ag BHK21 cells labelled with tritiated-thymior IB. Fusion of these cells does not stimudine were mixed with an equal number of late V-ag synthesis. In HEp2 cells and HEp2 cells and fused with Sendai virus. homokaryocytes only a negligible percentage The cells were seededon coverslips and 12 h of nuclei did not contain both V-ag and later infected with Ad. 12. Two days after IB, indicating a good correlation of the two infection the coverslips were stained with assay systems. fluorescein-labelled antibody against V-ag In general it appears that a low percentage by the indirect method and selected micro- of nuclei are V-ag positive but do not scope fields containing mono- and multinucontain IB. This suggests that the antigen cleate cells were drawn indicating the presence assay is somewhat more sensitive than the IB or absence of positive fluorescence within assay. the nuclei. The coverslips were subsequently As previously observed with IB, fusion Exptl Cell Res 74 c.1972)
426 J. Weber & S. Mak of the BHK21 cells with HEp2 cells induced the presence of V-ag in the BHK21 nuclei of the heterokaryocytes. Since Ad. 12 does not induce V-ag in mononucleated or multinucleate BHK21 cells, the presence of these antigens in the BHK21 nuclei of heterokaryocytes must be due to the effect of HEp2 nuclei or cytoplasm. The relative proportion of the two types of nuclei did not appear to affect the presence of antigen, as one HEp2 nucleus in the heterokaryocyte ensured the staining of all resident nuclei. The transport mechanism into the non-permissive nuclei appears therefore to function normally. In these respects the results are similar to those obtained with monkey-mouse heterokaryocytes infected with SV40 [9]. Viral DNA The results described so far have shown that in Ad. 12 infected heterokaryocytes the permissive HEp2 nuclei and the non-permissive BHK21 nuclei both contained IB, neoantigen and V-ag. Whether viral DNA replication also takes place in the induced BHK21 nuclei of heterokaryocytes, remains to be determined. In cells which support adenovirus replication, host DNA synthesis is progressively inhibited and shut off completely late in the infectious cycle, while viral DNA synthesis continues [3]. It follows that in an Ad. 12 infected permissive cell system, a 3H-thymidine pulse subsequent to the shutting off of host DNA synthesis would be
incorporated into newly synthesized viral DNA. Heterokaryocyte cultures on coverslips were infected with 1 PFU/cell of Ad. 12. Twenty-four and 36 h post-infection 4 ,uCi/ ml (18 Ci/mM) of 3H-thymidine was added and incubation continued for 2 h. The coverslips were then rinsed, fixed in acetic-alcohol for 10 min and autoradiographs prepared. After exposure, the slides were examined. The percentage of IB positive single cells (and therefore HEp2 since single BHK21 cells do not form IB) which were labelled specifically over the IB was scored. The results (table 3) show that virtually 100 % of IB containing cells are labelled and that the label is localized specifically over the IB. Fig. 3 shows the localization of the silver grains over the IB in a polykaryocyte. Both the labelling and the IB are removed when the cells are treated with deoxyribonuclease prior to autoradiography, as shown by fig. 4. The percentages of IB containing polykaryocytes of infected cultures which were labelled were compared with the percentages of labelled polykaryocytes of uninfected control cultures. A similar comparison was also made on the basis of the percentages of nuclei of uninfected versus infected (IB containing) polykaryocytes. The results are shown in table 4. All infected polykaryocytes and their IB contained label. Since in uninfected control cultures the proportion of cells and nuclei labelled by a short pulse of
Figs I-4. Polykaryocyte cultures of HEp2 and BHK21 cells infected 12 h after cell fusion with Ad.12. Fig. 1. Heterokaryocyte 48 h after infection with Ad.12, stained with antibodies to viral antigens by the indirect method. Each of the 8 nuclei in the cell shows positive fluorescence. x 400. Fig. 2. The same heterokaryo-
cyte as in fig. 1, after autoradiography and staining with aceto-orcein. The label identifies the BHK 21 nucleus, x 400. Fig. 3. Polykaryocyte labelled for 2 h with 4 &i/ml of 3H-thymidine at 36 h post-infection. The autoradiograph was stained with aceto-orcein. The nuclei show compartmentalization with label only over the inclusion bodies. Each nucleus appears to contain a single unlabelled nucleolus distinctly separated from the inclusions. x 600. Fig. 4. Polykaryocyte culture treated as in fig. 3, but digested with deoxyribonuclease prior to autoradiography. The digestion was carried on for 2 h, at 37°C with 10 pug/ml of enzyme (Nutritional Biochemicals) in Tris buffer, pH 7.0 containing 0.2 M MgCl,. X 400. Exptl
Cell Res 74 (1972)
Synthesis of adenoviral componentsin cell hybrids 421
Exptl Cell Res 74 (1972)
428
J. Weber & S. Mak
Table 3. Distribution
of pulse-labeled
DNA
at 24 and 36 h after infection of HEp2 cells with Ad.12 Time of 2 h pulse-labelling post-infection (hours)
IB positive cells labelled’ (%I
IB positive cells in which label is localized over IB* (%I
a At least 500 cells examined. * A minimum of 1 500 cells examined.
