deletion, double mutants, and the induction of host DNA synthesis

VIRObOGY

101,250-260

(1980)

The Isolation of SV40 ~sA/Deietion, Double Mutants, and the Induction of Host DNA Synthesis VALERIE CAROL

PETIT SETLOW, A. F. EDWARDS

MARIA

PERSICO-DILAURO, G. MARTIN’

AND ROBERT

Simian virus 40 mutants were constructed that conttin both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20-200 base pairs leading to a loss in the expression ofthe 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in Iytically infected cells. At nonpermissive temperatures, the &/deletion mutants stimulated the ineorporation of nueieosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutams resulted from semiconservative DNA replication, not repair synthesis. At 200 &g/ml caffeine the induction of host DNA by AS%%was inhibited by 30 to 5O%7 whereas induction by the ~~deIetion mut~ts was abolished. INTRODUCTION

The 92K protein but not the ZOK protein seems to be directly involved in the inducThe early region of SV40 codes for at least tion of host DNA synthesis: (i) The deletion two polypeptides, the 92K and 20K Tmutants that failed to synthesize the 20K antigens (Crawford and O’Farrell, 1979; protein were nondefe~tive for viral DNA Paucha and Smith, 1978; Paucha et al., 1978; replication (Shenk et ai., 1976). (ii) The Prives et nl., 1977; Tenen et al., 1975). Studies concerning the fun&ion of these induction of host DNA synthesis by the proteins have been aided by the isolation of deletion mutants in monkey cells appea~d mutants (Carbon et al., 1975; Chou and normal but could be distinguished from the virus in that the Martin, 1974; Cole et ai., 1971; Crawford et induction by wild-tie induction by the mutants was completely al., 1978; Feunteun et al,, 1978; Paucha inhibited by concentrations of theophyliine et al., 19’78; Sleigh et al., 1978; Tegtmeyer, that only inhibited the induction by wild1972). These mutants are either temperature type virus to 50% (Rundell and Cox, 1979). sensitive for the 92K protein (Jesse1 et al., (It was therefore proposed that the 20K 1975; Tenen et at?.r 19’75) or have deletions in protein played an indirect role in the inducthe SV40 genome between map positions 0.54 and 0.59 and make an abnormal or no tion process.) (iii) The target size by uv or X irradiation for the induction of DNA synthe20K protein (Crawford st a$,, 1978; Crawford and ~Fa~ell~ 1979; Pauehaet ai,) 1978; sis was considerably larger than the coding region for the 2OK protein in both Sleigh et al., 1978). Although these mutants permissive and transforming infections have been available for several years, con(Basilic0 and Di Mayorca, 1965; Chou and fusing and conflicting data exist concerning Martin, 1974; Dulbeccoet al., 1965; Gershon the need for the two early proteins for the et al., 1965). (iv) The induction of host DNA establishment and maintenance of transforsynthesis was somewhat impaired at 42.5” mation and for the induction of cellular when infection was carried out with tsA DNA synthesis. mutants (Chou and Martin, 1974), mutants that affect the 92K but not the ZOK pro’ To whom reprint requests should be addressed. tein (Jesse1 et al., 1975; Tenen et ~2. ) 1975). ~2*~/30/0302~-11~02.~/0 Copyright All rights

8 1980 by Aeademie Press, Inc. of reproduction in my form reserved.

250

SV40 t.sAIDELETION

AND

THE

INDUCTION

(v) Microinjection of a protein related to the 92K protein of SV40 induced DNA synthesis (Tijan et al., 19’78), whereas (vi) microinjection of that portion of the SV40 genome encoding the 20K protein did not (Mueller et al., 1979). On the other hand, none of these experiments are definitive. And furthermore, the tsA mutants induced host DNA synthesis at 40.5” (although viral DNA synthesis was completely abolished at this temperature) suggesting that the 92K protein also might not be essential for this induction (Chou and Martin, 19’74; Chou et al., 1974; Hiscott and Defendi, 1979). In an attempt to clarify the roles of each tumor antigen we have constructed mutants with deletions in the region of 0.540.59 map unit in viruses that already contain a temperature-sensitive mutation, A209 (Chou and Martin, 1974). This report concerns the iso.lation and partial characterization of the double mutants and their physiology in lytic infection. Transformation studies involving these mutants as well as the induction of DNA synthesis in transforming infection are described elsewhere (Martin et al., 1979; Seif and Martin, 1979). MATERIALS

