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Induction of cellular mRNA synthesis in BSC-1 cells infected by SV40
36.439-444 (1968)
VIROLOGY
Induction
of Cellular
mRNA
KINICHIRO The Salk Institute
Synthesis ODA’
AND
for Biological
in BSC-1 Cells Infected
RENATO
DULBECCO
Studies, San Diego,
Accepted April
by SV40
California
92112
18, 1968
A marked stimulation of cellular mRNA synthesis occurs during productive infection of BSC-1 cells with SV40. The synthesis of ribosomal and transfer RNA is not stimulated. INTRODUCTION
The small DNA-containing tumor-producing viruses, polyoma virus and SV40, cause striking changes in the functional state of the cells they infect. In productive infection, which is characterized by viral multiplication and ultimate cell death, these changes appear before the cells succumb to infection. It has been shown that in suitable systems both viruses induce the synthesis of cellular DNA and of several cellular enzymes (Dulbecco et al., 1965; Hatanaka and Dulbecco, 1966). An induction of the synthesis of cellular RNA by polyoma virus has been reported by Benjamin (1966). This paper will describe marked alterations of the synthesis of cellular RNA’s during t’he productive infection of BSC-1 cells by SV40. MATERIALS
AND
METHODS
BSC-1 cells, a permanent line of African green monkey kidney cells, were cultivated in reinforced Eagle’s medium with 10 % fet’al bovine serum and 10% tryptose phosphate in glass or plastic petri dishes at 37” in a humidified incubator flushed with a COz-air mixt,ure. SV40 virus stocks were prepared in BSC-1 cells and titrat’ed by plaque formation in t)he same cells. In t.he experiments t,o be described, monolayer cult,ures of BSC-1 cells were infected with
SV40
when
they
became
1 Present address: Virus Laboratory, Cancer Center Research Institute, chome, Chno-ku, Tokyo, Japan.
confluent,, National Tsukiji 5439
using 0.4 ml of virus stock on a 100 mm petri dish. After 60-90 minutes’ absorption at 37”, 10 ml of medium was added to each culture. Labeling of the cultures was carried out by adding to the medium uridine-3H, 24.7 C/ mmole, in the presence of 2 X 10-S M unlabeled thymidine . Cell fractionation into nuclear, cytoplasmic and membrane fractions, was carried out according to Attardi and Attardi (1967). The RlUA was extracted from whole cells or nuclear fractions in the following way. The cells or nuclei were suspended in 0.05 M sodium acetate pH 5.1, at a concentration of approximat’ely 1 to 2 X lO’/ml, and one-tenth volume of 10 % sodium dodecyl sulfate (SDS) was added. The mixture was heated at 60” for 10 minutes and one-fifth volume of 5 dl sodium perchlorate was added. Nucleic acids were extract’ed two to three times with an equal volume of chloroform-isoamyl alcohol (24: l), precipitated by adding two volumes of 95 % et’hanol, and kept at - 20” overnight. The suspension was then centrifuged; the precipitate was gently homogenized using a Dounce homogenizer and was resuspended in 0.05 A1 Tris, pH S.0, containing 0.01 M MgCls. Electrophoretically purified pancreatic DNase I (Worthingt,on) was added to a concentration of 20 pg/ml and incubated at 37” for 20 minutes. The RNA was again extracted two or three times with chloroform-amyl alcohol, precipitated with two volumes of ethanol, and stored at -20”. RNA was extracted from cytoplasmic fractions according to Oda and Joklik (1967).
