Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4508-4510, October 1977
Cell Biology
Helper factor(s) for growth of adeno-associated virus in cells transformed by adenovirus 12 (cell fusion/transformed cells/T antigen/restriction endonuclease)
HIROSHI HANDA, KAZUKO SHIROKI, AND HIROTO SHIMOJO Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan
Communicated by Wallace P. Rowe, August 8, 1977
ABSTRACT Evidence is presented that a helper factor(s) for growth of adeno-associated virus (AAV) is present in cells transformed by adenovirus type 12 (Adl2). The growth of AAV was observed in heterokaryons formed by fusion of human KB and Adl2-transformed rodent cells by using ultraviolet-inactivated Sendai virus without coinfection of cells with adenovirus. The presence of the helper factor(s) for AAV growth in rat cells transformed by the EcoRI-C fragment or the HindIII-G fragment of Adl2 DNA suggests that the helper factor(s) induced by infection with adenovirus is the Adl2-specific T antigen.
min, washed with Ca-balanced salt solution, soaked in lowcalcium (1 mM) Eagle's minimum essential medium supplemented with 2% fetal calf serum, and incubated at 370 in a CO2 incubator. Cell Lines. KB cells were used. A rat cell line, 3Y1, established from a Fischer rat embryo (9), was kindly provided by G. Kimura. The following Adl2-transformed cell lines were used. WY3 cells: 3Y1 cells transformed by the whole Adl2 DNA (10). CY1 cells: 3Y1 cells transformed by the EcoRI-C fragment of Adl2 DNA (10). GYl cells: 3Y1 cells transformed by the HindIII-G fragment of Adl2 DNA (11). JtsA3 cells: strain JAR rat embryo cells transformed by a temperature-sensitive mutant (ts A) of Adl2 (K. Shiroki, unpublished). HT4 cells were derived from an Adl2-induced tumor in a hamster (12). W-3Y-23 cells, 3Y1 cells transformed by the wild type of simian virus 40 (SV40) (13), were kindly provided by N. Yamaguchi. H5 cells, monkey cells transformed by an Ad7-SV40 hybrid virus (14), were used. H-NRK cells, nonproducer normal rat kidney cells infected and transformed by Harvey murine sarcoma virus (15), were kindly provided by Y. Yuasa. Virus Titration. The infected cells and medium were frozen and thawed three times. After centrifugation, 0.2 ml of the supernatant was titrated by production of AAV1 antigen-positive cells per cover slip as described previously (2). Virus titer was expressed by FIU/0.2 ml. Immunofluorescent Staining and Autoradiography. KB cells labeled with [3H]thymidine (0.5 ,uCi/ml, 22 Ci/mmol) for 48-72 hr before cocultivation were used to produce heterokaryons. At 20 hr after fusion, the heterokaryons on cover slips were fixed with acetone. Immunofluorescent staining was done by a direct method similar to that described by Ishibashi (16). After examination under a fluorescent microscope, the cover slips were washed with phosphate-buffered saline, treated with 2% perchloric acid, coated with Sakura NR-M2 emulsion, and kept in the dark for 10 days. The samples were developed with Konidol X, stained with Giemsa, and examined. Labeling, Extraction, and Analysis of Viral DNA. At 11 hr after cell fusion, the culture was labeled with [3H]thymidine (10 ,ACi/ml) for 1 hr. Viral DNA was selectively extracted by the Hirt procedure (17). The extracted DNA in Hirt supernatants was analyzed in 5-20% neutral sucrose gradients, containing 0.01 M Tris-HCI (pH 7.2), 0.1 M NaCl, 0.01 M EDTA, and 0.1% Sarkosyl, by centrifugation in a Beckman SW 41 rotor at 40 for 20 hr at 25,000 rpm. Fractions were assayed for radioactivity as described previously (2). RESULTS of AAV1 Growth. KB and JtsA3 Examination Miscroscopic cells were used to produce heterokaryons. After infection of the cocultured cells with AAV1 and subsequent cell fusion, the formation of AAV1 antigens was detected by immunofluoresAbbreviations: AAV, adeno-associated virus; Ad, adenovirus; FIU, fluorescent infectious units; UV, ultraviolet; SV40, simian virus 40.