AND

METHODS

Cells, virzcs, and media. CV-1 cells and their growth in Eagle’s media or Ham’s F-12 have been described (Chou and Martin, 1974) as has the SV40 mutant, A209 (Chou and Martin, 1975). Dulbecco-Vogt modified MEM medium (DV) or MEM medium was supplemented with fetal bovine serum (Flow Laboratories, Rockville, Md.) as indicated: DVlO (10% serum), DVO (no serum), etc. Viral stocks were prepared by infecting CV-1 cells in 150-cm2 flasks at m.o.i. = 0.01. Following adsorption -50 ml DV2 medium was added per flask. After a week at 33”, the medium was replaced with 10 ml of fresh DV2. Lysis was generally complete in a further week to 10 days. Control uninfected cultures were grown in parallel and all extracts prepared by freezing and thawing the cultures three times. Titers were generally between lo8 and lo9 PFU/ml. Viral stocks were diluted in PBS for adsorption. DNA preparation. SV40 tsA209 DNA was extracted by the method of Hirt (1967)

OF HOST

DNA

SYNTHESIS

251

from CV-1 cells infected with virus for 6072 hr at 33”. Closed-circular, viral DNA was isolated by centrifugation to equilibrium in 10 mJ4 Tris, 1 mM EDTA, pH 7.4 containing CsCl (p = 1.58) and ethidium bromide (300 pg/ml). Form I DNA was isolated from the gradient and ethidium bromide removed with Dowex-50, followed by dialysis against 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, and 100 m&Z NaCl or by extraction with CsCl-H,O saturated isopropanol. After precipitation with absolute ethanol, the DNA was resuspended in 10 miW Tris-HCl pH 7.4, 1 mM EDTA, and repurified on a CsCl gradient as before. Enzymes, enzymatic digestion and gel electrophoresis. Restriction endonucleases TuqI, HinfI, AluI, EcoRII, and HaeIII were obtained from Bethesda Research Laboratories (Bethesda, Md.) Endonuclease Mb01 was obtained from New England Biolabs (Beverly, Mass). h 5’-exonuclease was the gift of Dr. Kiyoshi Mizuuchi. The restriction endonuclease TuqI was used at 50” for 90-120 min to insure complete digestion. The extent of digestion was analyzed by electrophoresis of the DNA in 0.9-1.2% agarose gels in 40 mM Tris-HCl pH 7.8, 5 mM sodium acetate, and 1 mM EDTA at 90 V for 5 hr or 50 V for 12-14 hr. SV40 DNA digestions with HinfI, AZuI, and Hue111 were performed at 37” for 90 min in 6 n&! Tris-HCl pH 7.6,6 n-&f MgCl, and 6 mM 2-mercaptoethanol. The enzyme Mb01 was used at 37” for 90 min in 10 mM Tris-HCl pH 7.4, 10 mM MgC12, 20 mM KCl, 1 ti dithiothreitol, and 100 pg/ml BSA. EcoRII was used at 37” for 90 min in in 100 n$l4 Tris-HCl, pH 8.0, 75 mMNaC1, 5 mJ4 MgC&, and 100 pg/ml gelatin. The small DNA fragments, containing 1 mg/ml bromphenol blue, 12.5% glycerol, 2.5% SDS, 50 mM EDTA, and 10 mM Tris-HCl pH 8.9, were separated in 5% acrylamide slab gels (3O:l acrylamide: bisacrylamide) in Tris-borate-EDTA buffer (180 mM Tris, 180 mM boric acid, and 5 n&f EDTA, pH 8.6). Electrophoresis was carried out at 50 V for 15 min followed by 150 V for 90-120 min. DNA fragments were visualized by soaking the agarose or acrylamide gels in running buffer containing 2.5 pg/ml eithidium

