Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

J. Mol. Biol. (1974) 87, 275-288

Triplication of a Unique Genetic Segment in a Simian Virus 404ike Virus of Human Origin and Evolution of New Viral Genomes GEORGE C. FAREED?, JANET C. BYRNE AND MALCOLM A. WTIN

Laboratory of Biology of Viruses National, Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Md 20014, U.S.A. (Received 30 November 1973, and in revised form 18 March 1974) The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multiplicities in primary monkey kidney cells. The 32P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R, restriction endonuclease from EschericEa co.5 (Eco RI$ restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the EGORI restriction nuolease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive”$ (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive ” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.

1e Introduction A simian

virus 40-&e

virus,

originally

isolated

from the brain

of a patient

(DAR-

isolate) with progressive multifocal leukoencephalopathy (Weiner et al., 1972) was shown by Sack et al. (1973) to be a genetic variant of SV4Ojl. Martin et ai. (1974) identified two classes of particles produced following high multiplicity passage in primary African green monkey kidney cells. The light particles (DAR-light virions) were non-infectious, failed to induce SV40 viral capsid or tumor antigens and contained duplex superhehcal genomes which were about 80% the size of SV40 DNA (Martin et al., 1974). The DAR-heavy virions were infectious, cross-reacted immunologically with SV40 and contained duplex, circular genomes which were similar t Present address : Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02115, U.S.A. $ This designation for the restriction nuclease is from the proposed nomenclature of Smith & Nathans (1973). 8 This term is used only to distinguish the genomes containing multiple Em RI cleavage sites from those with only one site. j/ Abbreviation used: SV40, simian virus 40. 275

276

G. C. FAREED,

J. C. BYRNE

AND

M. A. MARTIN

in mass and polynucleotide sequence homology with those of SV40. DAR-heavy particles could be distinguished from SV40 by their ability to grow efficiently in human cells and the instability of their plaque morphology (Martin et al., 1974). Serial undiluted passage of SV40 results in the appearance of defective virions (Uchida et al., 1966; Yoshiike, 1968). Subsequent reports (Lavi & Winocour, 1972; Lavi et al., 1973) have focused on the incorporation of monkey DNA sequences into the circular SV40 genome as a consequence of high multiplicity passage. Brockman et al. (1973) and Rozenblatt et al. (1973) have demonstrated evolutionary changes in the DNA of serially passaged SV40 including duplication of certain DNA sequences and substitution of cellular DNA. Another report (Martin et al., 1973) describes the amplification of SV40 DNA sequences following high multiplicity passage. In order to evaluate evolutionary changes in DAR-viral DNA, we propagated DAR-heavy virus at high input multiplicities. In contrast to previous studies with SV40, serial passage of DAR-heavy particles results in alteration(s) and rearrangements of viral DNA without detectable incorporation of host cell sequences. This paper describes a highly specific and rapid evolution of new viral genomes in which a unique onethird of the original DAR-viral genome has been triplicated and two-thirds eliminated. This new population of viral genomes accumulates in vivo and becomes the predominant viral DNA species after a few passages. In the accompanying paper (Khoury et al., 1974) physical mapping studies on this variant genome are reported.

2. Materials and Methods (a) Cell culture and virus preparations Primary African green monkey kidney cells were cultured in 32-0~ prescription bottles in Eagle’s minimal essential medium (Eagle, 1959) supplemented with 50/6 fetal calf serum and 2 mna-L-glutamine. Confluent cellular monolayers were infected with purified, 3Hlabeled DAR-heavy virions as described below and in a previous paper (Martin et al., 1974). Progeny virions were purified from the cellular pellets by trypsin and sodium deoxycholate treatment, sedimentation onto a CsCl cushion, and isopycnic centrifugation which always appeared in CsCl (Yoshiike, 1968; Gelb et al., 1971). The progeny virions, as one discrete band, were dialyzed at 4°C against 100 vol. of sterile 0.01 M-sodium phosphate (PH 7.4), 0.15 M-NaCl with one change of dialysis buffer prior to estimation of viral concentration by optical density and DNA extraction. (b) Radio-labeling

