Induction of cellular DNA synthesis by adenovirus 12 in human embryo kidney cells

SHORT

strains of Pereira da Silva and Jacob (21). All other genetic work appears to originate with XPaPa.3 This divergence of lambda strains reveals a rapid process of genetic drift by the lambda prophage. Such drift occurred while the prophage genotype was not being screened. It is hoped that the strain variation documented here will allow proper precautions to be taken when isogenicity is important.

8. 4. 6. 6. 7. 8. 9. 10.

11. Id. 18. 14. 15. 16. lY. 18.

19. 20. 21.

(1951). L. Proc. (1944).

N&Z. 30, 404-410 VOGT, M., WITKIN, E., and APPLEYARD, R., personal communication. JACOB, F., and WOLLMAN, E-L. Ann. Inst. Pasteur 87. 653-673 (1954). FISCHER-FANTUZZI, L., and CALEF, E. Virology 23, 209-216 (1964). MARCHELLI, C., PICA, L., and SOLLER, A. Virology 34, 650-663 (1968), and CALEF, E., personal communication. PICA, L., and CALEF, E. J. Mol. Biol. 32, 513520 (1968). TATUM, E. L., and LEDERBERC~, J. J. Bacterial. 53, 673-684 (1947). KAISER, A. D. Virology 1, 424-443 (1955). KAISER, A. D. Virology 3, 42-61 (1957). KAISER, A. D., personal communication. GREEN, B., and BRACHET, P., personal communication. JACOB, F., personal communication. JACOB, F., and CAMPBELL, A. M. Compt. Rend. 248, 3219-3221 (1959). KELLENBERGER, G., ZICHICHI, M. L., and WEIGLE, J. Nature 187, 161-162 (1960). DOVE, W. F., and SZYBALSKI, W., unpublished measurements (1968). CAMPBELL, A. M. Virology 14. 22-32 (1961). EIS~N, H., FUERST, C. R., SIMINOVITCH, L., THOMAS, R., LAMBERT, L., PEREIRA DA SILVA, L., and JACOB, F. Virology 36, 224 241 (1966). SUSSMAN, R., and JACOB, F. Compt. Rend. 254, 1517-1520 (1962). JACOB, F., SUSSMAN, R., and MONOD, J. Compt. Rend. 254, 4214-4216 (1962). PEREIR-4 Dh SILVA, L., and JACOB, F. Bnn. Inst. Pasteur 115, 145-158 (1968). Acad.

Sci.

U.S.

WILLIAM F. DOVER McArdle Laboratory University of Wisconsin Madison, Wisconsin 63706 Accepted March 17, 1969 4 Supported by a program-project 07175) from the National Institutes the Alexander and Margaret Stewart

Induction

REFERENCES

1. LEDERBERG, E. Genetics 36,560 2. GRAY, C. H., and TATUM, E.

351

COMMIJNICATIONS

3 One further strain which is not in wide use is the lambda from Wollman’s E. coli K12 112-12. This pha,ge is reported to have the unique density of 1.512 (15).

of Cellular Adenovirus

DNA

grant (CAof Health and Trust Fund.

Synthesis

by

12 in Human

Embryo Kidney

Cells

It was shown in a previous report (1) that adenovirus 12 (Ad 12) induced cellular DNA synthesis in non-growing hamster cells in which the virus did not replicate. The present communication describes the induction of cellular DNA synthesis by Ad 12 in nongrowing human embryo kidney cells (HEK) in which the virus replicated efficiently. Ledinko reported the stimulation of DNA synthesis in HEK by Ad 12 (2) but the nature of DNA induced was not determined. The primary culture of HEK was prepared as described previously (3). Cells in the primary culture of HEK were harvested, suspended in growth medium (Eagle’s MEM, supplemented with 10% bovine serum), and cultured at 36” in 2-0~ bottles (lo6 cells in 5 ml per bottle). Confluent monolayers were usually formed 3 days after seeding of cells. Then, the growth medium was replaced with the maintenance medium (Eagle’s MEM without bovine serum) and the cultures were further incubated at 36”. DNA synthesis in the above culture was examined by the incorporation of 3H-thymidine into trichloroacetic acid (TCA) insoluble fractions. Every 24 hours after seedingof cells, aliquots of the cultures were pulselabeled with 3H-thymidine (The Radiochemical Center 5,000 mCi/mM, 10 &?i per bottle) for 30 minutes at 36”. Then, cells were harvested and cold 5 % TCA insoluble fraction of cells was collected on a filter paper and the radioactivity of the dried filter paper was measured as described previously (1). DNA synthesis in these cell cul-