252

SETLOW

bromide and the gels were photographed under short-wavelength uv light. Isolation of TaqZ resistant virus. Double mutants were isolated by conventional procedures (Carbon et al., 1975; Cole et al., 1971; Feunteun et al., 1978) starting with DNA from a tsA mutant. Concentrated form I A209 DNA was linearized by treatment with Z’uqI (4 units enzyme/pg DNA), phenol extracted, and precipitated with ethanol. A small portion of the total DNA was subjected to electrophoresis in agarose to determine the completeness of the reaction. After resuspending in 10 mit4 Tris, the DNA was briefly digested with A 5’exonuclease at 0” in a mixture containing 67 n&f glyeine-KOH, pH 9.4, and 3 mM MgCl,. The reaction was te~inated by the addition of 9 vol of 17 n&f EDTA and 1 mg/ml calf thymus DNA. The resulting mixture was serially diluted (1:lO) and each dilution added to 4 parts of a 4 mglml DEAE dextran suspension and 8 parts depleted medium (Chou and Martin, 1974). The DNA was then added to confluent CV-1 monolayers and allowed to adsorb at 33” for 30 min. The cells were washed twice with PBS, once with depleted medium, and overlayered with 1% agar in MEM with 3x vitamins and amino acids. The viruses from plaques arising after 3 weeks at 33” were isolated and used to infect CV-1 cells. Potential mutant viral DNA was recovered as Hirt supernatants (1967) and subjected to equilibrium centrifugation in CsCl-EtBr gradients. Recovered DNA was subjected to Tag1 digestion and resistant DNAs were re-plaque purified before characterization with other restriction endonucleases. Preparation of cell extracts, immunoprecipitation, and protein gel electrophoresis. T-Antigens were extracted from CV-1 cells during lytie viral infection. The labeling of cells with [?S]methionine, extraction of T-antigens, immunoprecipitation with anti-T serum and protein A-sepharose CL-4B beads, electrophoresis, and subsequent autoradiography of dried gels were carried out as previously described (Edwards et al., 1979). Viral DNA synthesis. Growing CV-1 cells were radioactively labeled with 0.5 $.Xml [14C]thymidine for 2-3 days. The

ET AL.

radioactive thymidine was removed, labeled cells were trypsinized and seeded onto 2-cm2 wells of 24-well Linbro plates and incubated either at 40” in DV2 or 33” in DVl, for 3 days. The medium was then removed, filtered, and reserved, while 2 x 105 PFU virus/well (m.o.i. = 1 PFUlcell) was adsorbed to the cells for 2 hr at 33 or 40”. The refiltered medium was replaced and the cells were incubated further at either 33 or 40”. At the indicated times, samples were pulse-labeled with 5 ~C~rnl r3H]thymidine for 60 min, all medium was removed, and the cell monolayer was washed once with cold PBS and cells were lysed with a solution of 0.1 N NaOH, 1 mM EDTA, and 0.6% SDS (lysing solution). The cell lysate was then precipitated with TCA. All acid-insoluble material was immobilized on filters and subjected to scintillation counting. Znduction of host DNA synthesis. CV-1 cells were seeded at confluence onto 2-em2 wells in 1 ml DV 0.5 and incubated for 3 days at 37”. Medium was removed and cells were either mock infected or infected with A209 or A209AW’ (m.o.i. = 5) in PBS for 3 hr. The viral titers were sufficiently high that only 1 ml of diluted viral stocks was required. The A209 virus was diluted 30fold; the double mutants 8-fold for ~~9~88 and A209A29t; lo-fold for A209AZ89, A209A290, and A209A29.2; and 12-fold for A209A287. Mock controls contained cell lysates at similar dilutions. All dilutions were in PBS. Virus was removed and half the cultures received 1 ml of DVO containing 200 yglml caffeine while the other half received DVO alone. Other cultures received DVlO + caffeine. All cells were then incubated at 40”. At various times after infection, cultures were pulse-labeled for 1 hr with 10 &X/ml ~3H]thymidine in DVlO prewarmed to 40”. Following the pulse, cells were washed with PBS, lysed with the lysis buffer, and precipitated with TCA. Acid-insoluble material was immobilized on filters and subjected to scintillation counting. Analysis of the nature of the induced host DNA synthesis. Virus, WT, ABOg, or A88.4 was adsorbed to CV-1 cells prelabeled with [14C]thymidine and-incubated 72 hr at 33” or 48 hr at 40”. Cells received a pulse of

SV40 tsA/DELETION

AND THE INDUCTION

253

OF HOST DNA SYNTHESIS RESULTS

of Mutants E nxyme Analysis

Isolation

FIG. 1. Electrophoresis patterns of AZWdeletion mutant DNA digested by various restriction endonucleases. DNA fragments were electrophoresed on 5% acrylamide slab gels as described under Materials and Methods. Upper left: HinfI patterns. Lanes 1 and 8 A209; lanes 2-7, A209M87, A209k88, A209k89, A209h290, AfO9A291, A209h292, respectively. Upper right: Mb01 patterns. Lane 1, A209; lanes 2-7, tsldl mutants in order. Lower left: Hoe111 patterns. Lane 1, A209; lanes 2-7, tsldl mutants; lane 8, unrelated sample. Lower right: AluI patterns. Lane 1, A209; lanes 2-7, tsldl mutants.