440
ODA
AND
DULBECCO
Cellular DNA was prepared from the nuclear fraction according to Rlarmur (1961), omitting RNase treatment. DNA-RNA hybridization was carried out essentially according to Gillespie and Spiegelman (1965). Hybridization was carried out in 6 X SSC containing 0.1% SDS at 66” for 24 hours. RSB is 0.01 M Tris-HCl, pH 7.4; 0.01 M NaCl; 0.0015 M RlgCL. SSC is 0.15 M NaCl; 0.015 M sodium citrate. Sucrose-SDS
gradients are 15-30 % (w/v) sucrose in 0.5 % SDS, 0.005 M Tris-HCl, pH 7.4, and 0.1 M KaCl. Gradients were collected through a Gilford automatic absorbancy recorder. RESULTS
Ej’ect of fT@ Infection on RNA Synthesis in the Infected Cells xine confluent loo-mm monolayer cultures of BSC-1 cells (10’ cells each) were
Cd
i
I
0,000
i
5,000
-
! 10,000
5,000
z E > E Fe .z .z 2 .g tr"
10,000
2.0
5,000
IO
IO
20
30
40 Fraction
I
1
IO
20
I
30
40
no
1. Sedimentation in a sucrose gradient of uridine-3H labeled cytoplasmic extract of SY40-in fected and mock-infected BSC-1 cultures. A = mock-infected cultures; B = SV40-infected cultures. (a) Labeling for 3 hours beginning 20 hours after infection; (b) labeling for 3 hours beginning at 48 hours; (c) labeling for 3 hours beginning at 94 hours. 0, Radioactivity; 0, absorbancy at 260 mu. FIG.
mRNA
SYSTHESIS
IX
SVJO-ISFECTED
441
over the gradient, but especially in t’he polysome region, and in the 74 S, 60 S, and 40 S peaks, which correspond to ribosomes and subribosomal particles. These differences begin to appear at 48 hours (Fig. lb). In order to determine the RNA species that were synthesized at an enhanced rate after infection, BSC-1 cultures were infected as described above, and, beginning at 10 and S-l hours after infection, were labeled for 16 hours with uridine-3H (0.1 PC/ml) in the presence of 1OW M cold uridine and 10m5M cold thymidine. The cytoplasmic fractions, made 1% with respect to SDS, were cent,rifuged in a lli-30% sucrose gradient cont,aining 0.5% SDS. Absorbancy at 260 rnp and radioactivity were determined in fractions collected from the bott’om of the t’ube; the data are given in Fig. 2. The long labeling period allowed labeling of all RNA species, including ribosomal and t8ransfer RNA. The data show that the rate of synthesis of these two species of RNA decreases somewhat with the age of the cultures, both in infect,cd and mock-infected cultures. The absorbancy pat,tern shows a greater re-
infcct,ed \vith SV40 at an input multiplicit,y of 100 I’l’L/cell. An equal number of cultures I\-we mock-infected, i.e., were handled cx:tct,ly like t,he infected ones, using medium instead of virus. Beginning at 20, 4S, and 94 hours after infection, groups of three infected or moclt-infected cultures were each labeled for 3 hours \vit,h uridine-“H (3 &/ml) in t,he presence of 2 X lo-” 3/ cold thymidine. At the end of the labeling period, the cultures lvc’rc \vashed and the cells were scraped off and fwctionated into a nuclear, a cytoplasmic, and :L membrane fr&ion. The cgtoplnsmic fraction w-as examined by sedimentation in a 1.5-30% (w/v) sucrose gradient, in RSB, with a 60 % sucrose cushion (3 ml), using an SW 2,?Spine0 rotor at 16,000 rpm for 15 hours. Fractions collected from the bottom of the tubes were counted for radioactivity after precipitation with 5 % t’richloroacet,ic acid. The results obtained are given in E’ig. 1. It was found that infected and mock-infected cultures have a similar distribution of label in the early stage (Fig. la), but differ markedly in late stages (Fig. lc), when t,he infected cultures have much more radioactivity. The excess is found all
lb)
BSC-1 CELLS
(b)
Fraction
no
FIG. 2. Sedimentat.ion ill an SDS sucrose gradient of labeled cytoplasmic RNA from SV40-infected and mock-ilrfected BSC-1 cultures. A = mock-infected cultures; B = 8\‘40-infected cultures. (a) Labeling betwee 10 and 26 holu-s after infection; (b) labeling between 84 and 100 hours. 0, Radioactivity; l , absorbancy at 260 rnp.
