- Email: [email protected]
Helper function for adenovirus replication in monkey cells by BK human papovavirus
VIROLOOY
98, 279-282 (1979)
Helper
Function
TATSUO National
Institute
for Adenovirus BK Human
MIYAMURA’
Replication Papovavirus
AND KENNETH
in Monkey
Cells by
K. TAKEMOT02
of Allergy and Infectious Diseases, National Bethesda, Maryland 20205
Institutes of Health,
Accepted July 15, 1979 CV-1 monkey kidney cells are semipermissive for BK human papovavirus at 37”. Although infected cells synthesize T antigen at this temperature, only a small percentage of the cells (less than 5%) produce viral antigen. However, when infected cells were incubated at 4W, characteristic CPE was observed with high virus yields. The inhibition of BKV growth in CV-1 cells was thus shown to be a temperature-dependent phenomenon. Experiments were then conducted to determine whether BKV provided a helper function for adenovirus growth in CV-1 cells at temperatures which were either permissive (40”) or semipermissive (3’7”) for BKV replication. At 37”, there was a low level of adenovirus enhancement of 0.5 to 1.0 log increase. However, at 4W, there was a 1.5 to 2.5 log increase in adenovirus yields, comparable to those obtained by coinfection with SV40 and adenovirus. These data provide additional information on common viral functions shared by BKV and SV40.
Since the discovery of BK human papovavirus in 1971 by Gardner et al. (1) detailed biological and biochemical studies have established its relatedness to the oncogenic simian papovavirus, SV40. Besides sharing cross-reacting viral antigens (V-Ag) demonstrable by various serological methods (Z), both viruses induce immunologically related nonstructural tumor antigens (T-Ag) (2). Biochemically, the shared sequences of the DNAs of BKV and SV40 were originally found to be primarily in the late regions, and relatively weak homology was observed in the early gene region coding for T-Ag (6). However, recent studies have shown extensive homology throughout the genomes of the two viruses and nucleotide sequencing studies of their DNAs have confirmed a high degree of relatedness of the genomes in both the early and late regions (4, 5). Since the early gene products (T-Ags) of BKV and SV40 are related, it is important to determine whether both viruses carry out similar biological functions. In a previous report from this laboratory, BKV was shown ’ Visiting Fellow, Department of Enteroviruses, National Institute of Health, Tokyo, Japan. 2 To whom reprint requests should be addressed. 279
to complement the growth of a tsA mutant of SV40 at restrictive temperature (6), indicating that some of the early functions of BKV and SV40 were similar. In other studies, however, the tumor specific transplantation antigens of BKV and SV40 were found to be different (2, 7, 8). Another early function attributed to SV40 is its ability to serve as a helper for adenovirus growth in monkey cells. Human adenoviruses do not usually replicate in monkey cells, although some of the early functions are expressed. For example, T-Ag (9), mRNA (IO), and viral DNA synthesis (11) are not impaired. The block in adenovirus replication in monkey cells has been reported to be at the post-transcriptional level (12, IS). Mixed infection of monkey cells with adenovirus and SV40 can convert this restrictive system to a permissive one for adenoviruses (14). The mechanism of enhancement of adenovirus growth by SV40 is not entirely understood. However, an early SV40 function appears to be essential (15) since some of the SV40 temperature-sensitive mutants can enhance adenovirus growth at nonpermissive temperatures (16,17). Furthermore, T-Ag-positive SV40-transformed monkey cells which do not release virus can support 0042~632zY79/130279-04$02.00/O Copyright AU rights
8 lS79 by Academic F’ress, Inc. of reproduction in any form reserved.