of

progeny viral DNA

and DNA

extraction

procedures

32P-labeled progeny viral DNA was prepared from purified virus grown in primary African green monkey kidney cells labeled 24 h after infection with [32P]orthophosphate (50 rCi/ml; International Chemical and Nuclear Corp.). Cells and medium were harvested 5 to 7 days after infection and labeled virus was prepared as previously detailed (Gelb et d., 1971). “H-labeled virus was prepared from infected monkey kidney cells exposed to L3H]thymidine (20 to 25 mCi/pmol; New England Nuclear Corp.) 24 h post infection at a final concentration of 2 $.?+nl. Progeny virions were harvested and purified 5 to 7 days after infection as described above. The radioactively labeled viral DNA was extracted from purified virions by treatment with 1% sodium dodecyl sulfate at 50°C for 30 min in the presence of 1 m&r-EDTA followed by precipitation of the sodium dodecyl sulfate in CsCl (1 g/ml) and subsequent isopycnic centrifugation in C&l containing 200 pg/ml of ethidium bromide (Radloff et al., 1967). Dye extraction and desalting of superhelical viral DNA was accomplished by filtration through a 1 cm x 1 cm layer of Dowex-50 resin on a 1 om x 15 cm column of Sephadex G50 (Gelb et al., 1971). The 32P-labeled DAR-viral DNAs from serial passaging in monkey kidney cells had specific activities of 4 to 6 x IO5 cts/min per pg.

TRIPLICATION

OF A UNIQUE

DNA

SEGMENT

277

Intracellular viral DNA molecules were labeled at 23, 32, 44 and 72 h after infection with [3H]thymidine (50 &i/ml) for 40 min at 37°C. In other experiments intracellular viral DNA was labeled with [14C]thymidine (1 #i/ml, 60 @i/mmol; New England Nuclear Corp.) for an 8-h period at 37°C. Intracellular viral DNAs were selectively extracted by the 0.6% sodium dodecyl sulfate/l M-N~CY treatment devised by Hirt (1967) which separates low molecular weight viral DNA from high molecular weight cellular DNA. The labeled, superhelical viral DNA molecules (DNA I molecules) were further purified by dye-density gradient centrifugation as described above. After dye extraction and vacuum dialysis against 0.01 M-Tris.HCl (pH 7.5), O-01 M-N&~, and 0.001 M-EDTA at 4”C, the radioactively labeled DNA I preparations were incubated with the EGO RI restriction endonuclease as described below.

(c) Serial passage of DAR-heavy

virions

3H-labeled DAR-heavy virions, purified from DAR-light virions by 2 cycles of isopycnic centrifugation in CsCI, were used to infect primary monkey kidney cells at a calculated multiplicity of infection of lo4 physical particles per cell. In order to estimate the amount of virus inoculum to be added to cell cultures, the number of physical particles was calculated based on the AZeonrn of the virus solution. The AaBOnm for 1 mg of SV40 was assumed to be 3.85 (Koch et al., 1967). The weight of one viral particle was calculated (Westphal & using the weight of 5~ IO-l2 pg as the weight of one viral DNA molecule Dulbecco, 1966) and a value of 11% for the proportion of DNA per viral particle. After a 2-h adsorption period the inoculum (5 ml/32-oz bottle) was removed and fresh medium (25 ml/bottle) was added containing 2% fetal calf serum, antibiotics and 2 mM-L-ghtamine. Infection of cells at each successive viral passage was carried out in this manner using purified, 3H-labeled progeny virions.

(d) Cleavage

of DAR-heavy

viral DNAs by the Eco RI restriction endonuclease

The EGORI restriction nuclease was the same preparation described previously (Martin et al., 1973) having 2 x lo5 SV40 DNA units/mg protein, We have defined one unit of nuclease activity as the amount of enzyme needed to convert O-1 pg of superhelical SV40 DNA to linear DNA III in 20 min at 37°C in the EGO RI nuclease reaction mixture. Reaction mixtures (0.2 ml) contained 5 to 10 pg of 32P-labeled DAR-viral DNA, 49 ng of 3H-labeled SV40 DNA I and II (9.6 x lo4 cts/min per pg) from plaque-purified SV40 propagated at a low input multiplicity of infection, 0.1 M-Tris*HCl (pH 7.5), 0.005 MMgCl,, and either 2 units of Eco RI nuclease or no enzyme addition (control). Following a 30-min incubation period at 37”C, the reactions were terminated by chilling to 0°C and adding 10 ~1 of 1% Sarkosyl and 10 ~1 of 0.2 M-EDTA (pH 8-O).