442
ODA
AND
DULBECCO
duction of the ribosomal RKA peaks in the infected cultures, suggesting a more rapid degradation of ribosomal R.XA’s after infection. Since the rsdioactivit’y sedimentation pattern shows no evidence of degradation of the recently synthesized RNA, only the RNA of old ribosomes appeared to be degraded. The increase in the rate of RNA synthesis in infecbed cultures (shown in Fig. 1) cannot be atrributed to increased synthesis of ribosomal or transfer RNA, but must be caused by synthesis of messenger RNA, either viral or cellular. In order to separate cellular from viral messenger RNA, which is synthesized after infection (Oda and Dulbecco, 1968), the synthesis of cellular mRNA was measured by hybridization with cellular DNA. Kinetics of Synthesis of Cellular mRNA afte? Infection BSC-1 cultures were infected as described above. At various times uridine-3H (final concentration 10 PC/ml) and cold thymidine (final concentration 2 X 10-j M) were added to the medium of both infected and mock-infected cultures. After 3 hours at 37”, the cultures were fractionated into a nuclear and a cytoplasmic fraction. The RNA was extracted from each fraction and dissolved in 1.3 ml of SSC/lO; 0.4 ml of each fraction was hybridized to 20 pg of BSC-1 cell DNA. The results, reported in Fig. 3, show t’hat in the mock-infected cultures there is a progressive decline in the rat’e of synt’hesis of mRNA, while in the infected cultures the rat,e of synthesis of cellular mR-UA increases, beginning about 24 hours after infection. The increase is more pronounced in the nuclear fraction probably because the labeling period was short. At this time, t’he rate of viral DNA synthesis, thymidine kinase, and SV40 T-antigen began to rise; also the synthesis of “late” viral messenger RNA began (Oda and Dulbecco, 1968). of Cellular mRNA, Acceding to Size, in XV@Infected BXC-1 Cells ,4n attempt was made to determine whether the cellular mRNA whose synthesis is induced by SV40 infection differs in size from the mRNA synthesized before inducFractionation
FIG. 3. Time course of synthesis of cellular mRNA in SIIO-infected BSC-1 cultures. 0, Nuclear RNA from infected cells; 0, nuclear RNA from uninfected cells; A, cytoplasmic RNA from infected cells; A, cytoplasmic RNA from uninfected cells.
tion occurs. Sixty loo-mm cultures of BSC-1 cells were infected at an input multiplicity of 100. The cultures were labeled wit)h uridine3H as follows. Batch a: 30 @C/ml for 12 hours, beginning at 10 hours after infection; batch b: 25 @Z/ml for 6 hours, beginning at, 50 hours; batch c: 25 &/ml for 6 hours, beginning at 93 hours. The RNA was extract’ed from whole cells at Dhe end of the labeling period. The RNA, dissolved in SSC/lO and 1% SDS, was centrifuged on a E-30% sucrose gradient with 0.5% SDS (SW 23,24,000 rpm, 19 hours). A sample of 0.005 ml of each fraction was counted aft,er precipitation with 5 % trichloroacetic acid; the remainder were pooled into six pools as indicated in Fig. 4. The RNA in each of the pools were precipitated with ethanol after addit’ion of 0.5 mg of cold yeast RNA as carrier. The data of Fig. 4 show that there was little radioact,ivity in ribosomal or transfer Rn’A in batches b or c, since t,he labeling period was sufficiently short; there was COINsiderable labeling of t,hese RNA’s in batch a, owing to the longer labeling period. There
mP\SA
SYNTlIkMIS
IS
S\‘JO-INFECTElI
USC-1 CELLS
443
was also a progressive decreaseof absorbancy in the ribosomal RNA peaks, as already indicated above. An aliquot of the RNA contained in each of t,he six pools was hybridized to 20 pg of BSGl cell DNA. The results are are shown in I$. 5. The data once more show the marked increase in cellular mRSA bet\veen 20 and 50 hours after infection. The size distribution is heterogeneous. The heterogeneity is not due to RNA breakdown during ext,raction, since the profile of radioactivit,y (Figs. 2 and 4a) gives no evidence of breakdown of the ribosomal ItKL4. The heterogeneit’y probably derives from t)he size variation of the messengers. As infection proceeds, the distribution of the label over the six pools does not change markedly, although there may be a slight increase in the proport’ion of short,er sizes at later times. DISCUSSION
Fraction
na
FIG. 1. Fractionation by size of uridine-3II labeled 1tNA in S\‘10-infected cells. (a) Labeling between 10 and 22 hours after infection; (b) labeling between 50 and 50 hours; (c) labeling between 93 and 99 hours. The variolls pools contained the following nllmbers of fractions (of constant size); pool I, 7 fractions; pool II, 5 fractions; pools III, I\‘, alld \., 1 fractions each; pool 1’1, 5 fractions. 0, Radioactivity; 0, absorbancy at, 200 m/l.