L3H]Budr (1 mCi/ml; 23.7 Ci/mmol; New England Nuclear, Boston, Mass.) for I hr at 40” or 2 hr at 33”. The washed cells were extracted by the procedure of Hirt (1967) and cellular DNA, recovered as the material precipitated by 1 M NaCl, was resuspended in 10 mM Tris, 1 mM EDTA, and sonicated for 1 min (maximum power with a microtip, Ultra-Sonics Inc., Plainview, N. Y.). CsCl was added to a final density of 1.74 g/cm2. Centrifugation was for ‘72 hr at 33K rpm and 20” in the fixed angle 40 rotor of a Spinco ultracentrifuge. Fractions were collected on glass microfiber filters (GF/B Whatman) saturated with 20% TCA and 1 fl cold Budr. The filters were washed five times with 5% TCA, dried and their radioactivity was determined.

and

Restriction

The method used to generate the double mutants was similar to published procedures used to construct viable deletion mutants (Carbon et al., 1975; Cole et al., 1977; Feunteun et aZ., 1978), but started with the DNA from a tsA mutant. Briefly, full-length linear SV40 tsA209 DNA was produced by digestion with Tag1 endonuclease and purified by phenol extraction and sedimentation through a sucrose gradient. After concentration, the DNA was digested with sufficient X 5’-exonuclease to digest approximately 10% of the total DNA in 20 min. Digestion was stopped by the addition of EDTA and CV-1 monolayers were infected directly with the A exonuclease, Tag1 digested linear DNA. (No attempt was made to recirculate the molecules because previous reports have shown that this function can be cell mediated (Carbon et al., 1975; Feunteun et al., 1978)) Of the 60 plaques picked, 60% contained DNA that was resistant to further Tag1 digestion. The six mutants reported here contained viral DNA in which the mutation could be explained by deletion of a single continuous piece of DNA. Restriction Endonuclease Deletion Mutants

Malrping

of A2091

Restriction endonuclease mapping of the tsldl mutants was carried out with the restriction enzymes, HinfI, AluI, MboI, HaeIII, and EcoRII. An analysis by gel electrophoresis of the restriction fragments generated by four of the enzymes is shown in Fig. 1. Digestion of the mutants with HinfI showed the D fragments (0.535-0.645 map position) to be smaller than that of the same fragment from A209. Comparison of the D fragments with deletions of known size gave estimates that the deletions ranged in size from 20-200 base pairs. All deletions were contained within the EcoRII B fragment (map position 0.4360.595). Digestion with Hue111 revealed that five of the six.mutants had deletions entirely within the A fragment (0.280-0.590 map

254

SETLOW

position) suggesting that the deleted portion was proximal to 0.59 map unit. The exception was AZ~9~9~ which had lost the A/E fragment junction implying that this deletion terminates between 0.59 and 0.595 map unit. Treatment with Mb01 revealed altered mobility of the D fragment (0.445-0.568) for A~O9~89 and AZO9~92 indicating that the missing fragment began proximal to map position 0.568, The B/D fragments of A209A287, A209A288, and AZOQA%9 were fused (the B fragment runs from map position 0.572 to 0.826 and contains 1264 bp) and were slightly larger than the Mb01 A fragment (1330 bp) indicating that the B/H and H/D junctions were absent and that the entire H fragment (0.562-0.5’72 = 60 bp) was eliminated. The mutant with the smallest deletion, A209M90, appeared to have no alteration in the mi~ation of either the B or D fragment as compared to Af09; we assume that the deletion is entirely within the 60-base pairMbo1 H fragment, although this particular fragment was not resolved in our gel conditions. Digestion with A2uI showed that four mutants, A20911287, AZO?~88, A209~89 and A209A292, had fused B/C fragments (B = 0.545-0.649, 483 bp; C = 0.4850.547, 329 bp), that were slightly smaller than the ALI A fragment (775 bp). The deletion in these mutants extends beyond 0.545, the proximal end of the B fragment. AZOgA290 and A209h291 had deletions entirely within the AM B fragment; the B fragment in AZO9A290 had a slightly increased mobility compared to A209, while the B fragment of A209A292 was significantly smaller and appeared as an apparent doublet with fragment C. Based on these data an estimate of the extent and position of the A209 deletion mutants is presented in Fig. 2.

CV-1 cells infected with WI’, A209, and the six AQOSldeletion mutants were labeled for 4 hr with [35S]methionine at 48 hr postinfection. Cell extracts were prepared and immunopreci~itated with sera from hamsters bearing an SV40 induced tumor,

ET AL. HinF HinF AluI II 1

Mb01 TaqIMboI 1 1 I

HadlI EcoRn 8 I - 160 b.p.

5287 &=*e

.-l

r,,,~,,m

4289

__L

rr~?

L:: -1 1 0.52

1 0.54

*

1

0.56 MAP

hp.

1

b.p.

- 150 b.p.

