Cancer Letters, 2 (1976) 53--58 © Elsevier/North-Holland BiomedicM Press
53
D E C R E A S E D T R A N S F O R M I N G ABILITY O F A T E M P E R A T U R E SENSITIVE M U T A N T O F H U M A N A D E N O V I R U S T Y P E 12
SEIJI HA/VIAand GENKI t[IMURA Department of Virology, Tottori University, Sd:hool of Medlcine, Yonago 683 (Japan) (Revised vers~vn received 25 June 1976)
SUMMARY A mutant of human adenovirus t y p e 12, temperat.ure~sensitive for virus replication and for the enhancement of thymidine incorporation in resting human embryonic kidney cells, is described. The muta.nt was also tempero attire-sensitive for transformation of rat cells. The ~esults indicate t h a t a viral gene required for the virus growth cycle: is also required for transformation.
INTRODUCTION Temperature-sensil~ive (ts) mutants of adenovirus :~ype 12 ( A d i 2 ) defective in transforming ability can be useful agents in exploring t h e mechanism of cell transformation b y this virus. In this paper we describe the isolation and some properties of such a mutant. MATERIALS AND METHODS A ts mutant (H12ts-505) of A d l 2 which produces plaques in human embryonic kidney (HEK) cells 34°C t o 37=C b u t n o t at 39.5°C was isolated from the wild type (WT) clone mutangenized with h y d r o x y l a m i n e by the method described previously [ 2 ] . The m u t a n t was plaque-purified 3 times at 34°C. The virus stocks used were clarified crude lysates prepared in HEK ceils at 34°C, ti~ering 1.0 X l 0 s P F U / m l (WT) and 3.5 X ! 0 ~ P F U / m l
(ts5o5). RESULTS
The ratio of number of plaques produced by ts505 at 39.5°C to t h a t a~ 34°C was less than 2.5 × 1 0 - s whereas for WT, this ratio was appro_,dmately 1. Growth curves of WT and ts505 at 39.5°C and 35°C i~.~HEK cells al~d the vkus yield after temperature shift (from 35°C to 39.5°C) ~ e shown tn Fig. 1. The growth of ts505 was markedly inhibited at 39.5°C (20,000-fold less yield
54
TS505
WILD TYPE
I0 ~
i
I0~
i
/
102
I01 0
48
0
2~4 -- ,¢18
HOURS AI:TER INFECTION Fig. 1. Gro~th curvesof wild type and HZ2~'s-505 adeno~irus12 at 39.5°Qand 35"C and virus yield after t e m p e r a t u r e shifts from 35°C ~o 39.5~C. Confluent secondary H EK cultures (1.5 × 10 s cells/tube) were infected w i t h 0.2 ml of virus at a m u l t i p l i c i t y of 5 PFU/cell. A f t e r i n c u b a t i o n for 1 h at 35°C, the inoeulu m ~vas discarded, the cull:ures treated with 1 :I 00 d i l u t i o n of anti-Ad12 r a b b i t serurn~ The cultures were washed 3 t i me s with 1 ml Dulbccco.Vogt's modification of Eagle's mediulm c ont a i ni ng 2% calf serum alad then incubated with this m e d i u m at 39.5°C and 35°C (for growth curves). At indicated times, the cultures were harvested and the virus c o n t e n t ir.~frozen and thz~wed (3 t i me s ) extracts was d c t e r m i n e d by plaque formation on HEK cultures at 34°C. For t e m p e r a t u r e shift-up curves, parallel cultures were shifted £rom 35°0 t~p 39.57C at the indicated times .~nd maintained at ~9.5~C ~ntil harvest. All culture9 ~.hiftcd from 35°C to 39.5°C were harvested 52 h post i n f e c t i o n and infectious viru'~ was assayed as above, o = 39.5°C., c = 35°C, 0 = shift from 35 to 39.5~C.