280
SHORT COMMUNICATIONS
DAYS AFTER INFECTION
FIG. 1. Kinetics of BKV T- and V-Ag synthesis in CV-1 cells at 37 and 40”. Monolayer cultures of CV-1 cells were infected with BKV at a multiplicity of approximately 50 PFU per cell. Cultures were kept at 37 (- - -1 or 40” (-), and T- and V-Ag were stained by indirect immunofluorescence at various times and the percentage positive cells determined.
adenovirus replication (l8,19). Since SV40 and BKV share early gene functions, experiments were conducted to determine whether BKV also has an adenovirus helper function in monkey cells. We have previously reported (6) that BKV infection of an established line of monkey cells, CV-1, was primarily an abortive one with most of the cells synthesizing T-Ag only. During the course of these experiments, we have discovered that when BKV-infected CV-1 cells were incubated at high temperature (40”) the cells became permissive for BKV growth and full yields of virus were obtained. The adenovirus helper experiments reported herein were therefore conducted under conditions where cells were either abortively (37”) or productively (40”) infected. BKV infection of CV-1 cells at 37 and 40”. Preliminary experiments were performed to study the growth of BKV in CV-1 cells at 37 and 40”. As previously noted (61, at 37” BKV infection of CV-1 cells was primarily abortive, with virtually all of the cells synthesizing T-antigen, but less than 5% were positive for viral antigen (Fig. 1). Cytopathic effects (cpe) characterized by cytoplasmic vacuolization were observed, but significant amounts of virus were not produced. However, when infected cells were incubated at 40”, a high percentage of cells (80%) were positive for T antigen by 3 days, and over 50% of cells were also positive for V antigen
at this time. The block in BKV replication in CV-1 cells was therefore a temperaturedependent phenomenon. When early passage African green monkey kidney cells were employed, similar results were obtained and viral replication was observed only in cultures incubated at 40”. Other strains of BKV isolated from patients with Wiskott-Aldrich syndrome also behaved like the prototype BKV strain in that they replicated in CV-1 cells at 40” but not at 37”. The results of studies on BKV replication at 37 and 40” obtained by fluorescent antibody staining was confirmed by measurement of actual viral yields at the two temperatures. CV-1 cells were infected with an input multiplicity of BKV of 10 and replicate cultures were incubated at the two temperatures. At various times after infection, cultures were removed, and frozen and thawed three times. The disrupted cells were treated with 100 units of receptordestroying enzyme at 37” for approximately 18 hr and virus yields were assayed by the plaque technique on human embryonic kidney (HEK) cells. Results of this experiment are presented in Fig. 2. Infectious virus was detected by 3 days in cultures incubated at 40” and increased to greater than 10’ PFU/culture by 7 days. On the other hand, at 37”, progeny virus was not detected until 4 days, and at 7 days, the virus yield was approximately 1.5 logs less than at 40”.
L>’ 0
1
2
I
I”
3
4
I 5
6
7
DAYS AFTER INFECTION
FIG. 2. Growth curve of BKV at 37 (- - -) and ) in CV-1 cells. Cultures infected at a multiplic40”(ity of 50 PFU/cell were harvested at designated times after infection. Cells were frozen and thawed, sonicated, and treated with receptor-destroying enzyme (100 unit/ml) for 18 hr at 37”. After the centrifugation of 3000 rpm for 15 min, supernatant fluids were titrated in HEK cells.
SHORT
281
COMMUNICATIONS
was a lo-fold increase in Ad-7 titer when the cells were incubated at 37”. There was a dramatic enhancement of Ad-7 growth (333-fold increase) when the culture which was doubly infected with BKV and Ad-7 was incubated at 40”. Control cultures infected with SV40 and Ad-7 showed enhancement of Ad-7 growth at both high and low temperatures. The greater degree of enhancement of Ad-7 growth in cultures coinfected with BKV and incubated at high temperature may be due to increased T-antigen production; BKV-infected CV-1 cells showed much brighter T-antigen staining when cultures were incubated at 40” as compared to cultures kept at 37”. The data presented in