Adeno-associated virus (AAV) is a defective parvovirus, which multiplies only in cells coinfected with a helper adenovirus (1). We have shown that adenovirus type 31 (Ad3l) induces a helper factor(s) for AAV growth as early as 6 hr after infection in human cells infected with Ad3l (2). The time of appearance of the helper factor(s) was similar to that of the Adl2-specific T antigen as reported by Shimojo et al. (3). However, neither Adl2-transformed cells (4) nor Ad2-transformed rat embryo cells (5) have been reported to support AAV growth, although these cells express early Adl2- or Ad2-specific functions such as synthesis of T antigen. The present study was undertaken to determine if a helper factor(s) for AAV growth is present in Adl2-transformed cells. Our object was successfully achieved by using heterokaryons made by fusion of Adl2-transformed cells with human KB cells, which support growth of AAV type 1 (AAV1) after coinfection with a helper adenovirus. We show in this report that small amounts of AAV are able to multiply in heterokaryons without coinfection with adenovirus. The results indicate the presence of the helper factor(s) for AAV growth in Adl2-transformed cells and suggest that the helper factor(s) is the Adl2-specific T antigen. MATERIALS AND METHODS Virus Infection and Cell Fusion. KB cells and Adl2-transformed cells from various species were cocultured in plastic petri dishes in low-calcium (1 mM) Eagle's minimum essential medium supplemented with 10% fetal calf serum. The cells were infected with AAV1 (6) at 10 fluorescent infectious units (FIU) (7) per cell after cocultivation at 370 for 4 hr. After adsorption for 2 hr at 370, the cells were incubated in medium containing 100-200 neutralizing units of anti-AAV1 guinea pig serum for 3Q min. Then, the cell fusion was performed by the method of Y. Okada. The cells were washed with Ca-balanced salt solution (0.0114 M NaCl/0.054 M KCI/0.34 mM Na2HPO4/0.44 mM KH2PO4/0.01 M Tris-HCI/1 mM CaCl2, at pH 7.6) and the ultraviolet (UV)-inactivated Sendai virus strain Z (8) suspension (500-3000 hemagglutinating units per dish) was added to the cells. The dishes were kept on ice for 10 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
4508
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Proc. Nati. Acad. Sci. USA 74 (1977)
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Table 1. Growth of AAV1 in heterokaryons formed by fusion of KB cells with JtsA3 cells at various ratios AAV1 titer, log (FIU/0.2 ml) KB cells/JtsA3 cells* 10:1 3:1 1:1 1:3 1:10 0 .
#
.40
6
FIG. 1. Microscopic examination of AAV1 growth in heterokaryons. (Upper) An autoradiogram of a heterokaryon. The KB cell nucleus is shown by an arrow. (X1500.) (Lower) Immunofluorescence of AAV1 virion antigens in a heterokaryon. (X750.) cence and the differentiation between KB and JtsA3 cell nuclei was made by autoradiography. The nuclei of KB cells in the heterokaryons were packed with grains (Fig. 1 upper ). The
results of autoradiography demonstrated that the KB cell nuclei than JtsA3 cell nuclei, indicating that KB cell nuclei could be differentiated from JtsA cell nuclei by their sizes. The formation of heterokaryons, which consisted of 2 to 20 nuclei, was confirmed by autoradiography. AAV1 virion antigens were detected only in heterokaryons, and neither in homokaryons nor in mononucleate cells. However, the proportion of AAV1 antigen-positive heterokaryons was small: only approximately 1% of all the heterokaryons. AAV1 virion antigens were detected in both KB and JtsA3 cell nuclei in some heterokaryons (Fig. 1 lower ) and in either KB or JtsA3 cell nuclei in other heterokaryons. These results indicate that a small number of nuclei in a small number of heterokaryons support the AAV1 growth under these conditions. Growth of AAV1 in Heterokaryons of KB and Adl2Transformed Cells. KB and JtsA3 cells were cocultured in various ratios. The cocultures infected with AAV1 were fused with UV-inactivated Sendai virus. The cultures were harvested 20 hr after fusion, and the virus yield in each culture was titrated (Table 1). The maximum virus yield was found in the coculture of KB and JtsA3 cells at the ratio of 3:1. This ratio of cells was used in the following experiments. Time Course of AAV1 Growth after Cell Fusion. Virus yields in heterokaryons of KB and JtsA3 cells were titrated at intervals of 4 hr after cell fusion (Fig. 2). The increase in virus titer was detected at 4 hr and reached its maximum at 12 hr after cell fusion. No virus growth was observed without the use of UV-inactivated Sendai virus. Analysis of DNA Synthesized in Heterokaryons. The were larger
3.6 3.9 3.8 3.3 3.0
* Ratio of the number of KB cells and JtsA3 cells used for fusion.