352

SHORT 3,000

COMMUNICATIONS

-

Medium

Oh

1

change to maintenance

medium

2345678 Days

after

after pulse-labeling with 3H-thymidine (10 &i per bottle). DNA synthesis in these cultures is shown in Fig. 2. DNA synthesis began to increase 16 hours postinfection (pi) and reached its maximum 24-28 hours pi, when cells were infected at higher multiplicities (4 and 20 TCIDS,, per cell). When cells were infected at a lower multiplicity (0.8 TCID50 per cell), a lower but definite synthesis of DNA was induced. It was confirmed that DNA synthesis was induced by Ad 12, since Ad 12 neutralized by antiAd 12 rabbit serum did not induce DNA synthesis in non-growing HEK, as shown in Fig. 3. The nature of induced DNA was examined with hybridization technic of Denhardt (4) between induced DNA (3H-labeled) and

seeding

3,000

1. DNA synthesis in cultured HEK cells. Cells in the primary culture of HEK were harvested, suspended in growth medium, and seeded into 2-0~ bottles. The cultures were incubated at 36”. After 4 days’ incubation, the growth medium was replaced with maintenance medium and the cultureswere incubatedat 36”. Every 24 hours, cells were harvested after pulse-labeling with 3Hthymidine (10 aCi per bottle) for 30 minutes. Radioactivities in TCA insoluble fractions of the harvested cells were measured and expressed in cpm per 106 cells. FIG.

tures is depicted in Fig. 1. The incorporation reached maximum 48 hours after cellseeding and then declined. A slight increase in the incorporation was noticed 24 hours after medium change. The incorporation then decreased and reached minimum after 48 hours. Thus, HEK became inactive in DNA synthesis and could be regarded to be in a non-growing state after the above incubation. DNA synthesis in non-growing HEK, prepared as described above, was then examined after infection with Ad 12. The cultures were infected with Ad 12 strain Huie at multiplicities of 20, 4, and 0.8 TCIDso per cell. To minimize the influence of the medium change on DNA synthesis, the medium was not changed either before nor after infection. Control cultures were inoculated with the same volume of Eagle’s MEM. After infection, cells were harvested every 4 hours

moi 20 i ._, :

’

A:

:

i

m

Peroid

of incubation

after

infection

(hrs)

2. DNA synthesis in non-growing HEK after infection with Ad 12. Confluent monolayers of HEK secondary cultures in 2-0~ bottles were maintained at 36” for 4 days in maintenance medium and then infected with Ad 12 at input multiplicities of 20, 4, and 0.8 TCID50 per cell. At S- or 4-hour interval, cells were harvested after pulselabeling with 3H-thymidine (10 pCi per bottle) for 30 minutes. Radioactivities in TCA insoluble fractions of the harvested cells were measured and expressed in cpm per lo6 cells. (0) Control cultures; (0) Ad la-infected at the multiplicity of 20 TCIDbo per cell, (X) Ad la-infected at the multiplicity of 4 TCID50 per cell, (A) Ad 12infected at the multiplicity of 0.8 TCIDso per cell. FIG.