tritiated thymidine was only 43 % (cells) and 22 % (nuclei), respectively, at 36 h after cell fusion, the increased percentage of IB containing labelled cell population is probably due to viral DNA synthesis. Although host DNA synthesis is shut off in the late phase of lytic systems,the existence of this mechanism in induced BHK21 nuclei of heterokaryocytes can only be inferred. The following experiment was designed to forego such an assumption and to provide evidence for viral DNA synthesis even if no inhibition of host DNA synthesis occurs in BHK21 cells. By means of an independent, but concurrent fusion experiment, using prelabelled BHK21 cells, the proportion of polykaryocytes which are heterokaryocytes, was determined (table 4). 28 % of the polykaryocytes in the infected culture are, by analogy, heterokaryocytes. It has previously been shown that under these conditions, about 90 % of the BHK21 nuclei in heterokaryocytes are induced to form IB [14]. By knowing that only HEp2/BHK21 and HEp2/HEp2 polykaryocytes contain IB, and by assuming that the number of BHK21 and HEp2 nuclei in the heterokaryocytes is about equal, the total number of IB containing BHK21 nuclei in the examined heterokaryocytes can be calculated from table 4. Since about 30 % of these BHK21 Exptl Cell Res 74 (1972)
nuclei may be labelled due to the S phase of the cell cycle, the actual number of BHK21 nuclei labelled which cannot be accounted for by cellular DNA synthesis is about 100. In the absence of induction of cellular DNA synthesis in these IB containing BHK21 nuclei, this number is too high to be attributed to experimental error. In addition, induction of cellular DNA synthesis by cell fusion can be excluded on the basis of the data in table 4. The proportion of uninfected polykaryocyte nuclei showing DNA synthesis decreasesfrom 38 % at 24 h to 22 % at 36 h whereas the proportion of infected polykaryocyte nuclei showing DNA synthesis remains 99-100 %. Although induction of cellular DNA synthesis [I 1, 121 in the BHK21 nuclei of polykaryocytes by virus infection cannot be entirely excluded, the following observations argue against such a possibility: (i) the label is always localized over the inclusion body, whereas the cellular DNA has been shown to be outside the inclusions in adenovirus infected nuclei [ 151; (ii) the presence of adenovirus-specific proteins in the BHK21 nuclei, as observed by immunofluorescence, may be expected to inhibit cellular DNA synthesis at 36 h post-infection [3, 6, 7, 81; (iii) Strohl [II] obtained, with an input multiplicity of 100, a maximum induction of cellular DNA synthesis in BHK21 cells of 70 % at 18 h, which subsequently declined to 45 % at 24 h post-infection, compared with our labelling frequency of 100% at both 24 and 36 h post-infection. The appearance of newly replicated viral DNA in the BHK21 nucleus of heterokaryocytes probably represents in situ synthesized molecules as there is at present no evidence to suspect transmission of DNA from the HEp2 to the BHK21 nuclei. The failure of the replication of Ad. 12 DNA in BHK21 cells [2] as the primary lesion leading
Synthesis of adenoviral componentsin cell hybrids 429 Table 4. Distribution of pulse-labelled DNA at 24 and 36 h after infection of fused culturc~J of HEp2 and BHK21 cells with Ad.12 Time of 2 hour pulse-lahelling post-infection (hours)
Polykaryocytes which are heterokaryocytes’
24 36
29 (S9)b 27 (75)
Polykaryocytes in S phase ( “/o)
IB positive Polykaryocyte polykaryocytes nuclei in S phase in S phase ( ‘%I ( %)
IB positive polykaryocytc nuclei with label over IB ( ‘!<>)
69 (175) 43 (217)
38 (492) 22 (566)
100 (181) 99 (473)
100 (62) 100 (166)
a As determined by a separate but concurrent experiment using Sendai virus from the same pool asin the other experiments. b Number of cells or nuclei.