(e) Characterization

of DAR-viral

DNA cleaved by the Eco RI wwlease

DNA samples were analyzed after treatment with the Eco RI restriction n&ease by velocity gradient sedimentation in 5% to 30% neutral or 10% to 30% alkaline sucrose density gradients using the Spin00 SW41 rotor (Fareed et ah, 1972). In order to further evaluate the DNA products of the EGO RI digestion, preparative neutral sucrose sedimentation was used and the s2P-labeled DNA products from a large-scale cleavage reaction (see the legend to Fig. 4) were identified by Cerenkov counting of gradient fractions. The pooled fractions containing the digestion products were dialyzed against 0.01 M-Tris~HCl (pH 7*5), 0.01 M-NaCl, 0.001 M-EDTA (pH 8.0) at 4°C and concentrated 5-fold by vacuum dialysis prior to further analysis. Gel electrophoresis of Eco RI digestion products in 0.5% agarose-2% polyacrylamide cylindrical gels was performed according to the procedure of Pettersson et al. (1973). Adenovirus type 2 DNA labeled with [“Hlthymidine had a specific activity of 1.05 x 105 ots/min per pg and was the gift of Drs B. Carter and J. Rose. Reassociation kinetic analysis of denatured, 32P-labeled viral DNAs was performed as described by Gelb et aZ. (1971).

278

G. C. FAREED,

J. C. BYRNE

AND

M. A. MARTIN 500

400

300.

700

I 00

-( 1 300’+

(b)

iO0

i

j

j

y+’

H-4 -1 100

?500 s d 111 t ii: -2 100 N P

2 1800 F 2 u ,I 1200

600

I00

-C ) I 600

I 280

-9 160

800

-6 i40

-2 i20

-C lb

ZO io Frocficnno.

4’0

FIG. 1. Sedimentation analysis of Eco RI cleavage products. The 32P-labeled, DAR-viral DNAs purified from progeny virions after the third (a), fourth (b) and fifth (c) serial passages in primary monkey kidney cells were cleaved by the Eco RI restriction endonuolertse as described in Materials and Methods. Each reaction mixture contained aH-labeled SV40 DNA I and II before cleavage and, after termination of the reactions, 14C-labeled SV40 DNA I and II (0.3 pg, 3.3 x lo3 cts/min per pg; Fareed et al., 1972) were added as sedimentation references. The DNA samples were layered onto 5% to 30% neutral sucrose gradients and centrifugation was carried out in an SW41 rotor for 7 to 8 h at 40,000 revs/min and 1O’C. At the end of the centrifugation period, the gradients were fractionated by collecting 0.3-ml samples dropwise from the bottom of each tube. The