55 l;h~,n a t 35°C), w h e r e a s WT grew well a t b o t h t e m p e r a t u r e s . I n t h e i n t e r p r e t a i:ion of ~he s h i f t . u p curve, it is a s s u m e d t h a t all ~he virus p a r t i c l e s w h o s e precursors have passed t h e t e m p e r a t u r e . s e n s i t i v e step b e f o r e t h e s h i f t will be infectious. Thus, t h e t i m e a t w h i c h t h e virus yield b e c o m e s r e s i s t a n t t o t h e shift f r o m 35~C is t h e t i m e a t w h i c h t h e t e m p e r a t u r e - s e n s i t i v e step b e g i n s t o b e c o m p l e t e d a t 35°C. T h e virus yield b e c a m e resist;mr t o t,he s h i f t 2 0 h aftex infection, w h i c h was early in t h e virus g r o w t h cycle, 8 h b e f o r e t h e a p p e a r a n c e of n e w infectious virus (Fig. 1). I n d u c t i o n of D N A ~;ynthesis in ~esting HEK ceils after i n f e c t i o n w i t h WT a n d t s 5 0 5 is s h o w n in Fig. 2. Cultures i n f e c t e d w i t h ts505 at 40.5°C s h o w e d a v e r y low rate o f [ ~ H l t h y m i d i n e i n c o r p o r a t i o n i n t o acid insoluble materials, w h e r e a s t h o s e a t 34°0, as well as c u l t u r e s i n f e c t e d w i t h WT at 40.5°(3 a n d 34°C, s h o w e d h i g h rotes of i n c o r p o r a t i o n . It is m o s t likely t h a t ts505 is d e f e c t i v e in viral D N A s y n t h e s i s at 40.5°C, since it has b e e n well established t h a t thymiLdine i n c o r p o r a t i o n in s e r u m - f r e e resting HEK cultures i n f e c t e d w i t h A d l 2 r e p r e s e n t s viral D N A s y n t h e s i s [ 8 , 9 ] , T h e transl'orming a':.flit.y of t s 5 0 5 was e x a m i n e d using clonal c u l t u r e s of r a t 3Y1 cells ~4] T h e ch.:rac~:existks of i n f e c t i o n o f 3Y1 cells w i t h A d 1 2 a n d t h o s e of t r a n s f o r m a t i o n b y A J 1 2 have; b e e n d e s c r i b e d [ 3 ] . Briefly, A d 1 2 carries o u t a n a b o r t i v e i n f e c t i o n o f 3YI cells leading t o t h e s y n t h e s i s o f T antigen b u t n o t of infectious virus. Morpholo~,~cal t r a n s f o r m a t i o n c a n b e assayed w i t h single h i t kinetics, o n e t r a n s f o r m a t i o n e v e n t b e i n g caused b y 104 t o 10 s p l a q u e f o r m i n g virions. As s h o w n in T a b l e 1, 3Y1 cells i n f e c t e d w i t h t s 5 0 5
NOT INFECTED
WILD TYPE
TS 5~5
V o ~7 u~ 5 ij 4
~3 '.J |
8
16 24 32 ~,0 HOURS
AF~fER I N F E C T I O N
Fig. 2. Thymidine incorporation in resting HEK cells after infection with wild type and t~505 Ad12 at 40.5°C and 34¢C. Confluent monolayer cultures of HEK cells w. !0 ml bottles (4 × I0 ~ cells/euIture) were maintained in a serum-free Dalbeeco-Voff.t's modification of Eagle's medium for 4 days a~ 37°C to become quiescent. For infection, the spent medium was kept aside, and the cultures were inoculated with 0.4 ml of ~he "~vildtype virus and tsS05 at an input multiplicity of inI'eetion of I0 PFU/eelL After in,:ubation at 34°C for 1 h, the inoeulum was discarded; the cultures received the old medium and were incubated at 40.5"C and at 34"C. At the ~imes indicated, a part of the cultures was pulselabelled with [~H]Lhymidine (2 gCi/ml, 10 -~ M) for 50 rain, the cells were lysed with 0.6% SDS and 570 cold TCA added. Precipitates were collected on GFF g~ass fiber filters, and w,dioactivi~y was determined in a scin~i]llation counter with a toluene scintillator. = at 34°C, o = at 40.5°C.