to abortive infection is thus overcome by the milieu of the heterokaryocyte. The nature of the mechanism or factor(s) provided by the permissive HEp2 component remains to be resolved. Experiments we have published elsewhere [13] suggest that the protection of the virus DNA from its normal fate of partial degradation in BHK21 cells [I], by the HEp2 component of the heterokaryocyte may be the crucial factor in the prevention of abortive infection.
This investigation was supported by grants from the National Research Council of Canada and Medical Research Council of Canada.
2. Doerfler, W & Lundholm, U, Virology 40 (1970) 754. Green, M, Ann rev biochem 39 (1970) 701. :: Green, M, Pina, M L Kimes, R C, Virology 31 (1967) 562. 5. zurHausen, H & Sokol, F, J virol 4 (1969) 256. 6. Hodge, L D & Scharff, M D, Virology 37 (1969) 554. 7. Ledinko, N & Fang, C K Y, J virol 4 (I 969) 123. 8. Levine, A J & Ginsberg, H S, J virol 2 ( 1968) 430. 9. Steplewski, Z & Koprowski, H, Exptl cell res 57 (1969) 433. 10. ~tepldwski, Z, Knowles, B B & Koprowski, H, Proc natl acad sci US 59 (1968) 769. 11. Strohl, W A, Virology 39 (1969) 653. 12. Takahashi, M, Ogino, T, Baba, K & Onaka, M, Virology 37 (1969) 513. 13. Weber, J & Mak, S, Virology 42 (1970) 540. 14. Weber, J & Stich, H F, Exptl cell res 56 (1969) 319. 15. Yamamoto, T & Shahrabadi, M S, Can j microbiol 17 (1971) 249.
REFERENCES 1. Doerfler, W, Proc natl acad sci US 60 (1968) 636.
Received February 15, 1972 Revised version received April 4, 1972
Exptl Cell Res 74 (1972)
Experimental
SYNTHESIS
Cell Research 74 (1972) 423-429
OF VIRAL
DIFFERENTIALLY
COMPONENTS PERMISSIVE
WITH ADENOVIRUS
IN HYBRIDS
OF
CELLS INFECTED TYPE 12
J. WEBER and S. MAK DEpartement de Microbiologic, Centre Hospitalier Universitaire, Universite’ de Sherbrooke, Sherbrooke, Q&bee, and Biology Department, McMaster University, Hamilton, Ont., Canada
SUMMARY Sendai virus induced heterokaryocytes of non-permissive BHK21 cells and permissive HEp2 cells were infected with adenovirus Type 12. Neoantigens, virus structural antigens and viral DNA were detected in the BHK21 nuclei as well as the HEp2 nuclei of heterokaryocytes, thus demonstrating positive control of these viral functions by the permissive cell component in the heterokaryocyte.
A complete understanding of the nature of the regulatory factors involved in the replication of a virus cannot be obtained without also understanding the nature of abortive infection by the same virus. BHK21 cells infected with human adenovirus type 12 (Ad. 12) represents an abortive system which allows only limited expression of viral functions, such as the synthesis of neoantigen and under appropriate conditions neoplastic transformation [3]. The limited response of hamster cells contrasts sharply with the response of HEp2 cells which facilitate the expression of a complete lytic cycle of the virus. In order to determine whether the permissive HEp2 cells contain some factor(s) which facilitate the replication of Ad. 12, HEp2 and BHK21 cells were fused together with inactivated Sendai virus and infected with Ad. 12. In a previous communication [14] it was shown that the normally non-permissive BHK21 cells become permissive as judged 28-
721810
by the formation of viral inclusion bodies (IB) in the hamster nuclei of heterokaryocytes. In this communication evidence is presented which demonstrates the presence of neoantigens, viral structural antigens and viral DNA in the induced BHK21 nuclei ot HEp2/BHK21 heterokaryocytes. MATERIAL
AND METHODS
Cells: Human epithelial (HEp2) and baby hamster kidney (BHK 21) cells were maintained in Eagle minimal essential medium with Hanks salts and 5 “b fetal calf serum. Human oral epidermoid carcinoma (KB) cells were grown in suspension with Joklik-modified minimum essential medium supplemented with 5 “6 horse serum. Viruses: Human adenovirus Type 12 (Ad.12, Huie) was grown in suspension cultures of KB cells purified by CsCl equilibrium centrifugation and titrated by the plaque method [4]. Sendai virus was grown in the allantoic cavity of 8 day old hens’ eggs, as described previously [14]. The infectious allantoic fluids were inactivated with ultraviolet light.