SHORT

filters. The preparation of these DNA’s and the details of the hybridization technic were described (1). The result is shown in Table 1. The amount of DNA hybridized was expressed by the percentage of DNA (cpm) fixed on a filter to the input DNA (cpm) and the extent of homology was expressed by the percentage of the amount of the test DNA hybridized to the amount of the homologous DNA hybridized. The ratio between viral and cellular DNA in induced DNA was roughly estimated from the extent of homology. As shown in Table 1 and Fig. 4, a small

1

3,000

01

8 Period

353

COMMUNICATIONS

of mcubofion

16

24 after

infection

Viral

ihrsl

3. DNA synthesis in non-growing HEK by Ad 12 neutralized with anti-Ad 12 rabbit serum. Ad 12 (107.’ TCIDso/ml) was mixed with an equal volume of anti-Ad 12 rabbit serum (40 neutralizing units per 0.1 ml, calculated from the neutralizing titer against 100 TCIDso of Ad 12). The mixture was kept at 36” for 2 hours and then 4” overnight. The neutralized virus was inoculated into nongrowing HEK as in Fig. 2 at the multiplicity of 26 TCIDso per cell, calculated from the virus titer before neutralization. Control cultures were inoculated similarly with unneutralized Ad 12, anti-Ad 12 serum without Ad 12 or the medium. At S-hour intervals, cells were harvested after pulse-labeling with 3H-thymidine (10 &i per bottle) for 30 minutes. Radioactivities in TCA insoluble fractions of the harvested cells were measured and expressed in cpm per lo6 cells. (0) Unneutralized Ad 12; (0) the medium without Ad 12; a) neutralized Ad 12; (0) anti-Ad 12 serum without Ad 12.

- 7.0 2 s

FIG.

cellular or viral DNA. Non-growing HEK were infected with Ad 12 at multiplicities of 20 and 0.8 TCIDbo per cell. At intervals of 8 hours, cells were collected after pulselabeling with 3H-thymidine for 30 minutes, and induced DNA was prepared therefrom. Cellular DNA was prepared from uninfected HEK and viral DNA was prepared from purified Ad 12 virions. Induced DNA (3H-labeled) was then hybridized to viral or cellular DNA, immobilized on Millipore

- 6.0

5.0

“0 <

0, 0

F ‘F = >. ,-2 2

-4.0

=

- 3.0

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Period

DN;

16 of incubation

24

32 offer

f

40

48

infection

lhrsi

1

FIG. 4. Viral replication and cellular and viral DNA synthesis in nongrowing HEK infected with Ad 12. Confluent monolayers of HEK were infected with Ad 12 at the multiplicity of 20 TCIDho per cell. After adsorption at 36” for 2 hours and then neutralization of unabsorbed virus by antiAd 12 rabbit serum at 36” for 60 minutes, cells were incubated at 36” in maintenance medium. Cells were harvested every 8 hours and suspended in PBS (2 X 106 cells per ml). After freezing and thawing 6 times, the infectivity was titrated in HEK tubes. Cellular and viral DNA synthesis in HEK infected with Ad 12 at the multiplicity of 20 TCIDS~ per cell was estimated from Fig. 2 and Table 1 and depicted separately. A small amount of DNA induced in mock-infected HEK (control) was subtracted in this figure.

354

SHORT

COMMUNICATIONS

INDUCED

BY Ad

TABLE HYBRIDIZATION

OF DNA

1 12 IN HEK 20 TCIDm

DNA

on a 6lter

HEK DNA 20 rg/filter

Ad 12 DNA 5 pg/filter

Input

3H-HEK” 3H-HEK 3H-Ad $H-Ad aH-Ad aH-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-Ad 3H-HEK

12-HEK 12-HEK 12-HEK 12-HEK 12-HEK 12-HEK 12-HEK 12b 12 12 12-HEK 12-HEK 12-HEK 12-HEK 12-HEK 12-HEK 12-HEK

TO

HEK

per cellc

AND

Ad

12 DNAQ

0.8 TCID60

per cellc

DNA

(16 (20 (24 (28 (32 (40 (48

hr hr hr hr hr hr hr

pi)& pi) pi) pi) pi) pi) pi)

y0 boundd

‘% homology”

y0 boundd

43.3 (41.4)f 34.0

100.0 (100.0)’ 78.5

40.4 -

100.0

9.3 1.3 0.4

21.5 3.0 1.0

34.5 20.9 -

85.4 51.7 -

0.3 0.4 0.4

0.8 1.0 1.0 (1.4)