AAV1-infected heterokaryons of KB and JtsA3 cells were pulse-labeled for 1 hr with [3H]thymidine (10 /Ci/ml) 11 hr after cell fusion. The Hirt supernatants prepared from the labeled cells were analyzed in neutral sucrose gradients. As a control, a similar analysis was performed without cell fusion (Fig. 3). The radioactive peak cosedimenting with 32P-labeled marker AAV1 DNA was detected in the coculture treated with UV-inactivated Sendai virus, but was not in the coculture without the treatment. The pattern shown in Fig. 3 is characteristic of the AAV1 DNA profile of the Hirt supernatants prepared from infected cells as described previously (2). The results indicate that AAV1 DNA is synthesized in heterokaryons without coinfection of cells with adenovirus. Similar results were obtained when GY1 cells were used instead of JtsA3 cells. Growth of AAV1 in Heterokaryons Formed with Various Lines of Transformed Cells and in Homokaryons. Growth of AAV1 in heterokaryons of KB and different Adl2-transformed cells was examined (Table 2). The result showed that all the heterokaryons of KB cells with WY3, CY1, GY1, or JtsA3 cells helped the AAV growth to the same extent. Similar results were obtained with HT4 cells, cells derived from a tumor induced in a hamster with Ad12. However, no AAV1 growth was found in heterokaryons of KB cells with SYl, W-3Y-23, or H-NRK cells. No AAV1 growth was detected in homokaryons of either KB or JtsA3 cells fused with UV-inactivated Sendai virus. No AAV1 virion antigen was detected in heterokaryons of KB and H5 cells, which were SV40 T antigen-positive and Adl2 T antigen-negative (14).
DISCUSSION AAV is a defective parvovirus, which can replicate only in cells coinfected with a helper adenovirus (5). It has been shown in our previous paper (2) that adenovirus induces a helper factor(s) for the AAV growth early after infection of human cells with adenovirus. This observation led us to examine whether or not 5
4 .
LL
..._
.4% ..5
9 .c
I
12 20 8 16 4 Hours after cell fusion FIG. 2. Growth of AAV1 after cell fusion. Cocultures of KB and JtsA3 cells with (0-0) or without (O---O) treatment with UV-inactivated Sendai virus. 0
Cell Biology: Handa et al.
4510
0-4
Proc. Nati. Acad. Sci. USA 74 (1977) Table 2. Growth of AAV1 in various types of heterokaryons and homokaryons AAV1 virion AAV1 titer, Combination of cells antigens* log (FIU/0.2 ml) KB WY3 + 3.9 KB CYM + N.D. KB GY1 + 3.8 KB JtsA3 + 3.9 KB HT4 + N.D. KB 3Y1 N.D. KB KB 1.9 JtsA3 JtsA3 1.8 KB W-3Y-23 1.8 KB H-NRK N.D. KB H5 N.D. N.D., not done. * +, AAV1 antigen-positive cells were detected; -, no AAV1 antigen-positive cells were detected.
10
so CD
x I
1
5
10 15 20 25 Fraction number FIG. 3. Analysis of DNA synthesized in heterokaryons. Cocultures of KB and JtsA cells with (0-0) or without (o---o) treatment with UV-inactivated Sendai virus were infected with AAV1 and labeled with [3H]thymidine 11 hr after infection. The Hirt supernatants extracted from the labeled cells were analyzed in sucrose gradients. The position of 32P-labeled AAV1 DNA (a marker) is shown by an arrow.