56
TABLE 1 TRANSFOIZMA'i"ION O F R A T 3YI-B C L O N E I-6 C E L L S W I T H W I L D T Y P E A N D taS05 A D E N O V I R U S T Y P E 12 a
Virus
Wild type
H12l::-505 None
Input virus multiplicity (PFU/cell) 45 22,5 4.5 48 25 4.9 0
:No. of transformed loci/dish
Efficiency of plating (%)
40.5~C
37°C
40.5°C
37°C
24 10 2.5 2.0 t.0 1.5 0
27 tl 7 25 [7.5 8 0
21
22
15
18
8
10
a 3YI cells (:tO~ cells) were mixed in suspension with 1 ml of virus fluid in a plastic tube and were incubated at 37~C for 1 h with occasional shakings. 10,000 infected ce:llswe:e seeded in each 50-ram plastic Petri dish and the cultures incubated at 37°12 or at 40.5~C in the calcium-free Dulbeeco-Vogt's modification of Eagle's medium contairting 10% fetal calf serum. A week after infection, the medium was replaced with the same medium .e~ngaining 0.27% Bacto agar. At 25 days, the agar medium was removed and the cultures fixed with 75% c~hanol and stained with hematoxyylin. The deeply stained, thick, p~led-up loci which appeared against the backgrou:ad of ligtstly stained untransformed 3Yl monolayer were counted visually Two Petri dishes were used for each determination. To determine the efficie:ncy of plating, an aliquot of the infected and mock irtfected 3YI cell suspensions used for the transformation assay was further diluted and seeded in calcium-free Dulbecco-Vogt's :modification of Eagle's medium containing 10% fetal calf serum at the density of 250 cells/50-mm plastic Pearl dish. Colonies were counted after 10 days of incubation at 40.5°C and 370C.
s h o w e d a l o w e r f r e q u e n c y o f t r a n s f o r m a t i o n at 40.5°C t h a n t h a t at 37~C. 3Y1 cells i n f e c t e d w i t h WT s h o w e d a similar efficiency o f t r a n s f o r m a t i o n at b o t h t e m p e r a t t t r e s . Essentially, t h e r e was n o d i f f e r e n c e in a c o l o n y los, m i n t efficiency b e t w e e n t h e celils i n f e c t e d w i t h WT and t h o s e i n f e c t e d w i t h tsS05 at b o t h t e m p e r a t u r e s (Table 1), suggesting t h a t cell d e a t h was n o t ~he cause o f t r a ~ M o r r n a t i o n i n h i b i t i o n in ts505 at 40,5°C. T h e d e c r e a s e d t r a n s f o r m i n g abiIity o f ts505 at 40.5°C was r e p r o d u c i b l y o b s e r v e d in 4 o f 4 o t h e r i n d e p e n d e n t experiment, s. F o u r ~adepender~tly t r a n s f o r m e d cell lines (2 WTt r a n s f o r m e d a n d 2 ~505-transformed lines) w e r e isolated at 37°C, g r o w n a t 37°C, and t e s t e d f o r T antige:n b y c o m p l e m e n t fi~cation. All t h e t r a n s f o r m e d lines, b u t n o t t h e p a r e n t a l 3Y1 cell, were f o u n d to c o n t a i n A d ! 2 T antigen. The a n t i s e r u m u s e d was t h e o n e f r o m h a m s t e r s be~$ing A d l 2 - i n d u c e d t.umor. T h e serum h a d , in o t h e r e x p e r i m e n t , t h e typical A d 1 2 T antigen staining p a t t e r n b y i r n m u n o f l u o r e s c e n c e in A d l 2 A n f e c t e d H E K cells ( f l u o r e s c e n t flecks [ 6 ] , a n d it d i d n o t have a n t i b o d y activity against purified A d l ' t virions as m e a s u r e d b y c o m p l l e m e n t f ~ a t i o n .