t t t ,fsI!, 6 2:sI!, t
2%
16.3
45
4s
FIG. 5. Size distribution of cellular mRNA in S\.iO-infected BSC-1 cells. The pools I to VI are defined in Fig. 4. (A) Labeling between 10 and 22 hours after infection; (B) labeling between 50 and 56: hours; (C) labeling between 93 and 99 hollrs. The radioactivity hybridizable to BSC-1 DN.4 in each pool was divided by t’he numbers of fractions composing each pool (see Fig. 4).
A marked stimulation of cellular mRNA synthesis occurs during the productive infection of BSC-1 cells \\-ith SV40. There are several interest’ing aspects of this phenomenon. One point is t)hat t,he st,imulation is not generalized to all cellular RSA’s, but is confined to messenger RNA. Clearly the synt,hesis of ribosomal and transfer RKA is not stimulat’ed; on t)he contrary, the ribosomal RKA of old ribosomes is broken down more rapidly after infection. The stimulation may affect preferentially cert’ain classes of messenger RNA. Thus the quest’ion arises of the mechanism of such sclect’ive induction. Another point’ is that stimulat’ion begins approximat,elp when the synt’hesis of viral DSA, virus-specific thvmidine kinase, and T-ant,igen begins. Thus‘induction of mRKA synthesis may be also the consequence of the expression of an “early” viral gene. -4 t>hird point, is the relationship between the virus-induced synthesis of cellular messenger RSA here described and t,he virusinduced synthesis of cellular DDlA and enzymes that both polyoma virus and SV40 cause in some cell systems. This relationship cannot be studied 111 the system employed in this work because no induction of cellular D?;,4 and enzymes is det’ected in t’he BSC-1 cells after SV40 infection. The infection, hojvever, causes the synthesis of a virus-
444
ODA
AND
specific thymidine kinase whose specification is unknown and may be partly cellular (Hatanaka and Dulbecco, 1967). The relationships of the synthesis of this enzyme to that of the cellular RiSA therefore needs investigation. Finally, it should be remarked t#hat the induction of cellular messenger RKA by SV40 and polyoma virus (Benjamin, 1966) adds to the differentiation of these viruses from cytopathic viruses, which usually cause inhibition of cellular messenger RNA synthesis. ACKNOWLEDGMENT This work was supported by Public Service Research Grant CA07592.
Health
REFERENCES
ATT~~RDI,B., and ATT~~RDI,G. (1967). Proc. Natl. Acad. Sci. 68, 1051-1058.
BENJAMIN, T. L. (1966). Virus-specific
RNA
in
DULBECCO cells productively infected or transformed by polyoma virus. J. &?oZ. Biol. 16, 359-373. DCTLBECCO,li., HARTWELL, L. H., and \-OGT, 11. (1965). Induction of cellular DNA synt,hesis by polyoma virus. Proc. Natl. Acad. Sci. U.S. 53,403-410. GILLESPIE, D., and SPIEGELMAN, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. ,Vfol. Bid.