TRIPLICATION

OF A UNIQUE

DNA

SEGMENT

279

3. Results (a) Cleavage of serially-passaged

DAR-viral DiVAs endonuclease

by the Eco RI restriction

The Eco RI restriction nuclease has been shown to introduce one unique doublestrand scission in SV40 DNA molecules (Morrow & Berg, 19’72; Mulder & Delius, 1972). We have previously observed that DAR-heavy viral genomes also contain one EGO RI cleavage site whereas the DAR-light viral genomes were resistant to this nuclease (Fareed et al., 1973). Serial passaging of SV40 at high multiplicities of infection results in alterations of viral DNA with introduction of cellular DNA sequences (Lavi & Winocour, 1972; Tai et al., 1972; Lavi et al., 1973; Rozenblat’t et al., 1973; Brockman et al., 1973). Concomitantly, a large proportion of the viral genomes become resistant to the Eco RI restriction nuclease (Mulder & Delius, 1972 ; Martin et al., 1973). The sensitivity of progeny DAR-heavy viral DNA molecules after serial passages in primary monkey kidney cells to the Eco RI restriction nuclease was analyzed by neutral sucrose gradient sedimentation after incubation in the presence or absence of the enzyme. Figure 1 illustrates three such analyses of the products of the nuclease digestion. DNAs, isolated from purified virions, were examined from the progeny of the third, fourth and fifth serial passages (Fig. l(a), (b) and (c)). In the control reaction mixtures (results not shown) all of the DAR-viral DNAs cosedimented with a 14C-labeled SV40 DNA I (21 9) marker. After cleavage, about 60% of the DNA from the third passage was found to be resistant to the Eco IR nuclease and about 40% was cleaved once to yield full-length linear molecules which sediment at 14.5 S (Fig. l(a)). A dramatic change in the population of viral genomes was observed after the fourth serial passage (Fig. l(b)). Three populations of viral DNA were observed with differing sensitivities to the Eco RI nuclease: a resistant fraction, a sensitive fraction with one cleavage site and a new “supersensitive” fraction giving rise to Eco RI fragments which sediment at 10.4 S. After the fifth passage, the “supersensitive ” DNA fraction becomes the predominant species (Fig. 1(c)). The experiment shown in Figure 2 compares the RDA-heavy viral DNA isolated from purified virions from the sixth serial passage, both before and after cleavage by the Eco RI nuclease, to the radioactively labeled viral DNA extracted from cells. In both cases the untreated DNA I samples (Fig. 2(a) and (c)) were physically indistinguishable from SV40 DNA I whereas, after cleavage by the Eco RI nuclease, significant differences were evident. The DNA prepared from purified virus following the sixth serial passage (Fig. 2(b)) contained 71 o/0Eco RI supersensitive genomes and 14% Eco RI resistant molecules. The intracellular viral DNA (Fig. 2(d)) from that infection consisted of 83 o/0EGORI super sensitive and 12 o/0Eco RI resistant molecules. A major difference between the viral encapsidated and intracellular DNAs thus appears to be a relative increase in the proportion of unencapsidated Eco RI supersensitive molecules. This difference was also observed during the fifth serial passage in which 64% of the intracellular viral DNA consisted of Eco :RI supersensitive molecules in contrast to 44% Eco RI supersensitive DNA molecules isolated from purified virions. The proportion of unencapsidated Eco RI sensitive DNA molecules arrows designate the positions of the W-labeled SV40 DNA as well as the position of linear DNA III. The recovery of labeled DNA in these and all other gradients shown in this paper was greater than 90%. -e--a--, 32P; --O---O--, 3H.

280

G. C. FAREED,

0

10

20

30

40

J. C. BYRNE

AND

0

50 Fraction

IO

M. A. MARTIN

20

30

40

59

lie.

Pm. 2. Sedimentation analysis of encapsidated and intracellular viral DNA I from the sixth serial passage before and after cleavage by the Eco RI restriction m&ease. DNA prepared from purified virus (sap-labeled; (a) and (b)) and intracellular viral DNA (“H and 14C-labeled; (c) and (d)) were obtained from the sixth serial passage of DAR-heavy virus. The samples were analyzed by neutral sucrose rate zonal sedimentation before ((a) and (0)) and after ((b) and (d)) cleavage by the Eco RI restriction nuclease as described in the legend to Fig. 1. The reaction mixtures analyzed in (a) and (b) contained 3H-labeled SV40 DNA II (9.6~ lo4 cts/min per pg) before incubation with the nuclease. The viral DNA analyzed in (c) and (d) was pulse-labeled with [“Hlthymidine (see Materials and Methods) for 5 min after the 8-h period of labeling with [‘%Ithymidine. -+-a---, 3aP; --A--A--, i4C; --O--O--, 3H.

(one cleavage site) was reduced compared to the proportion present in viral particles. The discrepancy between the proportion of Eco RI supersensitive molecules in the intracellular pool of molecules and that found in purified virions may be due to inefficient encapsidation of the supersensitive genomes. After the sixth serial passage of DAR-heavy virions in monkey cells, more than 70% of the genomes are cleaved to 10.4 S DNA fragments by the EGO RI nuelease. Furthermore, we have found the EGO RI cleavage profiles of progeny viral DNA from the seventh and eighth serial passages to be nearly identical to those from the sixth passage.

(b) Early appearance of the Eco RI supersensitive genomes in vivo At different times after infection of monkey cells by the third passage DAR-virions, the intracellular viral DNA was labeled with [3H]thymidine for a 40-minute period and then selectively extracted (Hirt, 1967). The 3H-labeled viral DNA molecules were primarily (>75%) DNA I molecules before Eco RI cleavage whereas, after

TRIPLICATION

OF A UNIQUE

DNA

281

SEGMENT

240 160

0

/ 0

,

IO

,

,

20

4

(

30

I

,

40

/

,

50

I

0

60

IO

20

30

40

--I 0 50

Fraction no.