the helper factor(s) is present in Adl2-transformed cells. The growth of AAV1 in heterokaryons of KB and Adl2-transformed rodent cells was shown by formation of the AAV1 antigen, by increase in AAV1 titers, and by synthesis of AAV1 DNA. These results indicate that the helper factor(s) is present in Adl2transformed cells and that AAV1 can replicate in heterokaryons without coinfection with adenovirus. It is also possible that the genetic information for a helper factor may not be expressed in transformed cells but only in heterokaryons. The replication of AAV in heterokaryons is not due to the replication of rescued adenovirus, because adenovirus virion antigen was never detected in heterokaryons by immunofluorescence. The growth of AAV1 is dependent on two factors, a hostrange factor and an adenovirus-induced helper factor. AAV1 used in this experiment is originally a human virus, which replicates in human cells coinfected with human adenovirus. AAV1 does not replicate in either rat cells coinfected with human adenovirus or rodent cells transformed by adenovirus (ref. 5; H. Handa, unpublished data). This restriction of AAV1 growth in rodent cells may be due to the lack of a host-range factor, which has not yet been clarified. The restriction was overcome by fusion of Adl2-transformed rodent cells with human KB cells, which supply a host-range factor. The nature of the adenovirus-induced helper factor is not known. However, it has been shown previously that the time of the appearance of the helper factor is quite similar to that of the Adl2-specific T antigen in Ad3l- or Adl2-infected cells (2, 3). This observation suggests that the helper factor is closely related to the Adl2-specific T antigen. This suggestion is supported by the present observations that heterokaryons of KB cells and either CY1 or GY1 cells support the growth of AAV1. CY1 cells are rat cells transformed by transfection with the EcoRI-C fragment (16%, the left end) of Adl2 DNA and contain only a portion of the EcoRI-C fragment as the integrated viral genome (10). GY1 cells are rat cells transformed by
transfection with the HindIII-G fragment (7.2%, the left end) of Adl2 DNA and contain only a portion of the HindIII-G fragment (11). It is unlikely that either CY1 or GY1 cells contain Adl2-specific early proteins other than T antigen, the product of the Adl2 transforming gene. The helper factor for the AAV1 growth may be the Adl2-specific T antigen, which helps the AAV1 growth either directly or indirectly. Although the Adl2 T antigen is regarded to play an important role in transformation of cells by Adl2, its biological function is still unknown. The helper function for the AAV1 growth may be a useful tool for analysis of the biological functions of the adenovirus T antigen. We are grateful to Dr. Yoshio Okada for kind instruction in cell fusion and to Dr. Barrie J. Carter for critical review of the manuscript. This work was supported by grants from Ministry of Education, Science and Culture, Japan, and from the Princess Takamatsu Fund for Cancer Research. 1. Atchison, R. W., Casto, B. C. & Hammon, W. M. (1965) Science
149,754-756. 2. Handa, H., Shimojo, H. & Yamaguchi, K. (1976) Virology 74, 1-15. 3. Shimojo, H., Yamamoto, H. & Abe, C. (1967) Virology 31, 748-752. 4. Hoggan, M. D., Blacklow, N. R. & Rowe, W. P. (1966) Proc. Natl. Acad. Sci. USA 55, 1467-1474. 5. Carter, B. J. (1977) in Parvoviruses: Replication and Biological Properties, eds. Ward, D. C. & Tattersall, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), in press. 6. Handa, H., Shiroki, K. & Shimojo, H. (1975) J. Gen. Virol. 29, 239-242. 7. Blacklow, N. R., Hoggan, M. D. & Rowe, W. P. (1967) J. Exp. Med. 123, 755-765. 8. Okada, Y. (1962) Exp. Cell Res. 26,98-107. 9. Kimura, G., Itagaki, A. & Summers, J. (1975) Int. J. Cancer 15, 694-706. 10. Yano, S., Ojima, S., Fujinaga, K., Shiroki, K. & Shimojo, H. (1977) Virology 82, 214-220. 11. Shiroki, K., Handa, H., Shimojo, H., Yano, S., Ojima, S. & Fujinaga, K. (1977) Virology, 82,462-471. 12. Nakajima, S., Hamada, C. & Uetake, H. (1973) Jpn. J. Microbiol.
17,303-311.
13. Segawa, K., Yamaguchi, N. & Oda, K. (1977) J. Virol. 22, 679-693. 14. Shiroki, K. & Shimojo, H. (1971) Virology 45, 163-171. 15. Levy, J. A. (1971) J. Natl. Cancer Inst. 46, 1001-1007. 16. Ishibashi, M. (1970) Proc. Natl. Acad. Sci. USA 65,304-309. 17. Hirt, B. (1967) J. Mol. Biol. 26, 365-369.