12,829~842. HATANAKA, JI., and DULBECCO, I<. (1966). Induction of DNA synthesis by ST’40. Proc. Satl. $cad. Sci. U.S. 66, 736-740. HATANAKA, PIT., and DULBECCO, 1%. (1967). Sly40specific thymidine kinase. Proc. Satl. ilcad. Sci. U.S. 68, 1888-1894. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Sol. 3, 208-218. ODA, K., and DULBECCO, R. (1968). Proc. Natl. Acad. Sci., in press. ODA, K., and JOKLIK, W. 1~. (1967). Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA. J. Mol. Biol. 27,395-419.
VIROLOGY
Induction
of Cellular
mRNA
KINICHIRO The Salk Institute
Synthesis ODA’
AND
for Biological
in BSC-1 Cells Infected
RENATO
DULBECCO
Studies, San Diego,
Accepted April
by SV40
California
92112
18, 1968
A marked stimulation of cellular mRNA synthesis occurs during productive infection of BSC-1 cells with SV40. The synthesis of ribosomal and transfer RNA is not stimulated. INTRODUCTION
The small DNA-containing tumor-producing viruses, polyoma virus and SV40, cause striking changes in the functional state of the cells they infect. In productive infection, which is characterized by viral multiplication and ultimate cell death, these changes appear before the cells succumb to infection. It has been shown that in suitable systems both viruses induce the synthesis of cellular DNA and of several cellular enzymes (Dulbecco et al., 1965; Hatanaka and Dulbecco, 1966). An induction of the synthesis of cellular RNA by polyoma virus has been reported by Benjamin (1966). This paper will describe marked alterations of the synthesis of cellular RNA’s during t’he productive infection of BSC-1 cells by SV40. MATERIALS
AND
METHODS
BSC-1 cells, a permanent line of African green monkey kidney cells, were cultivated in reinforced Eagle’s medium with 10 % fet’al bovine serum and 10% tryptose phosphate in glass or plastic petri dishes at 37” in a humidified incubator flushed with a COz-air mixt,ure. SV40 virus stocks were prepared in BSC-1 cells and titrat’ed by plaque formation in t)he same cells. In t.he experiments t,o be described, monolayer cult,ures of BSC-1 cells were infected with
SV40
when
they
became
1 Present address: Virus Laboratory, Cancer Center Research Institute, chome, Chno-ku, Tokyo, Japan.
confluent,, National Tsukiji 5439
using 0.4 ml of virus stock on a 100 mm petri dish. After 60-90 minutes’ absorption at 37”, 10 ml of medium was added to each culture. Labeling of the cultures was carried out by adding to the medium uridine-3H, 24.7 C/ mmole, in the presence of 2 X 10-S M unlabeled thymidine . Cell fractionation into nuclear, cytoplasmic and membrane fractions, was carried out according to Attardi and Attardi (1967). The RlUA was extracted from whole cells or nuclear fractions in the following way. The cells or nuclei were suspended in 0.05 M sodium acetate pH 5.1, at a concentration of approximat’ely 1 to 2 X lO’/ml, and one-tenth volume of 10 % sodium dodecyl sulfate (SDS) was added. The mixture was heated at 60” for 10 minutes and one-fifth volume of 5 dl sodium perchlorate was added. Nucleic acids were extract’ed two to three times with an equal volume of chloroform-isoamyl alcohol (24: l), precipitated by adding two volumes of 95 % et’hanol, and kept at - 20” overnight. The suspension was then centrifuged; the precipitate was gently homogenized using a Dounce homogenizer and was resuspended in 0.05 A1 Tris, pH S.0, containing 0.01 M MgCls. Electrophoretically purified pancreatic DNase I (Worthingt,on) was added to a concentration of 20 pg/ml and incubated at 37” for 20 minutes. The RNA was again extracted two or three times with chloroform-amyl alcohol, precipitated with two volumes of ethanol, and stored at -20”. RNA was extracted from cytoplasmic fractions according to Oda and Joklik (1967).