Cell fusion: The techniques of cell fusion, virus infection and autoradiography were described previously [141. Exptl Cell Res 74 (1972)
424
J. Weber & S. Mak
Table 1. Presence of neoantigens in mononucleate and multinucleate cells in Sendai virus fused cultures of HEp2 and BHK21 cells, 48 h after infection with Ad. 12
Cell type
Nuclear type
Nuclei of positive Cells cells containing containing neoantigen ( %) neoantigen ( %)
HEp2= BHK 21= HEp2/HEp2 BHK 21/BHK21 HEp2/BHK 2 1
HEp2 BHK 21 HEp2 BHK 21 HEp2 BHK 21
89 (500)b 30 (500) 91 (76) 35 (84) 57 (82) 57 (82)
89 (5OO)C 30 (500) 97 (301) 95 (324) 95 (183) 89 (120)
a Unfused cells in the same culture with multinucleate cells. b Number of cells examined. ’ Number of nuclei examined.
Zmmunofluorescence: Air dried cells were fixed for
were drawn, noting their position on the stage micrometer and indicating the presence or absence of neoantigens in the nuclei. The coverslips were subsequently rinsed, and autoradiographs prepared. The neoantigen positive cells were reIocated and the nuclei identified as HEp2 or BHK21 by RESULTS AND DISCUSSION the presence of silver grains. Neoantigens The results are shown in table 1. Almost Both the HEp2 and BHK21 cells infected without exception either all or none of the with Ad. 12 normally contain intranuclear nuclei of HEp2 or BHK21 homokaryocytes neoantigens. In the following experiments contained neoantigen. Among the positive the effect of the presence of several homo- homokaryocytes, only 2-5 %, in tota1, of logous or heterologous nuclei within the the nuclei appeared devoid of neoantigen. Cell fusion enhances the incidence of same cytoplasm on the synthesis of neoantigens and their appearance in the nuclei neoantigens in the nuclei of HEp2 homoof the different types of multinucleated cells karyocytes (from 89 % in monokaryocytes was examined. to 97 %), but especially in the nuclei of Aliquots of BHK21 cells which had been BHK21 homokaryocytes (from 30 % to previously labelled with 3H-thymidine for 95 %). The nuclei of the heterokaryocytes subsequent identification, and HEp2 cells responded similarly, with 95 % of HEp2 nuclei and 89 % of BKH21 nuclei (compared were mixed and fused with UV-inactivated Sendai virus. The cells were seededon cover- with 89 % and 30 % in the monokaryocytes, slips and 12 h later infected with 1 PFU/cell respectively) of positive cells containing of Ad. 12. Forty-eight hours thereafter the neoantigens. Since the multiplicity of infeccells were stained with fluorescein-labelled tion was 1 PFU/cell, the majority of multiantiserum against the neoantigens, and the nucleate cells would contain only one incidence of nuclei with fluorescence was ‘infected’ nucleus. The marked increase in scored. Randomly selected microscope fields the incidence of nuclei of multinucleate
20 min in carbon tetrachloride and stained for neoantigen by the direct method using fluorescein conjugated gamma globulin from Ad.12 tumorbearing Syrian hamsters. Virus structural antigens (V-ag) were stained by the indirect method using commercial antisera.
Synthesis of adenoviral components in cell hybrids
,125
Table 2. Percentages of V-antigen and IB positive nuclei of mononucleate and multinucleated cells containing at least one V-antigen or IB positive nucleus 48 h after infection with Ad. 12.