Cell Biology
Helper factor(s) for growth of adeno-associated virus in cells transformed by adenovirus 12 (cell fusion/transformed cells/T antigen/restriction endonuclease)
HIROSHI HANDA, KAZUKO SHIROKI, AND HIROTO SHIMOJO Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan
Communicated by Wallace P. Rowe, August 8, 1977
ABSTRACT Evidence is presented that a helper factor(s) for growth of adeno-associated virus (AAV) is present in cells transformed by adenovirus type 12 (Adl2). The growth of AAV was observed in heterokaryons formed by fusion of human KB and Adl2-transformed rodent cells by using ultraviolet-inactivated Sendai virus without coinfection of cells with adenovirus. The presence of the helper factor(s) for AAV growth in rat cells transformed by the EcoRI-C fragment or the HindIII-G fragment of Adl2 DNA suggests that the helper factor(s) induced by infection with adenovirus is the Adl2-specific T antigen.
min, washed with Ca-balanced salt solution, soaked in lowcalcium (1 mM) Eagle's minimum essential medium supplemented with 2% fetal calf serum, and incubated at 370 in a CO2 incubator. Cell Lines. KB cells were used. A rat cell line, 3Y1, established from a Fischer rat embryo (9), was kindly provided by G. Kimura. The following Adl2-transformed cell lines were used. WY3 cells: 3Y1 cells transformed by the whole Adl2 DNA (10). CY1 cells: 3Y1 cells transformed by the EcoRI-C fragment of Adl2 DNA (10). GYl cells: 3Y1 cells transformed by the HindIII-G fragment of Adl2 DNA (11). JtsA3 cells: strain JAR rat embryo cells transformed by a temperature-sensitive mutant (ts A) of Adl2 (K. Shiroki, unpublished). HT4 cells were derived from an Adl2-induced tumor in a hamster (12). W-3Y-23 cells, 3Y1 cells transformed by the wild type of simian virus 40 (SV40) (13), were kindly provided by N. Yamaguchi. H5 cells, monkey cells transformed by an Ad7-SV40 hybrid virus (14), were used. H-NRK cells, nonproducer normal rat kidney cells infected and transformed by Harvey murine sarcoma virus (15), were kindly provided by Y. Yuasa. Virus Titration. The infected cells and medium were frozen and thawed three times. After centrifugation, 0.2 ml of the supernatant was titrated by production of AAV1 antigen-positive cells per cover slip as described previously (2). Virus titer was expressed by FIU/0.2 ml. Immunofluorescent Staining and Autoradiography. KB cells labeled with [3H]thymidine (0.5 ,uCi/ml, 22 Ci/mmol) for 48-72 hr before cocultivation were used to produce heterokaryons. At 20 hr after fusion, the heterokaryons on cover slips were fixed with acetone. Immunofluorescent staining was done by a direct method similar to that described by Ishibashi (16). After examination under a fluorescent microscope, the cover slips were washed with phosphate-buffered saline, treated with 2% perchloric acid, coated with Sakura NR-M2 emulsion, and kept in the dark for 10 days. The samples were developed with Konidol X, stained with Giemsa, and examined. Labeling, Extraction, and Analysis of Viral DNA. At 11 hr after cell fusion, the culture was labeled with [3H]thymidine (10 ,ACi/ml) for 1 hr. Viral DNA was selectively extracted by the Hirt procedure (17). The extracted DNA in Hirt supernatants was analyzed in 5-20% neutral sucrose gradients, containing 0.01 M Tris-HCI (pH 7.2), 0.1 M NaCl, 0.01 M EDTA, and 0.1% Sarkosyl, by centrifugation in a Beckman SW 41 rotor at 40 for 20 hr at 25,000 rpm. Fractions were assayed for radioactivity as described previously (2). RESULTS of AAV1 Growth. KB and JtsA3 Examination Miscroscopic cells were used to produce heterokaryons. After infection of the cocultured cells with AAV1 and subsequent cell fusion, the formation of AAV1 antigens was detected by immunofluoresAbbreviations: AAV, adeno-associated virus; Ad, adenovirus; FIU, fluorescent infectious units; UV, ultraviolet; SV40, simian virus 40.