* ’

0.58

’

1

0.60

UNITS

2. Position and extent of the tsldl mutants. The minimum segment in the deletions is indicated by the the maximum extent of the deletions tbe clear areas. FIG.

bP.

-180

-210

V’fmmm’f~‘ 1

-175

- 23 b.p.

-

A290 A 291 A.292

-i-

deletions in the of DNA missing hatched areas; is indicated by

or with sera from normal hamsters. Figure 3A shows the analyses of the immunoprecipitates on 10% acrylamide gels after autoradiography. The anti-t-antisera specifically precipitated two peptides of molecular weight 92K (T-antigen) and ZOK (t-antigen) from cells infected with WT or A209 when grown at 33”. In addition a 92K T-antigen was produced in all tsldl mutant treated cells, and was similar in size and amount to the T-antigen produced by either WT or A209. The 20K t-antigen was not detected in any of the extracts from cells infected with the tsldl mutants. Several different antisera and times of labeling from 1 to 4 hr at 24 to 72 hr postinfection gave similar results, The absence of a precipitable small tantigen could result from the production of reduced amounts or a reduction in the size of the 20K t-antigen. Because we were unable to detect any appreciable amount oftantigen or t-antigen peptides in the tsldl mutants at the permissive tem~rat~e, cells were infected at the nonpermissive temperature and the extracts were immunoprecipitated. Tegtmeyer et al. (1975) and Alwine et al, (1975) have shown that at nonpermissive temperatures, a ts mutation in the A gene region causes an ove~~uetion of a heat-labile T-antigen. The phenomenon of overproduction might amplify the amounts of the 20K t-antigen or t-antigen fragments and increase our ability to detect them. Although the t-antigen was overproduced upon infection with a tsA mutant at 40”, t-antigen fragments remained undetected

SV40 tiA/DELETION

AND THE INDUCTION

OF HOST DNA SYNTHESIS

255

FIG. 3. Autoradiogram of SDS-acrylamide electrophoretic gel showing 35S-labeled immunoprecipitates from cells lytically infected with A909 and various deletion mutants. Immunoprecipitates made with normal hamster serum and anti-SV40 T-antigen serum are in pairs. (A) Leff panel, lanes a and b, WT; lanes c and d, A209; lanes e and f, A209h287, lanes g and h A209A.288; lanes i and j, A209 AZ89; lanes k and 1, A209 A990; lanes m and n, A209 A991; and lanes o and p, A909 h292. (B) Right panel, lanes a through d represent CV-1 cells infected withA at either 33” (lanes a and b) or IO”(lanes c and d). L#anes e-h are derived from cells infected withA209&87 at either 33” (e and f) or 40” (g and h). Lanes i-l are derived fromA209A290 infected cells at either 33” (i and j) or 40” (k and 1). Lanes m-p are derived from A209h292 infected cells at either 33” (m and n) or 40” (o and p). The arrows indicate the positions of the 92K and 20K antigens. The gels have been deliberately overexposed to detect any 20K t-antigen related peptides.

in the tsldl mutants (Fig. 3B). Proteins from cells infected with A209 show significantly more of both the 92K and 20K proteins produced at 40” (lane d) than at 33” (lane b). Similar treatment of CV-1 cells with A209A287, A209M90, and A209A292 showed that whereas the 92K T-antigen was overproduced at 40” vs 33”, the 20K t-antigen could not be detected at either temperature. (The overproduction of the 92K T-antigen cannot be judged from Fig. 3 which was TABLE VIRUS

Virus

WT A884

A209 A209A287 A209A288

A209 A289 A209A290 A209h291

A209A.292

TITERS 40

SV40 MUTANTS 33”

8 x 106 8 x
Viral DNA Synthesis with ts Mutants

1

OF VARIOUS

106

loz 102

102

deliberately overexposed in the attempt to detect any 20K protein peptides.) This result suggests that the 20K t-antigen is not made in these mutants. Other investigators have been able to detect t-antigens of reduced size made by other 0.54-0.59 viable deletion mutants (Crawford et al., 19’78; Paucha and Smith, 1978; Sleigh et al., 1978), however, not all mutants with deletions in this region produce detectable t-antigens (Feunteun et al., 19’78; Sleigh et al., 1978).