this report provide further evidence for common functions shared by the human and simian papovaviruses, BKV and SV40. Previous results have indicated that the SV40 T antigen plays an important role in adenovirus growth in monkey cells. For example, microinjection of SV40 T-antigen in monkey cells resulted in adenovirus growth (21). In addition, monkey cells transformed by SV40 which synthesized T antigen were capable of supporting adenovirus replication (18, 19). Similar results were recently obtained with BKV by Bradley and Dougherty (22) who reported that T antigen-positive BKVtransformed AGMK cells supported full yields of adenovirus. The T antigens of BKV and SV40 are immunologically related (2) and approxi-
BKV-cdenovimcs 7 mixed infection experiments. Based on the experiments described above, studies to determine whether BKV could serve as helper for adenovirus replication in CV-1 cells were performed at permissive (40”) and semipermissive (37”) temperatures. It has been shown previously that certain strains of adenovirus replicated poorly in HEK cells at 40” (20); therefore, adenovirus 7 (Ad-‘7) was selected for these experiments because it is capable of growing well at high temperature in HEK cells. To analyze BKV for helper function for adenovirus growth, CV-1 cultures were simultaneously infected with BKV and Ad-7 at input multiplicities of 50 and 20, respectively. As positive controls, other cultures were simultaneously infected with SV40 (5 PFU/cell) and Ad-7. Additional controls consisted of CV-1 cells infected with Ad-7 alone. Two hours after infection, the cultures were washed three times with medium, and fresh medium was added. One set of cultures infected with the different combinations of viruses was incubated at 37” and another set was incubated at 40”. After 3 days, all cultures were frozen and thawed four times, followed by low speed centrifugation (3000 rpm) to remove cell debris. The supernatant fluids were assayed for Ad-7 yields by the plaque technique on HEK monolayers. The results of this experiment are presented in Table 1. There was no detectable growth of Ad-7 in CV-1 cells at either temperature; however, when coinfected with BKV, there TABLE
1
REPLICATION OF Ad-7 IN CV-1 CELLS WITH OR WITHOUT BKV OR SV40 Cells infected with” Ad-7 only Ad-7 only Ad-7 only Ad-7
+ BKV
Ad-7 + BKV Ad-7 + BKV Ad-7
+ SV40
Ad-7 + SV40 Ad-7 + SV40 ” Multiplicity
Incubation temperature 37 37 40 37 37 40 37 37 40
Harvest time 2 hr 3 days 3 days 2 hr 3 days 3 days 2 hr 3 days 3 days
Yield of Ad-7 1.5 3.8 3.5 1.2 1.2 4.0 1.5 9.5 7.6
x x x x x x x x x
-Fold increase in titer
106 lo6 106 106 10’ 108 106 108 108
of infection: Ad-7, 20 PFUlcell; BKV, 50 PFUlcell; and SV40, 5 PFU/cell.
2 2 -
10 333 633 506
282
SHORT
COMMUNICATIONS
mately one-third of the tryptic peptides are shared (23). Although the early regions of the DNAs of both viruses were originally reported to have minimal shared sequences, recent studies have revealed more extensive homology (4, 5, 2.4 -26). Lai et al. (27) have also established that there are functional similarities of the early genes of BKV and SV40. In light of these more recent findings on a high degree of relatedness of the early regions of the DNAs of the two viruses, common viral functions shared by BKV and SV40 should be expected. A question still to be resolved is the lack of common transplantation antigens which has been observed by several investigators (2, 7’, 8). This antigen is also considered to be an early gene product of polyomaviruses. REFERENCES 1. GARDNER, S. D., FIELD, A. M., COLEMAN, D. V., and HULME, B., Lancet 1, 1253-1257 (1971). 2. TAKEMOTO, K. K., and MULLARKEY, M. F., J. Viral. 12, 625-631 (1973). 3. KHOURY, G., HOWLEY, P. M., GARON, C., MULLARKEY, M. F., TAKEMOTO, K. K., and MARTIN, M. A., Proc. Nat. Acad. Sci. USA 72, 2563-2567 (1975). N., LAI, C. J., KHOURY, G., and KELLY, 4. NEWELL, T. J., JR., J. Viral. 25, 193-201 (1978). 5. YANG, R. C. A., and WV, R.,Proc. Nat. Acad. Sei. USA 76, 1179-1183 (19’79). 6. MASON, D. M., JR., and TAKEMOTO, K. K., J. Viral. 17, 1060-1062 (1976). 7. PADGE~, B. L., HUNT, J. M., and WALKER, D. L., Zntewirology 8, 182-185 (1977). 8. KARJALAINEN, H. E., LAAKSONEN, A. M., and MANTYJARVI, R. A., J. Gen. Viral. 41,171-174 (1978).