440
ODA
AND
DULBECCO
Cellular DNA was prepared from the nuclear fraction according to Rlarmur (1961), omitting RNase treatment. DNA-RNA hybridization was carried out essentially according to Gillespie and Spiegelman (1965). Hybridization was carried out in 6 X SSC containing 0.1% SDS at 66” for 24 hours. RSB is 0.01 M Tris-HCl, pH 7.4; 0.01 M NaCl; 0.0015 M RlgCL. SSC is 0.15 M NaCl; 0.015 M sodium citrate. Sucrose-SDS
gradients are 15-30 % (w/v) sucrose in 0.5 % SDS, 0.005 M Tris-HCl, pH 7.4, and 0.1 M KaCl. Gradients were collected through a Gilford automatic absorbancy recorder. RESULTS
Ej’ect of fT@ Infection on RNA Synthesis in the Infected Cells xine confluent loo-mm monolayer cultures of BSC-1 cells (10’ cells each) were
Cd
i
I
0,000
i
5,000
-
! 10,000
5,000
z E > E Fe .z .z 2 .g tr"
10,000
2.0
5,000
IO
IO
20
30
40 Fraction
I
1
IO
20
I
30
40
no
1. Sedimentation in a sucrose gradient of uridine-3H labeled cytoplasmic extract of SY40-in fected and mock-infected BSC-1 cultures. A = mock-infected cultures; B = SV40-infected cultures. (a) Labeling for 3 hours beginning 20 hours after infection; (b) labeling for 3 hours beginning at 48 hours; (c) labeling for 3 hours beginning at 94 hours. 0, Radioactivity; 0, absorbancy at 260 mu. FIG.
mRNA
SYSTHESIS
IX
SVJO-ISFECTED
441
over the gradient, but especially in t’he polysome region, and in the 74 S, 60 S, and 40 S peaks, which correspond to ribosomes and subribosomal particles. These differences begin to appear at 48 hours (Fig. lb). In order to determine the RNA species that were synthesized at an enhanced rate after infection, BSC-1 cultures were infected as described above, and, beginning at 10 and S-l hours after infection, were labeled for 16 hours with uridine-3H (0.1 PC/ml) in the presence of 1OW M cold uridine and 10m5M cold thymidine. The cytoplasmic fractions, made 1% with respect to SDS, were cent,rifuged in a lli-30% sucrose gradient cont,aining 0.5% SDS. Absorbancy at 260 rnp and radioactivity were determined in fractions collected from the bott’om of the t’ube; the data are given in Fig. 2. The long labeling period allowed labeling of all RNA species, including ribosomal and t8ransfer RNA. The data show that the rate of synthesis of these two species of RNA decreases somewhat with the age of the cultures, both in infect,cd and mock-infected cultures. The absorbancy pat,tern shows a greater re-
infcct,ed \vith SV40 at an input multiplicit,y of 100 I’l’L/cell. An equal number of cultures I\-we mock-infected, i.e., were handled cx:tct,ly like t,he infected ones, using medium instead of virus. Beginning at 20, 4S, and 94 hours after infection, groups of three infected or moclt-infected cultures were each labeled for 3 hours \vit,h uridine-“H (3 &/ml) in t,he presence of 2 X lo-” 3/ cold thymidine. At the end of the labeling period, the cultures lvc’rc \vashed and the cells were scraped off and fwctionated into a nuclear, a cytoplasmic, and :L membrane fr&ion. The cgtoplnsmic fraction w-as examined by sedimentation in a 1.5-30% (w/v) sucrose gradient, in RSB, with a 60 % sucrose cushion (3 ml), using an SW 2,?Spine0 rotor at 16,000 rpm for 15 hours. Fractions collected from the bottom of the tubes were counted for radioactivity after precipitation with 5 % t’richloroacet,ic acid. The results obtained are given in E’ig. 1. It was found that infected and mock-infected cultures have a similar distribution of label in the early stage (Fig. la), but differ markedly in late stages (Fig. lc), when t,he infected cultures have much more radioactivity. The excess is found all
lb)
BSC-1 CELLS
(b)
Fraction
no
FIG. 2. Sedimentat.ion ill an SDS sucrose gradient of labeled cytoplasmic RNA from SV40-infected and mock-ilrfected BSC-1 cultures. A = mock-infected cultures; B = 8\‘40-infected cultures. (a) Labeling betwee 10 and 26 holu-s after infection; (b) labeling between 84 and 100 hours. 0, Radioactivity; l , absorbancy at 260 rnp.