Adeno-associated virus (AAV) is a defective parvovirus, which multiplies only in cells coinfected with a helper adenovirus (1). We have shown that adenovirus type 31 (Ad3l) induces a helper factor(s) for AAV growth as early as 6 hr after infection in human cells infected with Ad3l (2). The time of appearance of the helper factor(s) was similar to that of the Adl2-specific T antigen as reported by Shimojo et al. (3). However, neither Adl2-transformed cells (4) nor Ad2-transformed rat embryo cells (5) have been reported to support AAV growth, although these cells express early Adl2- or Ad2-specific functions such as synthesis of T antigen. The present study was undertaken to determine if a helper factor(s) for AAV growth is present in Adl2-transformed cells. Our object was successfully achieved by using heterokaryons made by fusion of Adl2-transformed cells with human KB cells, which support growth of AAV type 1 (AAV1) after coinfection with a helper adenovirus. We show in this report that small amounts of AAV are able to multiply in heterokaryons without coinfection with adenovirus. The results indicate the presence of the helper factor(s) for AAV growth in Adl2-transformed cells and suggest that the helper factor(s) is the Adl2-specific T antigen. MATERIALS AND METHODS Virus Infection and Cell Fusion. KB cells and Adl2-transformed cells from various species were cocultured in plastic petri dishes in low-calcium (1 mM) Eagle's minimum essential medium supplemented with 10% fetal calf serum. The cells were infected with AAV1 (6) at 10 fluorescent infectious units (FIU) (7) per cell after cocultivation at 370 for 4 hr. After adsorption for 2 hr at 370, the cells were incubated in medium containing 100-200 neutralizing units of anti-AAV1 guinea pig serum for 3Q min. Then, the cell fusion was performed by the method of Y. Okada. The cells were washed with Ca-balanced salt solution (0.0114 M NaCl/0.054 M KCI/0.34 mM Na2HPO4/0.44 mM KH2PO4/0.01 M Tris-HCI/1 mM CaCl2, at pH 7.6) and the ultraviolet (UV)-inactivated Sendai virus strain Z (8) suspension (500-3000 hemagglutinating units per dish) was added to the cells. The dishes were kept on ice for 10 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
4508
Cell Biology: Handa et al.
Proc. Nati. Acad. Sci. USA 74 (1977)
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Table 1. Growth of AAV1 in heterokaryons formed by fusion of KB cells with JtsA3 cells at various ratios AAV1 titer, log (FIU/0.2 ml) KB cells/JtsA3 cells* 10:1 3:1 1:1 1:3 1:10 0 .
#
.40
6
FIG. 1. Microscopic examination of AAV1 growth in heterokaryons. (Upper) An autoradiogram of a heterokaryon. The KB cell nucleus is shown by an arrow. (X1500.) (Lower) Immunofluorescence of AAV1 virion antigens in a heterokaryon. (X750.) cence and the differentiation between KB and JtsA3 cell nuclei was made by autoradiography. The nuclei of KB cells in the heterokaryons were packed with grains (Fig. 1 upper ). The
results of autoradiography demonstrated that the KB cell nuclei than JtsA3 cell nuclei, indicating that KB cell nuclei could be differentiated from JtsA cell nuclei by their sizes. The formation of heterokaryons, which consisted of 2 to 20 nuclei, was confirmed by autoradiography. AAV1 virion antigens were detected only in heterokaryons, and neither in homokaryons nor in mononucleate cells. However, the proportion of AAV1 antigen-positive heterokaryons was small: only approximately 1% of all the heterokaryons. AAV1 virion antigens were detected in both KB and JtsA3 cell nuclei in some heterokaryons (Fig. 1 lower ) and in either KB or JtsA3 cell nuclei in other heterokaryons. These results indicate that a small number of nuclei in a small number of heterokaryons support the AAV1 growth under these conditions. Growth of AAV1 in Heterokaryons of KB and Adl2Transformed Cells. KB and JtsA3 cells were cocultured in various ratios. The cocultures infected with AAV1 were fused with UV-inactivated Sendai virus. The cultures were harvested 20 hr after fusion, and the virus yield in each culture was titrated (Table 1). The maximum virus yield was found in the coculture of KB and JtsA3 cells at the ratio of 3:1. This ratio of cells was used in the following experiments. Time Course of AAV1 Growth after Cell Fusion. Virus yields in heterokaryons of KB and JtsA3 cells were titrated at intervals of 4 hr after cell fusion (Fig. 2). The increase in virus titer was detected at 4 hr and reached its maximum at 12 hr after cell fusion. No virus growth was observed without the use of UV-inactivated Sendai virus. Analysis of DNA Synthesized in Heterokaryons. The were larger
3.6 3.9 3.8 3.3 3.0
* Ratio of the number of KB cells and JtsA3 cells used for fusion.