0.5 0.5 0.4

1.3 1.3 1.0

100.0 (100.0) 13.0 75.5 95.5 96.5 96.0 98.5 99.5 (1.7)

38.3

100.0

(0.6)

(16 (20 (24 (28 (32 (40 (48

hr hr hr hr hr hr hr

pi) pi) pi) pi) pi) pi) pi)

37.1 (35.2) 4.8 28.0 35.4 35.8 35.6 35.5 36.9

QJ.6)

y0 homologye

-

5.4 18.9 -

11.4 46.8 -

37.3 37.8 36.5

97.4 98.8 97.9

D Heat-denatured HEK-DNA (20 pg) or Ad 12 DNA (5 rg) was fixed to a Millipore filter (HAWP, 25 mm). After preincubation of the filter at 60” for 6 hours in 6x SSC containing 0.1% bovine serum albumin, heat-denatured DNA (200&20,006 cpm) to be hybridized was put into the vial containing the filter and the annealing was carried out at 60” for 24 hours. After washing and drying of the filter, radioactivity of the filter was measured. A duplicate of filters was used, and non-specific binding to a DNAfree filter (less than 0.8% of the input DNA) was subtracted. b 3H-Ad 12-HEK: 3H-labeled DNA induced in HEK by Ad 12 at the time indicated in the parenthesis. The specific activities of labeled DNA’s induced at the multiplicity of 20 TCIDso per cell were 16 hours pi, 880 cpm; 20 hours pi, 3180 cpm; 24 hours pi, 3240 cpm; 28 hours pi, 3270 cpm; 32 hours pi, 2560 cpm; 46 hours pi, 1810 cpm; and 48 hours pi, 1510 cpm/pg, respectively. The specific activities of labeled DNA’s induced at the multiplicity of 0.8 TCIDso per cell were 20 hours pi, 630 cpm; 24 hours pi, 1030 cpm; 32 hours pi, 1390 cpm; 40 hours pi, 1130 cpm; and 48 hours pi, 986 cpm/pg, respectively. 3H-HEK: 3H-labeled DNA prepared from non-infected HEK (the specific activity 5540 cpm/pg).aH-Ad 12: 3H-labeled DNA prepared from purified Ad 12 virions (the specific activity 2060 cpm/& . c Input multiplicity of infection. d The amount of DNA hybridized (70 bound) was expressed by the percentage of DNA (cpm) trapped on the filter to the input DNA (cpm). c The extent of homology (% homology) was expressed by the percentage of the amount of the test DNA hybridized (To bound) to the amount of the homologous DNA hybridized. f Figures in parentheses were obtained in a separate experiment.

of cellular DNA was induced at the early stage of viral replication, although major part of DNA induced 24 hours pi and thereafter were viral. It was shown previously by immunofluorescence that all cells in a HEK culture induced viral antigens after infection with Ad 12 at the similar amount

multiplicity (30 TCID,,, per cell) (5). It is, therefore, inferred that cells which were stimulated to synthesize cellular DNA may be the cells that produced virus. The replication of Ad 12 in non-growing HER was examined. HEK was infected with Ad 12 at the multiplicity of 20 TCIDSo