9. MALMGREN. R. A., RABSON, A. S., CARNEY, P. G., and PA&, F. G., J. Bacterial. 91, 262 (1966). 10. BAUM, S. G., WIESE, W. H., and REICH, P. R., Virology 34, 373-376 (1968). 11. REICH, P. R., BAUM, S. G., ROSE, J. A., ROWE, W. P., and WEISSMAN, S. M., Proc. Nat. Acad. Sci. USA 55, 336-341(1966). 12. Fox, R. I., and BAUM, S. G., Virology 60.45-53 (1974). 13. ERON, L., WESTPHAL, H., and KHOURY, G., J. Viral. 15, 1256-1261 (1975). G. T., BEREZESKY, lh. RABSON, A. S., O’CONNOR, I. K., and PAUL, F. J., Proc. Sot. Exp, Biol. Med. 116, 187-190 (1964). 15. FRIEDMAN, M. P., LYONS, M. J., and GINSBERG, H. S., J. Viral. 5, 586-597 (1970). M., and RAPP, F., Virology 51,46616. JERKOFSKY, 473 (1973). 17. KIMURA, G., Nature (London) 248, 590-592 (1974). 18. RAPP, F., and TRULOCK, S. C., Virology 40, 961-970 (1970). 19. SHIROKI, K., and SHIMOJO, H., Virology 45, 163171 (1971). 20. JERKOFSKY, M., and RAPP, F., J. Viral. 2, 670677 (1968). A., Proc. 21. TJIAN, R., FEY, G., and GRAESSMANN, Nat. Acad. Sci. USA 75, 1279-1283 (1978). 22. BRADLEY, M. K., and DOUGHERTY, R. M., Virology 85, 231-240 (1978). D., and MARTIN, M. A., Proc. Nat. 23. SIMMONS, Acad. Sci. USA 75, 1131-1135 (1978).
2.4. YANG, USA 25.
R. C. A., and WV, R., Proc. 75, 2150-2154 (1978).
Nat.
Acad.
Sci.
DHAR, R., LAI, C. J., and KHOURY, G., Cell 13, 345-358 (1978). G., Proc. Nat. 26. DHAR, R., SEIF, I., and KHOURY, Acad. Sci. USA 76, 565-569 (1979). 27. LAI, C-J., GOLDMAN, N. D., and KHOURY, G., J. Viral. 30, 141-147 (1979).
98, 279-282 (1979)
Helper
Function
TATSUO National
Institute
for Adenovirus BK Human
MIYAMURA’
Replication Papovavirus
AND KENNETH
in Monkey
Cells by
K. TAKEMOT02
of Allergy and Infectious Diseases, National Bethesda, Maryland 20205
Institutes of Health,
Accepted July 15, 1979 CV-1 monkey kidney cells are semipermissive for BK human papovavirus at 37”. Although infected cells synthesize T antigen at this temperature, only a small percentage of the cells (less than 5%) produce viral antigen. However, when infected cells were incubated at 4W, characteristic CPE was observed with high virus yields. The inhibition of BKV growth in CV-1 cells was thus shown to be a temperature-dependent phenomenon. Experiments were then conducted to determine whether BKV provided a helper function for adenovirus growth in CV-1 cells at temperatures which were either permissive (40”) or semipermissive (3’7”) for BKV replication. At 37”, there was a low level of adenovirus enhancement of 0.5 to 1.0 log increase. However, at 4W, there was a 1.5 to 2.5 log increase in adenovirus yields, comparable to those obtained by coinfection with SV40 and adenovirus. These data provide additional information on common viral functions shared by BKV and SV40.