AAV1-infected heterokaryons of KB and JtsA3 cells were pulse-labeled for 1 hr with [3H]thymidine (10 /Ci/ml) 11 hr after cell fusion. The Hirt supernatants prepared from the labeled cells were analyzed in neutral sucrose gradients. As a control, a similar analysis was performed without cell fusion (Fig. 3). The radioactive peak cosedimenting with 32P-labeled marker AAV1 DNA was detected in the coculture treated with UV-inactivated Sendai virus, but was not in the coculture without the treatment. The pattern shown in Fig. 3 is characteristic of the AAV1 DNA profile of the Hirt supernatants prepared from infected cells as described previously (2). The results indicate that AAV1 DNA is synthesized in heterokaryons without coinfection of cells with adenovirus. Similar results were obtained when GY1 cells were used instead of JtsA3 cells. Growth of AAV1 in Heterokaryons Formed with Various Lines of Transformed Cells and in Homokaryons. Growth of AAV1 in heterokaryons of KB and different Adl2-transformed cells was examined (Table 2). The result showed that all the heterokaryons of KB cells with WY3, CY1, GY1, or JtsA3 cells helped the AAV growth to the same extent. Similar results were obtained with HT4 cells, cells derived from a tumor induced in a hamster with Ad12. However, no AAV1 growth was found in heterokaryons of KB cells with SYl, W-3Y-23, or H-NRK cells. No AAV1 growth was detected in homokaryons of either KB or JtsA3 cells fused with UV-inactivated Sendai virus. No AAV1 virion antigen was detected in heterokaryons of KB and H5 cells, which were SV40 T antigen-positive and Adl2 T antigen-negative (14).
DISCUSSION AAV is a defective parvovirus, which can replicate only in cells coinfected with a helper adenovirus (5). It has been shown in our previous paper (2) that adenovirus induces a helper factor(s) for the AAV growth early after infection of human cells with adenovirus. This observation led us to examine whether or not 5
4 .
LL
..._
.4% ..5
9 .c
I
12 20 8 16 4 Hours after cell fusion FIG. 2. Growth of AAV1 after cell fusion. Cocultures of KB and JtsA3 cells with (0-0) or without (O---O) treatment with UV-inactivated Sendai virus. 0
Cell Biology: Handa et al.
4510
0-4
Proc. Nati. Acad. Sci. USA 74 (1977) Table 2. Growth of AAV1 in various types of heterokaryons and homokaryons AAV1 virion AAV1 titer, Combination of cells antigens* log (FIU/0.2 ml) KB WY3 + 3.9 KB CYM + N.D. KB GY1 + 3.8 KB JtsA3 + 3.9 KB HT4 + N.D. KB 3Y1 N.D. KB KB 1.9 JtsA3 JtsA3 1.8 KB W-3Y-23 1.8 KB H-NRK N.D. KB H5 N.D. N.D., not done. * +, AAV1 antigen-positive cells were detected; -, no AAV1 antigen-positive cells were detected.
10
so CD
x I
1
5
10 15 20 25 Fraction number FIG. 3. Analysis of DNA synthesized in heterokaryons. Cocultures of KB and JtsA cells with (0-0) or without (o---o) treatment with UV-inactivated Sendai virus were infected with AAV1 and labeled with [3H]thymidine 11 hr after infection. The Hirt supernatants extracted from the labeled cells were analyzed in sucrose gradients. The position of 32P-labeled AAV1 DNA (a marker) is shown by an arrow.