Cell type
Nuclear type
V-antigen only ( X)
HEp2 BHK 21 HEp2/HEp2 BHK 21/BHK 21 HEpZ/BHK 21 HEp2/BHK 21
HEp2 BHK 21 HEp2 BHK 21 HEp2 BHK 21
1.3 0 7.3 0 3.1 4.4
IB only (o/o)
V-antigen and IB ( %>
No. of nuclei examined
0.7 0 0.6 0 0 0
41.4 0 89.4 0 95.4 91.1
140 151 116 100 66 47
a Presence of viral antigens (V-antigen) and inclusion bodies (IB) were determined on the same cells.
cells which are positive for neoantigen suggests that the neoantigens may be transported into nuclei devoid of infectious viral DNA. A similar observation has previously been made in SV40 infected multinucleated cells [lo]. Enhancement is observable largely in the BHK21 nuclei, as the HEp2 nuclei already exhibit near maximum levels of neoantigen positive nuclei.
prepared for autoradiography. The microscope fields previously drawn were relocated, the BHK21 nuclei were identified by the silver grains, and the presence of IB in the different types of nuclei was scored. Figs 1 and 2 show the fluorescence and the autoradiography on the same positive heterokaryocyte. The results (table 2) are expressedin terms of the percentage of nuclei of infected cells Viral antigens which contained at least one nucleus positive In a previous communication it was already for either V-ag or IB, or both. Since the shown that viral inclusion bodies (IB) are same cells were scored for the expression of induced in the BHK21 nuclei of Ad. 12 both types of cytologically assayable viral infected heterokaryocytes [14]. In order to functions the degree of correlation would determine if such IB also contain viral incidentally indicate the specificity of the structural antigens (V-ag) the following IB assay. As expected none of the BHK21 experiments were performed. cells or homokaryocytes had either V-ag BHK21 cells labelled with tritiated-thymior IB. Fusion of these cells does not stimudine were mixed with an equal number of late V-ag synthesis. In HEp2 cells and HEp2 cells and fused with Sendai virus. homokaryocytes only a negligible percentage The cells were seededon coverslips and 12 h of nuclei did not contain both V-ag and later infected with Ad. 12. Two days after IB, indicating a good correlation of the two infection the coverslips were stained with assay systems. fluorescein-labelled antibody against V-ag In general it appears that a low percentage by the indirect method and selected micro- of nuclei are V-ag positive but do not scope fields containing mono- and multinucontain IB. This suggests that the antigen cleate cells were drawn indicating the presence assay is somewhat more sensitive than the IB or absence of positive fluorescence within assay. the nuclei. The coverslips were subsequently As previously observed with IB, fusion Exptl Cell Res 74 c.1972)
426 J. Weber & S. Mak of the BHK21 cells with HEp2 cells induced the presence of V-ag in the BHK21 nuclei of the heterokaryocytes. Since Ad. 12 does not induce V-ag in mononucleated or multinucleate BHK21 cells, the presence of these antigens in the BHK21 nuclei of heterokaryocytes must be due to the effect of HEp2 nuclei or cytoplasm. The relative proportion of the two types of nuclei did not appear to affect the presence of antigen, as one HEp2 nucleus in the heterokaryocyte ensured the staining of all resident nuclei. The transport mechanism into the non-permissive nuclei appears therefore to function normally. In these respects the results are similar to those obtained with monkey-mouse heterokaryocytes infected with SV40 [9]. Viral DNA The results described so far have shown that in Ad. 12 infected heterokaryocytes the permissive HEp2 nuclei and the non-permissive BHK21 nuclei both contained IB, neoantigen and V-ag. Whether viral DNA replication also takes place in the induced BHK21 nuclei of heterokaryocytes, remains to be determined. In cells which support adenovirus replication, host DNA synthesis is progressively inhibited and shut off completely late in the infectious cycle, while viral DNA synthesis continues [3]. It follows that in an Ad. 12 infected permissive cell system, a 3H-thymidine pulse subsequent to the shutting off of host DNA synthesis would be