57 DISCUSSION T h e results suggest t h a t t h e a f f e c t e d geve f u n c t i o n ila ts505 i~ r e q u i r e d b o t h for virus r e p l i c a t i o n ( p e r h a p s l~or viral D N A synthesis) in p r o d u c t i v e i n f e c t i o n and f o r ceil t r a n s f o r m a t i o n in a b o r t i v e i n f e c t i o n , O t h e r ts m u t a n t s o f A d l 2 have b e e n k n o w n w h i c h are t s in viral D N A synthesis a n d w h i c h a p p a r e n t l y have either a decreased [1,7] o r a n increased [5,7] t r a n s f o r m i n g ability at t h e restrictive t e m p e r a t u r e . Shiroki a n d S h i m o j o [ 8 ] , u s i n g ts mutar~ts o f A d 1 2 , have su[,,gested t h a t a t least 3 viral genes are involved in t h e i n i t i a t i o n o~ viral D N A replication. T h e r e l a t i o n s h i p b e t w e e n t h e viral ger.es r e p r e s e n t e d b y t h e s e m u t a n t s a n d t h e gene r e p r e s e n t e d b y o u r ts505 r e m a i n s t o b e determined. ACKNOWLEDGMENTS We t h a n k Dr. Eigo Nakaso for his ~ o n t i n u e d c o o p e r a t i o n in p r e p a r a t i o n of cell cultures. This w o r k was s u p p o r t e d b y ~ a n t s for cancer research f r o m t h e Ministry of Educatio¢~ o f t h e J a p a n e s e G o v e r n m e n t a n d b y t h e Asahi News Co. REFERENCES 1 Ginsberg, H.S., Ensinger, M.J., Kauffman, ~'~.S., Mayer, A.J. and Lundholm, U. (1974) Cell transformation: a study of regulation with types 5 and 12 adenovir~ temperaturesensitive mutants. Cold Spring Harbor Syrup. Quant. Biol., 39, 419--426. 2 Hama, S. and Kimura, G. (1972) Temperature-sensitive mutants o!" human adenovirus type 12. Japan. J. Microbiol., 9, 969--'970. 3 Hama, S. and Kimura, G. (1973) Infection of rat cell line 2Y1 by adenovirus type 12. Virus, 23, 389 (in JapaJ~se). 4 Kimura, G., Itagaki, A. and Summers, J. (1975) Rat celi line 3YI and its virogenic polyoma and SV40-transformed derivatives. Int. J. Cancer, 15,694--706. 5 Ledinko, N. (1974) Temperature-sensitive mutants of adenovirus 12 defective in viral DNA synthesis, J. ViroL, 14, 457--468. 6 Pope, J.H. and Rowe, W.P. (1964) Immuno:iluoreseen~: studies of adenovirus 12 tumors and of" cells transformed or infected by adenovirus. J. Exp. Med., 120, 577--588. 7 Rubestein, F.E. and Ginsberg, H.S. (1974) q~-ansformation eh:aracteristicz of temperature-sensitive mutants ¢~ftype 12 adenovirus. Intervirology, 3, t70--174. S Sbi~'oki, K. and Sh[mojo, H. (I974) Analysis of adenovirus 12 Semperature-zensitive mutants defective in viral DNA replication. Vlromgy, ~il, 474--485. 9 Yamashita, T. and Shimojo, H. (1969) Induction of cellutar DNA synthesis by adenoviru~ 12 in human embryo kidney cells. Virology, 38,351--355.
53
D E C R E A S E D T R A N S F O R M I N G ABILITY O F A T E M P E R A T U R E SENSITIVE M U T A N T O F H U M A N A D E N O V I R U S T Y P E 12
SEIJI HA/VIAand GENKI t[IMURA Department of Virology, Tottori University, Sd:hool of Medlcine, Yonago 683 (Japan) (Revised vers~vn received 25 June 1976)
SUMMARY A mutant of human adenovirus t y p e 12, temperat.ure~sensitive for virus replication and for the enhancement of thymidine incorporation in resting human embryonic kidney cells, is described. The muta.nt was also tempero attire-sensitive for transformation of rat cells. The ~esults indicate t h a t a viral gene required for the virus growth cycle: is also required for transformation.
INTRODUCTION Temperature-sensil~ive (ts) mutants of adenovirus :~ype 12 ( A d i 2 ) defective in transforming ability can be useful agents in exploring t h e mechanism of cell transformation b y this virus. In this paper we describe the isolation and some properties of such a mutant. MATERIALS AND METHODS A ts mutant (H12ts-505) of A d l 2 which produces plaques in human embryonic kidney (HEK) cells 34°C t o 37=C b u t n o t at 39.5°C was isolated from the wild type (WT) clone mutangenized with h y d r o x y l a m i n e by the method described previously [ 2 ] . The m u t a n t was plaque-purified 3 times at 34°C. The virus stocks used were clarified crude lysates prepared in HEK ceils at 34°C, ti~ering 1.0 X l 0 s P F U / m l (WT) and 3.5 X ! 0 ~ P F U / m l