3 x 107 2.5 x 10’ 10’ 9 x 106 4 x 106 5 x 106 106 5 x 106 5 x 106

40”/33” 0.3 0.3 <10-s t1.1 x <5 x <4 x <10-d <2 x
10-S 10-S 10-S 10-S

The tsldl mutants retained the temperature-sensitive mutation as determined by plaque assay (Table 1). All mutants tested exhibited a difference in titer of at least lo4 when titers were compared at 33 and 40”. The decrease in titer observed was of the same magnitude as that for the parental strain, tsA209. The ability of the ts/dZ mutants to induce total DNA synthesis (host plus viral) in monkey cells at 33 and 40” is shown in Fig. 4. The values plotted in Fig. 4 represent the difference between virus-treated and mock-

256

SETLOW

treated cultures. CV-1 cells infected with WT or various SV40 mutants at 33” showed little difference in the rate or extent of total DNA synthesis. There was a slight lag in the peak of synthesis by the tsA mutants compared to WT or the viable deletion mutant A88.4. At 40” the rate of DNA synthesis was greatly reduced for both A209 and A209A287; the little DNA synthesis observed was primarily due to the induction of host DNA synthesis. This experiment has been repeated several times and similar results were obtained with each of the six double mutants. At the permissive temperature viral DNA synthesis accounted for >70% of the total counts incorporated in several control experiments in which the complete extracts were centrifuged to equilibrium in CsCl-EtBr gradients (data not shown). In similar control experiments at the nonpermissive temperature, viral DNA synthesis accounted for ~10% of the total counts incorporated. Induction of Host DNA Synthesis

Since >90% of the DNA synthesized following infection by the tsA or tsA/dl mutants at 40” was due to the induction of host DNA, it was possible to follow this induction without fractionation. This experiment has been performed many times with AZO9~8~ and at least once with each of the other five double mutants with identical results. The background incorporation from the cells in DVO medium (mock infected with the uninfected cell lysate) that was subtracted from the data presented was low, amounting, for example, to less than 1.5% of that for the cells in DVlO medium at 24 hr. Both the tsA and the tsA/dE mutants stimulated host DNA synthesis at the restrictive temperature (Fig. 5A). The maximum stimulation by AgO9 (at m.o.i, -5) was between 30 and 50% that observed when the cells were incubated in medium with 10% serum. The maximum stimulation in DVO medium by the double mutants was between 15 and 25% of cells in DVlO medium, or approximately half that of AZ09 alone. Rundell and Cox (1979) have reported that xanthine derivatives, at concentrations that only pa~i~ly inhibited the induction

ET AL.

0

20

40 n&E

60

80

100

omurr)

FIG. 4. Induction of total DNA syntheses in (X-1 cells infected at either 33 or 40”. As indicated under Materials and Methods CV-1 cells were preIabeled with ‘*C and seeded at confluence onto 2-cm2 weIls. The specific activity of the cells were approximately 0.1 cpm/cell so that from each well l-2 x l@ cpm of “C was recovered. After incubation for 3 days in DV2 medium at 40” or DVl medium at 33” the medium was removed and saved. The cells were infected with virus and following 2 hr for adsorption the old medium was replaced. At the times indicated the medium was replaced with DVZ prewarmed medium wantoning 5 $X/ ml [3~]thymid~ne and incubated at the appropriate temperature for 1 hr. Samples were then Iysed and precipitated with TCA. The mock-infected cultures incorporated CO.1 cpm 13H]thymidine per cpm ]l*C]thymidine at each time point. (A) WT; (A) d1384; (0) A209;

(0) A209Az287.

of host DNA synthesis by wild-type virus, completely inhibited the induction by deletion mutants that synthesize defective 20K t-proteins. When 200 fig/ml caffeine was added to the medium following infection, no effect on the stimulation of host DNA was observed in the presence of 10% serum. In A209 infected cultures incubated at 40” in DVO medium with caffeine, the maximum induction was reduced by -50% as compared with caffeine-free cultures (compare Figs. 5A and B). However, the induction of DNA synthesis by A209A%W was virtually eliminated by 200 pg/ml caffeine.

SV40 ISA/DELETION

AND THE INDUCTION

2 0

0

20

40

60 TIME

600

20

40

60

60

(hours)

FIG. 5. Induction of host DNA synthesis. CV-1 cells were either mock infected, or infected with A209 (A) orA209A287 (V). One set of mock-infected cultures was then stimulated with medium containing 10% serum, (0). The remaining mock-infected cultures and the infected cultures were changed to DVO. The cultures were incubated at 40”, pulse-labeled, and analyzed at the indicated times. Open symbols, without caffeine; filled symbols incubation in DVO containing 200 pgiml (caffeine.