Since the discovery of BK human papovavirus in 1971 by Gardner et al. (1) detailed biological and biochemical studies have established its relatedness to the oncogenic simian papovavirus, SV40. Besides sharing cross-reacting viral antigens (V-Ag) demonstrable by various serological methods (Z), both viruses induce immunologically related nonstructural tumor antigens (T-Ag) (2). Biochemically, the shared sequences of the DNAs of BKV and SV40 were originally found to be primarily in the late regions, and relatively weak homology was observed in the early gene region coding for T-Ag (6). However, recent studies have shown extensive homology throughout the genomes of the two viruses and nucleotide sequencing studies of their DNAs have confirmed a high degree of relatedness of the genomes in both the early and late regions (4, 5). Since the early gene products (T-Ags) of BKV and SV40 are related, it is important to determine whether both viruses carry out similar biological functions. In a previous report from this laboratory, BKV was shown ’ Visiting Fellow, Department of Enteroviruses, National Institute of Health, Tokyo, Japan. 2 To whom reprint requests should be addressed. 279
to complement the growth of a tsA mutant of SV40 at restrictive temperature (6), indicating that some of the early functions of BKV and SV40 were similar. In other studies, however, the tumor specific transplantation antigens of BKV and SV40 were found to be different (2, 7, 8). Another early function attributed to SV40 is its ability to serve as a helper for adenovirus growth in monkey cells. Human adenoviruses do not usually replicate in monkey cells, although some of the early functions are expressed. For example, T-Ag (9), mRNA (IO), and viral DNA synthesis (11) are not impaired. The block in adenovirus replication in monkey cells has been reported to be at the post-transcriptional level (12, IS). Mixed infection of monkey cells with adenovirus and SV40 can convert this restrictive system to a permissive one for adenoviruses (14). The mechanism of enhancement of adenovirus growth by SV40 is not entirely understood. However, an early SV40 function appears to be essential (15) since some of the SV40 temperature-sensitive mutants can enhance adenovirus growth at nonpermissive temperatures (16,17). Furthermore, T-Ag-positive SV40-transformed monkey cells which do not release virus can support 0042~632zY79/130279-04$02.00/O Copyright AU rights
8 lS79 by Academic F’ress, Inc. of reproduction in any form reserved.
280
SHORT COMMUNICATIONS
DAYS AFTER INFECTION
FIG. 1. Kinetics of BKV T- and V-Ag synthesis in CV-1 cells at 37 and 40”. Monolayer cultures of CV-1 cells were infected with BKV at a multiplicity of approximately 50 PFU per cell. Cultures were kept at 37 (- - -1 or 40” (-), and T- and V-Ag were stained by indirect immunofluorescence at various times and the percentage positive cells determined.
adenovirus replication (l8,19). Since SV40 and BKV share early gene functions, experiments were conducted to determine whether BKV also has an adenovirus helper function in monkey cells. We have previously reported (6) that BKV infection of an established line of monkey cells, CV-1, was primarily an abortive one with most of the cells synthesizing T-Ag only. During the course of these experiments, we have discovered that when BKV-infected CV-1 cells were incubated at high temperature (40”) the cells became permissive for BKV growth and full yields of virus were obtained. The adenovirus helper experiments reported herein were therefore conducted under conditions where cells were either abortively (37”) or productively (40”) infected. BKV infection of CV-1 cells at 37 and 40”. Preliminary experiments were performed to study the growth of BKV in CV-1 cells at 37 and 40”. As previously noted (61, at 37” BKV infection of CV-1 cells was primarily abortive, with virtually all of the cells synthesizing T-antigen, but less than 5% were positive for viral antigen (Fig. 1). Cytopathic effects (cpe) characterized by cytoplasmic vacuolization were observed, but significant amounts of virus were not produced. However, when infected cells were incubated at 40”, a high percentage of cells (80%) were positive for T antigen by 3 days, and over 50% of cells were also positive for V antigen
at this time. The block in BKV replication in CV-1 cells was therefore a temperaturedependent phenomenon. When early passage African green monkey kidney cells were employed, similar results were obtained and viral replication was observed only in cultures incubated at 40”. Other strains of BKV isolated from patients with Wiskott-Aldrich syndrome also behaved like the prototype BKV strain in that they replicated in CV-1 cells at 40” but not at 37”. The results of studies on BKV replication at 37 and 40” obtained by fluorescent antibody staining was confirmed by measurement of actual viral yields at the two temperatures. CV-1 cells were infected with an input multiplicity of BKV of 10 and replicate cultures were incubated at the two temperatures. At various times after infection, cultures were removed, and frozen and thawed three times. The disrupted cells were treated with 100 units of receptordestroying enzyme at 37” for approximately 18 hr and virus yields were assayed by the plaque technique on human embryonic kidney (HEK) cells. Results of this experiment are presented in Fig. 2. Infectious virus was detected by 3 days in cultures incubated at 40” and increased to greater than 10’ PFU/culture by 7 days. On the other hand, at 37”, progeny virus was not detected until 4 days, and at 7 days, the virus yield was approximately 1.5 logs less than at 40”.