the helper factor(s) is present in Adl2-transformed cells. The growth of AAV1 in heterokaryons of KB and Adl2-transformed rodent cells was shown by formation of the AAV1 antigen, by increase in AAV1 titers, and by synthesis of AAV1 DNA. These results indicate that the helper factor(s) is present in Adl2transformed cells and that AAV1 can replicate in heterokaryons without coinfection with adenovirus. It is also possible that the genetic information for a helper factor may not be expressed in transformed cells but only in heterokaryons. The replication of AAV in heterokaryons is not due to the replication of rescued adenovirus, because adenovirus virion antigen was never detected in heterokaryons by immunofluorescence. The growth of AAV1 is dependent on two factors, a hostrange factor and an adenovirus-induced helper factor. AAV1 used in this experiment is originally a human virus, which replicates in human cells coinfected with human adenovirus. AAV1 does not replicate in either rat cells coinfected with human adenovirus or rodent cells transformed by adenovirus (ref. 5; H. Handa, unpublished data). This restriction of AAV1 growth in rodent cells may be due to the lack of a host-range factor, which has not yet been clarified. The restriction was overcome by fusion of Adl2-transformed rodent cells with human KB cells, which supply a host-range factor. The nature of the adenovirus-induced helper factor is not known. However, it has been shown previously that the time of the appearance of the helper factor is quite similar to that of the Adl2-specific T antigen in Ad3l- or Adl2-infected cells (2, 3). This observation suggests that the helper factor is closely related to the Adl2-specific T antigen. This suggestion is supported by the present observations that heterokaryons of KB cells and either CY1 or GY1 cells support the growth of AAV1. CY1 cells are rat cells transformed by transfection with the EcoRI-C fragment (16%, the left end) of Adl2 DNA and contain only a portion of the EcoRI-C fragment as the integrated viral genome (10). GY1 cells are rat cells transformed by
transfection with the HindIII-G fragment (7.2%, the left end) of Adl2 DNA and contain only a portion of the HindIII-G fragment (11). It is unlikely that either CY1 or GY1 cells contain Adl2-specific early proteins other than T antigen, the product of the Adl2 transforming gene. The helper factor for the AAV1 growth may be the Adl2-specific T antigen, which helps the AAV1 growth either directly or indirectly. Although the Adl2 T antigen is regarded to play an important role in transformation of cells by Adl2, its biological function is still unknown. The helper function for the AAV1 growth may be a useful tool for analysis of the biological functions of the adenovirus T antigen. We are grateful to Dr. Yoshio Okada for kind instruction in cell fusion and to Dr. Barrie J. Carter for critical review of the manuscript. This work was supported by grants from Ministry of Education, Science and Culture, Japan, and from the Princess Takamatsu Fund for Cancer Research. 1. Atchison, R. W., Casto, B. C. & Hammon, W. M. (1965) Science
149,754-756. 2. Handa, H., Shimojo, H. & Yamaguchi, K. (1976) Virology 74, 1-15. 3. Shimojo, H., Yamamoto, H. & Abe, C. (1967) Virology 31, 748-752. 4. Hoggan, M. D., Blacklow, N. R. & Rowe, W. P. (1966) Proc. Natl. Acad. Sci. USA 55, 1467-1474. 5. Carter, B. J. (1977) in Parvoviruses: Replication and Biological Properties, eds. Ward, D. C. & Tattersall, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), in press. 6. Handa, H., Shiroki, K. & Shimojo, H. (1975) J. Gen. Virol. 29, 239-242. 7. Blacklow, N. R., Hoggan, M. D. & Rowe, W. P. (1967) J. Exp. Med. 123, 755-765. 8. Okada, Y. (1962) Exp. Cell Res. 26,98-107. 9. Kimura, G., Itagaki, A. & Summers, J. (1975) Int. J. Cancer 15, 694-706. 10. Yano, S., Ojima, S., Fujinaga, K., Shiroki, K. & Shimojo, H. (1977) Virology 82, 214-220. 11. Shiroki, K., Handa, H., Shimojo, H., Yano, S., Ojima, S. & Fujinaga, K. (1977) Virology, 82,462-471. 12. Nakajima, S., Hamada, C. & Uetake, H. (1973) Jpn. J. Microbiol.
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13. Segawa, K., Yamaguchi, N. & Oda, K. (1977) J. Virol. 22, 679-693. 14. Shiroki, K. & Shimojo, H. (1971) Virology 45, 163-171. 15. Levy, J. A. (1971) J. Natl. Cancer Inst. 46, 1001-1007. 16. Ishibashi, M. (1970) Proc. Natl. Acad. Sci. USA 65,304-309. 17. Hirt, B. (1967) J. Mol. Biol. 26, 365-369.