incorporated into newly synthesized viral DNA. Heterokaryocyte cultures on coverslips were infected with 1 PFU/cell of Ad. 12. Twenty-four and 36 h post-infection 4 ,uCi/ ml (18 Ci/mM) of 3H-thymidine was added and incubation continued for 2 h. The coverslips were then rinsed, fixed in acetic-alcohol for 10 min and autoradiographs prepared. After exposure, the slides were examined. The percentage of IB positive single cells (and therefore HEp2 since single BHK21 cells do not form IB) which were labelled specifically over the IB was scored. The results (table 3) show that virtually 100 % of IB containing cells are labelled and that the label is localized specifically over the IB. Fig. 3 shows the localization of the silver grains over the IB in a polykaryocyte. Both the labelling and the IB are removed when the cells are treated with deoxyribonuclease prior to autoradiography, as shown by fig. 4. The percentages of IB containing polykaryocytes of infected cultures which were labelled were compared with the percentages of labelled polykaryocytes of uninfected control cultures. A similar comparison was also made on the basis of the percentages of nuclei of uninfected versus infected (IB containing) polykaryocytes. The results are shown in table 4. All infected polykaryocytes and their IB contained label. Since in uninfected control cultures the proportion of cells and nuclei labelled by a short pulse of
Figs I-4. Polykaryocyte cultures of HEp2 and BHK21 cells infected 12 h after cell fusion with Ad.12. Fig. 1. Heterokaryocyte 48 h after infection with Ad.12, stained with antibodies to viral antigens by the indirect method. Each of the 8 nuclei in the cell shows positive fluorescence. x 400. Fig. 2. The same heterokaryo-
cyte as in fig. 1, after autoradiography and staining with aceto-orcein. The label identifies the BHK 21 nucleus, x 400. Fig. 3. Polykaryocyte labelled for 2 h with 4 &i/ml of 3H-thymidine at 36 h post-infection. The autoradiograph was stained with aceto-orcein. The nuclei show compartmentalization with label only over the inclusion bodies. Each nucleus appears to contain a single unlabelled nucleolus distinctly separated from the inclusions. x 600. Fig. 4. Polykaryocyte culture treated as in fig. 3, but digested with deoxyribonuclease prior to autoradiography. The digestion was carried on for 2 h, at 37°C with 10 pug/ml of enzyme (Nutritional Biochemicals) in Tris buffer, pH 7.0 containing 0.2 M MgCl,. X 400. Exptl
Cell Res 74 (1972)
Synthesis of adenoviral componentsin cell hybrids 421
Exptl Cell Res 74 (1972)
428
J. Weber & S. Mak
Table 3. Distribution
of pulse-labeled
DNA
at 24 and 36 h after infection of HEp2 cells with Ad.12 Time of 2 h pulse-labelling post-infection (hours)
IB positive cells labelled’ (%I
IB positive cells in which label is localized over IB* (%I
a At least 500 cells examined. * A minimum of 1 500 cells examined.
tritiated thymidine was only 43 % (cells) and 22 % (nuclei), respectively, at 36 h after cell fusion, the increased percentage of IB containing labelled cell population is probably due to viral DNA synthesis. Although host DNA synthesis is shut off in the late phase of lytic systems,the existence of this mechanism in induced BHK21 nuclei of heterokaryocytes can only be inferred. The following experiment was designed to forego such an assumption and to provide evidence for viral DNA synthesis even if no inhibition of host DNA synthesis occurs in BHK21 cells. By means of an independent, but concurrent fusion experiment, using prelabelled BHK21 cells, the proportion of polykaryocytes which are heterokaryocytes, was determined (table 4). 28 % of the polykaryocytes in the infected culture are, by analogy, heterokaryocytes. It has previously been shown that under these conditions, about 90 % of the BHK21 nuclei in heterokaryocytes are induced to form IB [14]. By knowing that only HEp2/BHK21 and HEp2/HEp2 polykaryocytes contain IB, and by assuming that the number of BHK21 and HEp2 nuclei in the heterokaryocytes is about equal, the total number of IB containing BHK21 nuclei in the examined heterokaryocytes can be calculated from table 4. Since about 30 % of these BHK21 Exptl Cell Res 74 (1972)