(ts5o5). RESULTS
The ratio of number of plaques produced by ts505 at 39.5°C to t h a t a~ 34°C was less than 2.5 × 1 0 - s whereas for WT, this ratio was appro_,dmately 1. Growth curves of WT and ts505 at 39.5°C and 35°C i~.~HEK cells al~d the vkus yield after temperature shift (from 35°C to 39.5°C) ~ e shown tn Fig. 1. The growth of ts505 was markedly inhibited at 39.5°C (20,000-fold less yield
54
TS505
WILD TYPE
I0 ~
i
I0~
i
/
102
I01 0
48
0
2~4 -- ,¢18
HOURS AI:TER INFECTION Fig. 1. Gro~th curvesof wild type and HZ2~'s-505 adeno~irus12 at 39.5°Qand 35"C and virus yield after t e m p e r a t u r e shifts from 35°C ~o 39.5~C. Confluent secondary H EK cultures (1.5 × 10 s cells/tube) were infected w i t h 0.2 ml of virus at a m u l t i p l i c i t y of 5 PFU/cell. A f t e r i n c u b a t i o n for 1 h at 35°C, the inoeulu m ~vas discarded, the cull:ures treated with 1 :I 00 d i l u t i o n of anti-Ad12 r a b b i t serurn~ The cultures were washed 3 t i me s with 1 ml Dulbccco.Vogt's modification of Eagle's mediulm c ont a i ni ng 2% calf serum alad then incubated with this m e d i u m at 39.5°C and 35°C (for growth curves). At indicated times, the cultures were harvested and the virus c o n t e n t ir.~frozen and thz~wed (3 t i me s ) extracts was d c t e r m i n e d by plaque formation on HEK cultures at 34°C. For t e m p e r a t u r e shift-up curves, parallel cultures were shifted £rom 35°0 t~p 39.57C at the indicated times .~nd maintained at ~9.5~C ~ntil harvest. All culture9 ~.hiftcd from 35°C to 39.5°C were harvested 52 h post i n f e c t i o n and infectious viru'~ was assayed as above, o = 39.5°C., c = 35°C, 0 = shift from 35 to 39.5~C.
55 l;h~,n a t 35°C), w h e r e a s WT grew well a t b o t h t e m p e r a t u r e s . I n t h e i n t e r p r e t a i:ion of ~he s h i f t . u p curve, it is a s s u m e d t h a t all ~he virus p a r t i c l e s w h o s e precursors have passed t h e t e m p e r a t u r e . s e n s i t i v e step b e f o r e t h e s h i f t will be infectious. Thus, t h e t i m e a t w h i c h t h e virus yield b e c o m e s r e s i s t a n t t o t h e shift f r o m 35~C is t h e t i m e a t w h i c h t h e t e m p e r a t u r e - s e n s i t i v e step b e g i n s t o b e c o m p l e t e d a t 35°C. T h e virus yield b e c a m e resist;mr t o t,he s h i f t 2 0 h aftex infection, w h i c h was early in t h e virus g r o w t h cycle, 8 h b e f o r e t h e a p p e a r a n c e of n e w infectious virus (Fig. 1). I n d u c t i o n of D N A ~;ynthesis in ~esting HEK ceils after i n f e c t i o n w i t h WT a n d t s 5 0 5 is s h o w n in Fig. 2. Cultures i n f e c t e d w i t h ts505 at 40.5°C s h o w e d a v e r y low rate o f [ ~ H l t h y m i d i n e i n c o r p o r a t i o n i n t o acid insoluble materials, w h e r e a s t h o s e a t 34°0, as well as c u l t u r e s i n f e c t e d w i t h WT at 40.5°(3 a n d 34°C, s h o w e d h i g h rotes of i n c o r p o r a t i o n . It is m o s t likely t h a t ts505 is d e f e c t i v e in viral D N A s y n t h e s i s at 40.5°C, since it has b e e n well established t h a t thymiLdine i n c o r p o r a t i o n in s e r u m - f r e e resting HEK cultures i n f e c t e d w i t h A d l 2 r e p r e s e n t s viral D N A s y n t h e s i s [ 8 , 9 ] , T h e transl'orming a':.flit.y of t s 5 0 5 was e x a m i n e d using clonal c u l t u r e s of r a t 3Y1 cells ~4] T h e ch.:rac~:existks of i n f e c t i o n o f 3Y1 cells w i t h A d 1 2 a n d t h o s e of t r a n s f o r m a t i o n b y A J 1 2 have; b e e n d e s c r i b e d [ 3 ] . Briefly, A d 1 2 carries o u t a n a b o r t i v e i n f e c t i o n o f 3YI cells leading t o t h e s y n t h e s i s o f T antigen b u t n o t of infectious virus. Morpholo~,~cal t r a n s f o r m a t i o n c a n b e assayed w i t h single h i t kinetics, o n e t r a n s f o r m a t i o n e v e n t b e i n g caused b y 104 t o 10 s p l a q u e f o r m i n g virions. As s h o w n in T a b l e 1, 3Y1 cells i n f e c t e d w i t h t s 5 0 5
NOT INFECTED
WILD TYPE
TS 5~5
V o ~7 u~ 5 ij 4
~3 '.J |
8
16 24 32 ~,0 HOURS
AF~fER I N F E C T I O N