L>’ 0
1
2
I
I”
3
4
I 5
6
7
DAYS AFTER INFECTION
FIG. 2. Growth curve of BKV at 37 (- - -) and ) in CV-1 cells. Cultures infected at a multiplic40”(ity of 50 PFU/cell were harvested at designated times after infection. Cells were frozen and thawed, sonicated, and treated with receptor-destroying enzyme (100 unit/ml) for 18 hr at 37”. After the centrifugation of 3000 rpm for 15 min, supernatant fluids were titrated in HEK cells.
SHORT
281
COMMUNICATIONS
was a lo-fold increase in Ad-7 titer when the cells were incubated at 37”. There was a dramatic enhancement of Ad-7 growth (333-fold increase) when the culture which was doubly infected with BKV and Ad-7 was incubated at 40”. Control cultures infected with SV40 and Ad-7 showed enhancement of Ad-7 growth at both high and low temperatures. The greater degree of enhancement of Ad-7 growth in cultures coinfected with BKV and incubated at high temperature may be due to increased T-antigen production; BKV-infected CV-1 cells showed much brighter T-antigen staining when cultures were incubated at 40” as compared to cultures kept at 37”. The data presented in this report provide further evidence for common functions shared by the human and simian papovaviruses, BKV and SV40. Previous results have indicated that the SV40 T antigen plays an important role in adenovirus growth in monkey cells. For example, microinjection of SV40 T-antigen in monkey cells resulted in adenovirus growth (21). In addition, monkey cells transformed by SV40 which synthesized T antigen were capable of supporting adenovirus replication (18, 19). Similar results were recently obtained with BKV by Bradley and Dougherty (22) who reported that T antigen-positive BKVtransformed AGMK cells supported full yields of adenovirus. The T antigens of BKV and SV40 are immunologically related (2) and approxi-
BKV-cdenovimcs 7 mixed infection experiments. Based on the experiments described above, studies to determine whether BKV could serve as helper for adenovirus replication in CV-1 cells were performed at permissive (40”) and semipermissive (37”) temperatures. It has been shown previously that certain strains of adenovirus replicated poorly in HEK cells at 40” (20); therefore, adenovirus 7 (Ad-‘7) was selected for these experiments because it is capable of growing well at high temperature in HEK cells. To analyze BKV for helper function for adenovirus growth, CV-1 cultures were simultaneously infected with BKV and Ad-7 at input multiplicities of 50 and 20, respectively. As positive controls, other cultures were simultaneously infected with SV40 (5 PFU/cell) and Ad-7. Additional controls consisted of CV-1 cells infected with Ad-7 alone. Two hours after infection, the cultures were washed three times with medium, and fresh medium was added. One set of cultures infected with the different combinations of viruses was incubated at 37” and another set was incubated at 40”. After 3 days, all cultures were frozen and thawed four times, followed by low speed centrifugation (3000 rpm) to remove cell debris. The supernatant fluids were assayed for Ad-7 yields by the plaque technique on HEK monolayers. The results of this experiment are presented in Table 1. There was no detectable growth of Ad-7 in CV-1 cells at either temperature; however, when coinfected with BKV, there TABLE
1
REPLICATION OF Ad-7 IN CV-1 CELLS WITH OR WITHOUT BKV OR SV40 Cells infected with” Ad-7 only Ad-7 only Ad-7 only Ad-7
+ BKV
Ad-7 + BKV Ad-7 + BKV Ad-7
+ SV40
Ad-7 + SV40 Ad-7 + SV40 ” Multiplicity
Incubation temperature 37 37 40 37 37 40 37 37 40
Harvest time 2 hr 3 days 3 days 2 hr 3 days 3 days 2 hr 3 days 3 days
Yield of Ad-7 1.5 3.8 3.5 1.2 1.2 4.0 1.5 9.5 7.6
x x x x x x x x x
-Fold increase in titer
106 lo6 106 106 10’ 108 106 108 108
of infection: Ad-7, 20 PFUlcell; BKV, 50 PFUlcell; and SV40, 5 PFU/cell.