SHORT

per cell in the same manner. After adsorption of virus for 2 hours at 36”, cells were washed with phosphate buffered saline (PBS) and 100 neutralizing units of antiAd 12 rabbit serum (neutralizing antibody titer against 100 TCIDjo of Ad 12, 1: 5,400) was added in 5 ml per bottle and unadsorbed virus was neutralized at 36” for 1 hour. Then, cells were washed twice with PBS and kept at 36” in fresh maintenance medium. Infected cells were harvested every 8 hours and suspended in PBS (2 X lo6 cells per ml). After freezing and thawing 6 times, the infectivity was titrated in HEK tubes (Fig. 4). The infectivity began to increase 24 hours pi, showing the efficient replication of Ad 12 in HEK. These observations show that Ad 12 induced cellular DNA synthesis in nongrowing HEK in which the virus replicated efficiently. In contrast to the induction of cellular DNA synthesis by adenovirus in hamster cells, the cellular DNA synthesis thus induced shifted to viral DNA synthesis as soon as the viral replication progressed. The shift from cellular to viral DNA synthesis, which was induced by adenovirus in growing cells, has already been described by Green et ab. (6) or by Ginsberg et al. (7). The entire course of DNA synthesis may be explained by the combination of the induction, followed by the suppression of cellular DNA synthesis and the replication of viral DNA. It remains to be determined whether or not the induction of cellular DNA synthesis is a prerequisite for the replication of viral DNA. REFERENCES H., and YAMASHITA, T., Virology 36, (1968). N., Cancer Res. 27, 1459-1469 (1967). H., SAWAMURA, K., WADA, R., and YOSHIKAWA, E., Japan. J. Med. Sci. Biol. 19, 1-8 (1966). DENHARDT, n. T., Bioch,em. Biophys. Res. Cmmun. 23,641-646 (1966). SHIMOJO, H., YAMAMOTO, H., and ABE, C., Virology 31, 748752 (1967). GREEN, M., PINA, M., and CHAGOYA, V., J. Biol. Chem. 239, 1188-1197 (1964). GINSBERG, H. S., BELLO, L. J., and LEVINE, A. J., In “The Molecular Biology of Viruses”

1. SHIMOJO, 422433 2. LEDINKO, 3. SHIMOJO,

1. 6. 6. 7.

355

COMMUNICATIONS (J. S. Colter and W. Paranchych, ed.), Academic Press, New York (1967). TADASHI HIROTO

Department Xational Murayama, Accepted

p. 547.

YAMASHITA SHIMOJO

of Enteroviruses Institute of Health Tokyo, Japan March

Ribonucleic Venezuelan

25, 1969

Acids

Synthesized

in

Equine Encephalomyelitis

Virus-Infected

Cells

The ribonueleic acids of some arboviruses have been recently studied. RNA’s from virions of Western Equine Encephalomyelitis (WEE) and from Sindbis and Semliki forest (SF) viruses appear to have similar sedimentation characteristics (40-425) and several virus-specific RNA’s (15-428) were found in virus-infected cells (l-3) including RNAse-resistant replicative intermediates (RI). This paper reports a study of the ribonucleic acids synthesized in Venezuelan Equine Encephalitis (VEE) virus-infected cells. A strain of VEE virus was obtained from the collection of the Rockefeller Institute in 1944. Since the strain formed plaques of various size, we attempted to isolate a genetically pure clone of the virus. A stable variant which formed small plaques was isolated, cloned 5 times from a single plaque, and then passaged 28 times in chick embryo fibroblast monolayers. The titer of the stock was 5 X log to 1O’OPFU/ml. Monolayers of chick embryo fibroblasts were grown for 2-3 days in ESLA medium (Earle’s solution, 2.5% of lactalbumin hydrolyzate) in a CO2 incubator. HTLA medium (Hanks’ solution, Tris 0.03 M, 0.5 % of lactalbumin hydrolyzate) was used as maintenance medium without CO2 . Actinomycin D (Lyovac, Cosmegen), 2 pg/ml, was added to the maintenance medium after 1 hour’s contact of the virus with the cells (100 PFU/cell at 4”). 3H-5-uridine (Radioisotope Centre, Leningrad, specific activity 0.5 Ci/mmole), 5 pCi/ ml was added to the medium for 1 hour at various times after infection. RNA extrac-