Fig. 2. Thymidine incorporation in resting HEK cells after infection with wild type and t~505 Ad12 at 40.5°C and 34¢C. Confluent monolayer cultures of HEK cells w. !0 ml bottles (4 × I0 ~ cells/euIture) were maintained in a serum-free Dalbeeco-Voff.t's modification of Eagle's medium for 4 days a~ 37°C to become quiescent. For infection, the spent medium was kept aside, and the cultures were inoculated with 0.4 ml of ~he "~vildtype virus and tsS05 at an input multiplicity of inI'eetion of I0 PFU/eelL After in,:ubation at 34°C for 1 h, the inoeulum was discarded; the cultures received the old medium and were incubated at 40.5"C and at 34"C. At the ~imes indicated, a part of the cultures was pulselabelled with [~H]Lhymidine (2 gCi/ml, 10 -~ M) for 50 rain, the cells were lysed with 0.6% SDS and 570 cold TCA added. Precipitates were collected on GFF g~ass fiber filters, and w,dioactivi~y was determined in a scin~i]llation counter with a toluene scintillator. = at 34°C, o = at 40.5°C.
56
TABLE 1 TRANSFOIZMA'i"ION O F R A T 3YI-B C L O N E I-6 C E L L S W I T H W I L D T Y P E A N D taS05 A D E N O V I R U S T Y P E 12 a
Virus
Wild type
H12l::-505 None
Input virus multiplicity (PFU/cell) 45 22,5 4.5 48 25 4.9 0
:No. of transformed loci/dish
Efficiency of plating (%)
40.5~C
37°C
40.5°C
37°C
24 10 2.5 2.0 t.0 1.5 0
27 tl 7 25 [7.5 8 0
21
22
15
18
8
10
a 3YI cells (:tO~ cells) were mixed in suspension with 1 ml of virus fluid in a plastic tube and were incubated at 37~C for 1 h with occasional shakings. 10,000 infected ce:llswe:e seeded in each 50-ram plastic Petri dish and the cultures incubated at 37°12 or at 40.5~C in the calcium-free Dulbeeco-Vogt's modification of Eagle's medium contairting 10% fetal calf serum. A week after infection, the medium was replaced with the same medium .e~ngaining 0.27% Bacto agar. At 25 days, the agar medium was removed and the cultures fixed with 75% c~hanol and stained with hematoxyylin. The deeply stained, thick, p~led-up loci which appeared against the backgrou:ad of ligtstly stained untransformed 3Yl monolayer were counted visually Two Petri dishes were used for each determination. To determine the efficie:ncy of plating, an aliquot of the infected and mock irtfected 3YI cell suspensions used for the transformation assay was further diluted and seeded in calcium-free Dulbecco-Vogt's :modification of Eagle's medium containing 10% fetal calf serum at the density of 250 cells/50-mm plastic Pearl dish. Colonies were counted after 10 days of incubation at 40.5°C and 370C.
s h o w e d a l o w e r f r e q u e n c y o f t r a n s f o r m a t i o n at 40.5°C t h a n t h a t at 37~C. 3Y1 cells i n f e c t e d w i t h WT s h o w e d a similar efficiency o f t r a n s f o r m a t i o n at b o t h t e m p e r a t t t r e s . Essentially, t h e r e was n o d i f f e r e n c e in a c o l o n y los, m i n t efficiency b e t w e e n t h e celils i n f e c t e d w i t h WT and t h o s e i n f e c t e d w i t h tsS05 at b o t h t e m p e r a t u r e s (Table 1), suggesting t h a t cell d e a t h was n o t ~he cause o f t r a ~ M o r r n a t i o n i n h i b i t i o n in ts505 at 40,5°C. T h e d e c r e a s e d t r a n s f o r m i n g abiIity o f ts505 at 40.5°C was r e p r o d u c i b l y o b s e r v e d in 4 o f 4 o t h e r i n d e p e n d e n t experiment, s. F o u r ~adepender~tly t r a n s f o r m e d cell lines (2 WTt r a n s f o r m e d a n d 2 ~505-transformed lines) w e r e isolated at 37°C, g r o w n a t 37°C, and t e s t e d f o r T antige:n b y c o m p l e m e n t fi~cation. All t h e t r a n s f o r m e d lines, b u t n o t t h e p a r e n t a l 3Y1 cell, were f o u n d to c o n t a i n A d ! 2 T antigen. The a n t i s e r u m u s e d was t h e o n e f r o m h a m s t e r s be~$ing A d l 2 - i n d u c e d t.umor. T h e serum h a d , in o t h e r e x p e r i m e n t , t h e typical A d 1 2 T antigen staining p a t t e r n b y i r n m u n o f l u o r e s c e n c e in A d l 2 A n f e c t e d H E K cells ( f l u o r e s c e n t flecks [ 6 ] , a n d it d i d n o t have a n t i b o d y activity against purified A d l ' t virions as m e a s u r e d b y c o m p l l e m e n t f ~ a t i o n .