2 2 -
10 333 633 506
282
SHORT
COMMUNICATIONS
mately one-third of the tryptic peptides are shared (23). Although the early regions of the DNAs of both viruses were originally reported to have minimal shared sequences, recent studies have revealed more extensive homology (4, 5, 2.4 -26). Lai et al. (27) have also established that there are functional similarities of the early genes of BKV and SV40. In light of these more recent findings on a high degree of relatedness of the early regions of the DNAs of the two viruses, common viral functions shared by BKV and SV40 should be expected. A question still to be resolved is the lack of common transplantation antigens which has been observed by several investigators (2, 7’, 8). This antigen is also considered to be an early gene product of polyomaviruses. REFERENCES 1. GARDNER, S. D., FIELD, A. M., COLEMAN, D. V., and HULME, B., Lancet 1, 1253-1257 (1971). 2. TAKEMOTO, K. K., and MULLARKEY, M. F., J. Viral. 12, 625-631 (1973). 3. KHOURY, G., HOWLEY, P. M., GARON, C., MULLARKEY, M. F., TAKEMOTO, K. K., and MARTIN, M. A., Proc. Nat. Acad. Sci. USA 72, 2563-2567 (1975). N., LAI, C. J., KHOURY, G., and KELLY, 4. NEWELL, T. J., JR., J. Viral. 25, 193-201 (1978). 5. YANG, R. C. A., and WV, R.,Proc. Nat. Acad. Sei. USA 76, 1179-1183 (19’79). 6. MASON, D. M., JR., and TAKEMOTO, K. K., J. Viral. 17, 1060-1062 (1976). 7. PADGE~, B. L., HUNT, J. M., and WALKER, D. L., Zntewirology 8, 182-185 (1977). 8. KARJALAINEN, H. E., LAAKSONEN, A. M., and MANTYJARVI, R. A., J. Gen. Viral. 41,171-174 (1978).
9. MALMGREN. R. A., RABSON, A. S., CARNEY, P. G., and PA&, F. G., J. Bacterial. 91, 262 (1966). 10. BAUM, S. G., WIESE, W. H., and REICH, P. R., Virology 34, 373-376 (1968). 11. REICH, P. R., BAUM, S. G., ROSE, J. A., ROWE, W. P., and WEISSMAN, S. M., Proc. Nat. Acad. Sci. USA 55, 336-341(1966). 12. Fox, R. I., and BAUM, S. G., Virology 60.45-53 (1974). 13. ERON, L., WESTPHAL, H., and KHOURY, G., J. Viral. 15, 1256-1261 (1975). G. T., BEREZESKY, lh. RABSON, A. S., O’CONNOR, I. K., and PAUL, F. J., Proc. Sot. Exp, Biol. Med. 116, 187-190 (1964). 15. FRIEDMAN, M. P., LYONS, M. J., and GINSBERG, H. S., J. Viral. 5, 586-597 (1970). M., and RAPP, F., Virology 51,46616. JERKOFSKY, 473 (1973). 17. KIMURA, G., Nature (London) 248, 590-592 (1974). 18. RAPP, F., and TRULOCK, S. C., Virology 40, 961-970 (1970). 19. SHIROKI, K., and SHIMOJO, H., Virology 45, 163171 (1971). 20. JERKOFSKY, M., and RAPP, F., J. Viral. 2, 670677 (1968). A., Proc. 21. TJIAN, R., FEY, G., and GRAESSMANN, Nat. Acad. Sci. USA 75, 1279-1283 (1978). 22. BRADLEY, M. K., and DOUGHERTY, R. M., Virology 85, 231-240 (1978). D., and MARTIN, M. A., Proc. Nat. 23. SIMMONS, Acad. Sci. USA 75, 1131-1135 (1978).
2.4. YANG, USA 25.
R. C. A., and WV, R., Proc. 75, 2150-2154 (1978).
Nat.
Acad.
Sci.
DHAR, R., LAI, C. J., and KHOURY, G., Cell 13, 345-358 (1978). G., Proc. Nat. 26. DHAR, R., SEIF, I., and KHOURY, Acad. Sci. USA 76, 565-569 (1979). 27. LAI, C-J., GOLDMAN, N. D., and KHOURY, G., J. Viral. 30, 141-147 (1979).