A low level of incorporation was only observed starting -50 hr postinfection. However, considerable cell necrosis under these conditions of incubation was obvious by 60 hr at 40” even in the mock-infected cultures. Similar results were also observed with the other five double mutants (data not shown). Under our conditions (which differ somewhat from those of Rundell and Cox (19’79)) the only difference between the induction by h884 and wild-type virions in the presence of caffeine was a 24-hr delay in the maximum induction by A884 (data not presentsed). On the Nature of the Host Induced DNA

The preceding results clearly confirm that host DNA synthesis is induced by tsA mutants at 40”. However, to our knowledge no one has analyzed the nature of the DNA synthesized at 40”. One possible explanation for these results was that the nucleotide incorporation at 33” resulted from semiconservative replication, whereas that at 40” represented repair synthesis. We have therefore examined host DNA synthesis in the presence of Budr. Cells prelabeled with

OF HOST DNA SYNTHESIS

257

[14C]thymidine were infected with A209 or with ~i88.4 or wild-type virions at 33 and 40”. The cells were then labeled with rH]Budr after 72 hr at 33” or 48 hr at 40” and the cellular DNA was analyzed. If the host synthesis were repair synthesis, then the [3H]13udr-labeled material would appear in the low-density region of the gradient i.e., the peak of 3H label would be superimposed on the 14C peak. If, however, semiconservative replication occurred at the nonpermissive temperature, a mixed population of DNA fragments would result after sonication and two distinct peaks of labeled material would result after equilibrium centrifugation. All cultures infected at 33” showed two distinct radiolabeled peaks as expected for semiconservative DNA replication. At 40”, the same separation was evident, again indicating semiconservative replication (Fig. 6). DISCUSSION

We have isolated a number of SV40 double mutants that contain both a deletion in the region specific to the 20K t-antigen and a temperature-sensitive mutation in the 92K T-antigen. The mutants produce no detectable 20K t-antigen and are temperature sensitive for growth and viral DNA replication. These mutants have proven useful for the clarification of a number of questions concerning the physiology of lytic and transforming infection. The latter results will be reported elsewhere. It has been demonstrated by Alwine et al. (19’75) and Tegtmeyer et al. (1975) that the 92K T-antigen is a self-regulatory protein that is overproduced by tsA mutants at the nonpermissive temperature. Our results with tsA209 (Fig. 3), in agreement with the more extensive work of Alwine and Khoury (manuscript in preparation), demonstrate that the rate of synthesis of the 20K tantigen is also increased at 40” on infection with tsA mutants. The additional information derived here from the double mutants is that the 20K t-antigen is not required for the autoregulatory process; that is, the rate of synthesis of the 92K T-antigen also increases at 40” in the absence of the 20K t-antigen.

SETLOW

258

0

0

25

500

25

FRACTION

ET AL.

500

25

50 0

23

SO

NUMBER

FIG. 6. Equilibrium density centrifugation of host DNA from infected cultures containing Budr. Infected cultures were prelabeled with [%]thymidine (0) and pulse-labeled with [3H]Budr (0) before analysis.

The most remarkable observation with the double mutants was that host DNA stimulation still occurred at 40”. At least two interpretations of this result are possible: (i) neither early protein is required for the induction of host DNA synthesis; and (ii) the temperature-sensitive 92K T-antigen is temperature sensitive for viral DNA replication but not for host DNA replication. There are a plethora of nondefinitive reasons for excluding the first possibility (see Introduction). One plausible explanation consistent with the second interpretation is to suppose that the tsA mutations, all of which are clustered in one section of the A gene (Lai and Nathans, 19’75), make a 92K protein that is primarily deficient in site-specific binding to SV40 DNA at the restrictive temperature. The 92K T-antigen binds site specifically (Jesse1 et ccl., 1975; Tijan, 1978) near the viral origin of replication with a dissociation constant of -lo-l2 and nonspecifically to any DNA (including host DNA) with a dissociation constant of -lo-lo

(Jesse1 et al., 1975). If one assumes that binding is prerequisite to the physiologically important enzymatic activity of the 92K protein (protein kinase? (Griffin et al., 1979; Tijan and Roggjns, 1979)) then one may readily understand why the tsA mutant (if primarily deficient in site-specific binding) is temperature sensitive for viral replication but not for host replication. This is not to deny that the 92K protein is unstable at 40” (Jesse1 et al., 1975; Tenen et al., 1975) but only to imply that compensatory overproduction may result in the lesion being manifest more as a defect in the K ,,, than in the V,,, or active concentration of the 92K protein. Some explanation is also required as to why caffeine inhibits DNA induction in the tsldl mutants more effectively than in tsA mutants alone. One possible explanation is based on the work of Stiles et al. (in press), who have demonstrated that cells have at least two arrest points between mitosis and DNA synthesis. The second