442
ODA
AND
DULBECCO
duction of the ribosomal RKA peaks in the infected cultures, suggesting a more rapid degradation of ribosomal R.XA’s after infection. Since the rsdioactivit’y sedimentation pattern shows no evidence of degradation of the recently synthesized RNA, only the RNA of old ribosomes appeared to be degraded. The increase in the rate of RNA synthesis in infecbed cultures (shown in Fig. 1) cannot be atrributed to increased synthesis of ribosomal or transfer RNA, but must be caused by synthesis of messenger RNA, either viral or cellular. In order to separate cellular from viral messenger RNA, which is synthesized after infection (Oda and Dulbecco, 1968), the synthesis of cellular mRNA was measured by hybridization with cellular DNA. Kinetics of Synthesis of Cellular mRNA afte? Infection BSC-1 cultures were infected as described above. At various times uridine-3H (final concentration 10 PC/ml) and cold thymidine (final concentration 2 X 10-j M) were added to the medium of both infected and mock-infected cultures. After 3 hours at 37”, the cultures were fractionated into a nuclear and a cytoplasmic fraction. The RNA was extracted from each fraction and dissolved in 1.3 ml of SSC/lO; 0.4 ml of each fraction was hybridized to 20 pg of BSC-1 cell DNA. The results, reported in Fig. 3, show t’hat in the mock-infected cultures there is a progressive decline in the rat’e of synt’hesis of mRNA, while in the infected cultures the rat,e of synthesis of cellular mR-UA increases, beginning about 24 hours after infection. The increase is more pronounced in the nuclear fraction probably because the labeling period was short. At this time, t’he rate of viral DNA synthesis, thymidine kinase, and SV40 T-antigen began to rise; also the synthesis of “late” viral messenger RNA began (Oda and Dulbecco, 1968). of Cellular mRNA, Acceding to Size, in XV@Infected BXC-1 Cells ,4n attempt was made to determine whether the cellular mRNA whose synthesis is induced by SV40 infection differs in size from the mRNA synthesized before inducFractionation
FIG. 3. Time course of synthesis of cellular mRNA in SIIO-infected BSC-1 cultures. 0, Nuclear RNA from infected cells; 0, nuclear RNA from uninfected cells; A, cytoplasmic RNA from infected cells; A, cytoplasmic RNA from uninfected cells.