nuclei may be labelled due to the S phase of the cell cycle, the actual number of BHK21 nuclei labelled which cannot be accounted for by cellular DNA synthesis is about 100. In the absence of induction of cellular DNA synthesis in these IB containing BHK21 nuclei, this number is too high to be attributed to experimental error. In addition, induction of cellular DNA synthesis by cell fusion can be excluded on the basis of the data in table 4. The proportion of uninfected polykaryocyte nuclei showing DNA synthesis decreasesfrom 38 % at 24 h to 22 % at 36 h whereas the proportion of infected polykaryocyte nuclei showing DNA synthesis remains 99-100 %. Although induction of cellular DNA synthesis [I 1, 121 in the BHK21 nuclei of polykaryocytes by virus infection cannot be entirely excluded, the following observations argue against such a possibility: (i) the label is always localized over the inclusion body, whereas the cellular DNA has been shown to be outside the inclusions in adenovirus infected nuclei [ 151; (ii) the presence of adenovirus-specific proteins in the BHK21 nuclei, as observed by immunofluorescence, may be expected to inhibit cellular DNA synthesis at 36 h post-infection [3, 6, 7, 81; (iii) Strohl [II] obtained, with an input multiplicity of 100, a maximum induction of cellular DNA synthesis in BHK21 cells of 70 % at 18 h, which subsequently declined to 45 % at 24 h post-infection, compared with our labelling frequency of 100% at both 24 and 36 h post-infection. The appearance of newly replicated viral DNA in the BHK21 nucleus of heterokaryocytes probably represents in situ synthesized molecules as there is at present no evidence to suspect transmission of DNA from the HEp2 to the BHK21 nuclei. The failure of the replication of Ad. 12 DNA in BHK21 cells [2] as the primary lesion leading
Synthesis of adenoviral componentsin cell hybrids 429 Table 4. Distribution of pulse-labelled DNA at 24 and 36 h after infection of fused culturc~J of HEp2 and BHK21 cells with Ad.12 Time of 2 hour pulse-lahelling post-infection (hours)
Polykaryocytes which are heterokaryocytes’
24 36
29 (S9)b 27 (75)
Polykaryocytes in S phase ( “/o)
IB positive Polykaryocyte polykaryocytes nuclei in S phase in S phase ( ‘%I ( %)
IB positive polykaryocytc nuclei with label over IB ( ‘!<>)
69 (175) 43 (217)
38 (492) 22 (566)
100 (181) 99 (473)
100 (62) 100 (166)
a As determined by a separate but concurrent experiment using Sendai virus from the same pool asin the other experiments. b Number of cells or nuclei.
to abortive infection is thus overcome by the milieu of the heterokaryocyte. The nature of the mechanism or factor(s) provided by the permissive HEp2 component remains to be resolved. Experiments we have published elsewhere [13] suggest that the protection of the virus DNA from its normal fate of partial degradation in BHK21 cells [I], by the HEp2 component of the heterokaryocyte may be the crucial factor in the prevention of abortive infection.
This investigation was supported by grants from the National Research Council of Canada and Medical Research Council of Canada.
2. Doerfler, W & Lundholm, U, Virology 40 (1970) 754. Green, M, Ann rev biochem 39 (1970) 701. :: Green, M, Pina, M L Kimes, R C, Virology 31 (1967) 562. 5. zurHausen, H & Sokol, F, J virol 4 (1969) 256. 6. Hodge, L D & Scharff, M D, Virology 37 (1969) 554. 7. Ledinko, N & Fang, C K Y, J virol 4 (I 969) 123. 8. Levine, A J & Ginsberg, H S, J virol 2 ( 1968) 430. 9. Steplewski, Z & Koprowski, H, Exptl cell res 57 (1969) 433. 10. ~tepldwski, Z, Knowles, B B & Koprowski, H, Proc natl acad sci US 59 (1968) 769. 11. Strohl, W A, Virology 39 (1969) 653. 12. Takahashi, M, Ogino, T, Baba, K & Onaka, M, Virology 37 (1969) 513. 13. Weber, J & Mak, S, Virology 42 (1970) 540. 14. Weber, J & Stich, H F, Exptl cell res 56 (1969) 319. 15. Yamamoto, T & Shahrabadi, M S, Can j microbiol 17 (1971) 249.
REFERENCES 1. Doerfler, W, Proc natl acad sci US 60 (1968) 636.
Received February 15, 1972 Revised version received April 4, 1972
Exptl Cell Res 74 (1972)