SV40 tsA/DELETION

AND

THE

INDU

arrest point, the competence point, can be reached by treating G, cells with certain competence-,promoting serum growth factors. Our results are explicable if one assumes that the 92K T-antigen has low competence-promoting activity and that this activity is inhibitable by caffeine. The reason why the tsA mutants by themselves are not inhibited by caffeine is that the 20K t-antigen is also a &ompetence-promoting agent and a more potent one than the 92K T-antigen (Martin et al., 1979). Finally, our results demonstrate that the induction of nucleotide incorporation into host DNA by tsA mutants at the restrictive temperature is due to semiconservative DNA replication, not repair synthesis. REFERENCES

ALWINE, J. C., REED, S. I., FERGUSON, J., and STARK, G. R. (1975). Properties of T-antigens indueed by wild type and ts-A mutants in &tic infection. Cell 6, 529-533. BASILICO, C., and DIMAYORCA, G. (1965). Radiation target size of the lytic and tr~sfo~ing ability of polyoma virus. Proc. Nat. Acad. Sci. USA 54, 125- 127. CARBON, J., SI-IENR, T. E., and BERG, P. (1975). Biochemical procedure for production of small deletions in simian virus 40 DNA. Proc. Nat. Acad. Sei. USA 71, 1392-1396. CHOU, J., AVILA, J., and NVIARTIN, R. G. (1974). Viral DNA synthesis in cells infected by temperature-sensitive mutants of simian virus 40. J. Viral. 14, 116-124. CHOU, J., and MARTIN, R. G. (1974). Complementation analysis of simian virus 40 mutants. J. Viral. 13, 1101-1109. CHOU, J., and MARTIN, R. G. (1975). DNA infectivity and the induction of host DNA synthesis with temperature sensitive mutants. J. Viral. 15, 145150. COLE, C. N., LANDERS, T., GOFF, S. P., MANTENIL BRUTLAG, S..,and BERG, P. (1977). Physical and genetic characterization of deletion mutants of simian virus 40 constructed in &ro. J. Viral. 24,227-294. CRAWFORD, L. V., COLE, C. N., SMITH, A., PAUCHA, E., TEGTMEYER, P., RUNDELL, K., and BERG, P. (1978). Organi~tion and expression of early genes of simian virus 40. Proc. Nut. Acad. Sci. USA 75, 117-121. CRAWFORD, L,. V., and O’FARRELL, P. Z. (1979). Effect of alkylation on the physical properties of simian virus 40 T-antigen species. J. Viral. 29, 587-596.

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RUNDELL, K., and COX, J. (1979). Simian virus 40 t antigen affects the sensitivity of cellular DNA synthesis to theophylline. J. Viral. 30, 394-396. SEIF, R., and MARTIN, R. G. (1979). SV40 small t-antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells. J. Vid. 32, 979-988. SHENK, T. E., CARBON, J., and BERG, P. (1976). Construction and analysis of viable deletion mutants of SV40. J. Vid. 18, 644-671. SLEIGH, M., TOPP, W. C., HANICH, R., and SAMBROOK, J. E. (1978). Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell 14, 79-88. STILES, C. D., PLEDGER, W. J., VANWYK, J. J., ANTONIADES, H., and SCHER, C. D. Hormonal control of early events in the Balbk-3T3 cell cycle: Commitment to DNA synthesis. In “Cold Spring Harbor Conference on Cell Proliferation: Hormones and Cell Culture. A Tribute to Gordon Tomkins” (G. Sato and R. Ross, eds.), pp. 425-439. Cold Spring Harbor Laboratory, New York, 1979.

ET AL. TEGTMEYER, P. (1972). Simian virus 40 deoxyribonucleic acid synthesis. The viral replicon. J. Viral. 10, 591-598. TEGTMEYER, P., SCHWARTZ, M., COLLINS, J. K., and RUNDELL, K. (1975). Regulation of tumor antigen synthesis by simian virus 40 A gene. J. Vi&. 16, 168-178. TENEN, D. G., BAYGELL, P., and LIVINGSTON, D. M. (1975). Thermolabile T (tumor) antigen from cells transformed by a temperature-sensitive mutant of simian virus 40. Proc. Nat. Acad. Sci. USA 72, 4351-4355. TIJAN, R. (1978). The binding site on SV40 DNA for a T-antigen related protein. Cell 13, 165-179. TIJAN, R., FEY, G., and GRAESSMANN, A. (1978). Biological activity of purified simian virus 40 T antigen proteins. Proc. Nat. Acad. Sci. USA 75,1279-1288. TIJAN, R., and ROGGINS, A. (1979). Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc. Nat. Acad. Sci. USA 76, 610-614.