tion occurs. Sixty loo-mm cultures of BSC-1 cells were infected at an input multiplicity of 100. The cultures were labeled wit)h uridine3H as follows. Batch a: 30 @C/ml for 12 hours, beginning at 10 hours after infection; batch b: 25 @Z/ml for 6 hours, beginning at, 50 hours; batch c: 25 &/ml for 6 hours, beginning at 93 hours. The RNA was extract’ed from whole cells at Dhe end of the labeling period. The RNA, dissolved in SSC/lO and 1% SDS, was centrifuged on a E-30% sucrose gradient with 0.5% SDS (SW 23,24,000 rpm, 19 hours). A sample of 0.005 ml of each fraction was counted aft,er precipitation with 5 % trichloroacetic acid; the remainder were pooled into six pools as indicated in Fig. 4. The RNA in each of the pools were precipitated with ethanol after addit’ion of 0.5 mg of cold yeast RNA as carrier. The data of Fig. 4 show that there was little radioact,ivity in ribosomal or transfer Rn’A in batches b or c, since t,he labeling period was sufficiently short; there was COINsiderable labeling of t,hese RNA’s in batch a, owing to the longer labeling period. There
mP\SA
SYNTlIkMIS
IS
S\‘JO-INFECTElI
USC-1 CELLS
443
was also a progressive decreaseof absorbancy in the ribosomal RNA peaks, as already indicated above. An aliquot of the RNA contained in each of t,he six pools was hybridized to 20 pg of BSGl cell DNA. The results are are shown in I$. 5. The data once more show the marked increase in cellular mRSA bet\veen 20 and 50 hours after infection. The size distribution is heterogeneous. The heterogeneity is not due to RNA breakdown during ext,raction, since the profile of radioactivit,y (Figs. 2 and 4a) gives no evidence of breakdown of the ribosomal ItKL4. The heterogeneit’y probably derives from t)he size variation of the messengers. As infection proceeds, the distribution of the label over the six pools does not change markedly, although there may be a slight increase in the proport’ion of short,er sizes at later times. DISCUSSION
Fraction
na
FIG. 1. Fractionation by size of uridine-3II labeled 1tNA in S\‘10-infected cells. (a) Labeling between 10 and 22 hours after infection; (b) labeling between 50 and 50 hours; (c) labeling between 93 and 99 hours. The variolls pools contained the following nllmbers of fractions (of constant size); pool I, 7 fractions; pool II, 5 fractions; pools III, I\‘, alld \., 1 fractions each; pool 1’1, 5 fractions. 0, Radioactivity; 0, absorbancy at, 200 m/l.
t t t ,fsI!, 6 2:sI!, t
2%
16.3
45
4s
FIG. 5. Size distribution of cellular mRNA in S\.iO-infected BSC-1 cells. The pools I to VI are defined in Fig. 4. (A) Labeling between 10 and 22 hours after infection; (B) labeling between 50 and 56: hours; (C) labeling between 93 and 99 hollrs. The radioactivity hybridizable to BSC-1 DN.4 in each pool was divided by t’he numbers of fractions composing each pool (see Fig. 4).
A marked stimulation of cellular mRNA synthesis occurs during the productive infection of BSC-1 cells \\-ith SV40. There are several interest’ing aspects of this phenomenon. One point is t)hat t,he st,imulation is not generalized to all cellular RSA’s, but is confined to messenger RNA. Clearly the synt,hesis of ribosomal and transfer RKA is not stimulat’ed; on t)he contrary, the ribosomal RKA of old ribosomes is broken down more rapidly after infection. The stimulation may affect preferentially cert’ain classes of messenger RNA. Thus the quest’ion arises of the mechanism of such sclect’ive induction. Another point’ is that stimulat’ion begins approximat,elp when the synt’hesis of viral DSA, virus-specific thvmidine kinase, and T-ant,igen begins. Thus‘induction of mRKA synthesis may be also the consequence of the expression of an “early” viral gene. -4 t>hird point, is the relationship between the virus-induced synthesis of cellular messenger RSA here described and t,he virusinduced synthesis of cellular DDlA and enzymes that both polyoma virus and SV40 cause in some cell systems. This relationship cannot be studied 111 the system employed in this work because no induction of cellular D?;,4 and enzymes is det’ected in t’he BSC-1 cells after SV40 infection. The infection, hojvever, causes the synthesis of a virus-
444
ODA
AND
specific thymidine kinase whose specification is unknown and may be partly cellular (Hatanaka and Dulbecco, 1967). The relationships of the synthesis of this enzyme to that of the cellular RiSA therefore needs investigation. Finally, it should be remarked t#hat the induction of cellular messenger RKA by SV40 and polyoma virus (Benjamin, 1966) adds to the differentiation of these viruses from cytopathic viruses, which usually cause inhibition of cellular messenger RNA synthesis. ACKNOWLEDGMENT This work was supported by Public Service Research Grant CA07592.
Health
REFERENCES
ATT~~RDI,B., and ATT~~RDI,G. (1967). Proc. Natl. Acad. Sci. 68, 1051-1058.
BENJAMIN, T. L. (1966). Virus-specific
RNA
in
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