57 DISCUSSION T h e results suggest t h a t t h e a f f e c t e d geve f u n c t i o n ila ts505 i~ r e q u i r e d b o t h for virus r e p l i c a t i o n ( p e r h a p s l~or viral D N A synthesis) in p r o d u c t i v e i n f e c t i o n and f o r ceil t r a n s f o r m a t i o n in a b o r t i v e i n f e c t i o n , O t h e r ts m u t a n t s o f A d l 2 have b e e n k n o w n w h i c h are t s in viral D N A synthesis a n d w h i c h a p p a r e n t l y have either a decreased [1,7] o r a n increased [5,7] t r a n s f o r m i n g ability at t h e restrictive t e m p e r a t u r e . Shiroki a n d S h i m o j o [ 8 ] , u s i n g ts mutar~ts o f A d 1 2 , have su[,,gested t h a t a t least 3 viral genes are involved in t h e i n i t i a t i o n o~ viral D N A replication. T h e r e l a t i o n s h i p b e t w e e n t h e viral ger.es r e p r e s e n t e d b y t h e s e m u t a n t s a n d t h e gene r e p r e s e n t e d b y o u r ts505 r e m a i n s t o b e determined. ACKNOWLEDGMENTS We t h a n k Dr. Eigo Nakaso for his ~ o n t i n u e d c o o p e r a t i o n in p r e p a r a t i o n of cell cultures. This w o r k was s u p p o r t e d b y ~ a n t s for cancer research f r o m t h e Ministry of Educatio¢~ o f t h e J a p a n e s e G o v e r n m e n t a n d b y t h e Asahi News Co. REFERENCES 1 Ginsberg, H.S., Ensinger, M.J., Kauffman, ~'~.S., Mayer, A.J. and Lundholm, U. (1974) Cell transformation: a study of regulation with types 5 and 12 adenovir~ temperaturesensitive mutants. Cold Spring Harbor Syrup. Quant. Biol., 39, 419--426. 2 Hama, S. and Kimura, G. (1972) Temperature-sensitive mutants o!" human adenovirus type 12. Japan. J. Microbiol., 9, 969--'970. 3 Hama, S. and Kimura, G. (1973) Infection of rat cell line 2Y1 by adenovirus type 12. Virus, 23, 389 (in JapaJ~se). 4 Kimura, G., Itagaki, A. and Summers, J. (1975) Rat celi line 3YI and its virogenic polyoma and SV40-transformed derivatives. Int. J. Cancer, 15,694--706. 5 Ledinko, N. (1974) Temperature-sensitive mutants of adenovirus 12 defective in viral DNA synthesis, J. ViroL, 14, 457--468. 6 Pope, J.H. and Rowe, W.P. (1964) Immuno:iluoreseen~: studies of adenovirus 12 tumors and of" cells transformed or infected by adenovirus. J. Exp. Med., 120, 577--588. 7 Rubestein, F.E. and Ginsberg, H.S. (1974) q~-ansformation eh:aracteristicz of temperature-sensitive mutants ¢~ftype 12 adenovirus. Intervirology, 3, t70--174. S Sbi~'oki, K. and Sh[mojo, H. (I974) Analysis of adenovirus 12 Semperature-zensitive mutants defective in viral DNA replication. Vlromgy, ~il, 474--485. 9 Yamashita, T. and Shimojo, H. (1969) Induction of cellutar DNA synthesis by adenoviru~ 12 in human embryo kidney cells. Virology, 38,351--355.