FIG. 3. Analysis of intracellular DAR-viral DNA I at different times during the fourth serial passage. The replicating DAR-viral DNA isolated from cells infected with progeny virions from the third serial passage was labeled with [3H]thymidine for 40 min (see Materials and Methods). The viral DNA was selectively extracted (Hirt, 1967) and analyzed by neutral sucrose gradient centrifugation after cleavage by the Eco RI restriction nuclease. SV40 DNA I and II (0.3 pg) labeled with [r*C]thymidine were added to the reaction mixture analyzed in (a) after terminating the reaction, whereas the same amount of WXabeled DNA I and II were added to the reaction mixture with the 73-h DNA sample prior to theadditionof enzyme(d).--A--A--,8H;-.w-.m-., I‘%.

cleavage, resistant, sensitive, and supersensitive forms of viral DNA could be identified as early as 23 hours after infection (Pig. 3(a)). L evine et al. (1970) have shown that labeling of viral specific DNA in primary monkey kidney cells with [3H]thymidine begins about 15 hours after infection. At later times (Fig. 3(b) and (c)), the proportion of 21 S DNA molecules cleaved to 10.4 S fragments increases and by 72 hours (Pig. 3(d)), the sedimentation profile of cleaved intracellular molecules resembles that found with DNA obtained from purified virions (Fig. l(b)). (c) Electron microscopic examination of replicating DAR-viral

DNA molecules

In order to evaluate the structure of replicating molecules containing reiterated Eco RI cleavage sites, the replicative intermediates from the sixth serial passage were 1-a

282

G. C. FAREED,

.I. C. BYRNE

AND

M. 9.

MARTIN

160

(b) 800

16s t

(cl

10.6s +

800 600 -

16s

I

300

t

t 4

200

100

0

5(1 Fraction

no.

FIG. 4. Alkaline sucrose sedimentation analysis of Eco RI cleavage products of DAR-viral DNA from the fourth’serial passage. A reaction mixture similar to that shown in Fig. l(b) containing Eco RI cleavage products of 32P-lebeled DAR-viral DNA’from the fourth serial passage in monkey kidney cells was analyzed by alkaline sucrose sedimentation (a). Sedimentation through the 10% to 30% alkaline sucrose gradient was for 18 h at 10°C in the SW41 rotor. In order to purify the DNA w&s incubated DNA III and 10.4 S reaction products, 100 ng of the sZP-labeled DAR-viral in the nuclease reaction mixture (see Mrtterials and Methods) with 10 units of Eco RI mstriotion nuolease. The 14-5 S (DNA III) and 10.4 S reaction products were isolated from a preparative neutral sucrose gmdient and, after dialysis snd concentration against 0.01 M-Tris.HCl (pH 7.5), 0.01 M-N&I and 0.002 M-EDTA at 4”C, the purified 14.5 S (b) and 10.4 S (c) products were

TRIPLICATION

OF A UNIQUE

DNA

SEGMENT

283

prepared for electron microscopy as described by Sebring et al. (1971). The twisted replicating molecules were opened with the addition of 1 pg ethidium bromide/ml in the spreading solution. Of 300 molecules at all stages of replication examined, more than 95% had the same configuration (one replication loop per molecule) as replicating molecules of SV40 DNA (Sebring et al., 1971). Only one molecule was observed with two replication loops and none was observed to have three of these regions. 12,600

Fraction no.

FIG. 6. Analysis of the 10.4 S DNA segments by polyaorylamide-agarose gel electrophoresis. Adenovirus type 2 DNA (1.1 pg), labeled with [“H]thymidine, was cleaved by the Eco RI restriction endonuolease (15 units) and a portion of this reaction mixture was combined with a sample of the purified 32P-labeled 10.4 S DNA (5500 cts/min). The two DNAs were subjected to gel electrophoresis in a 12-cm cylindrical, 2% polyacrylemide-0*6% isgarose gel (Pettersson et al., 1973). After 12.5 h of electrophoresis at 3 V/cm and 2O”C, the gel w&s frozen and fractionated into l-mm slices which were treated with 50 ~1 of 30% HzOz (6O”C, 10 h) and then counted in s liquid scintillation spectrometer. The disproportionate amounts of radioactivity in fragments C and E es compared to fragments B end D of adenoviral DNA is due to differences in the A-T content among these fragments (B. Carter, unpublished data). ---a--, 3zP; --O--O--, 3H.

(d) Characterization of the IO.4 X DNA segments When the cleavage products of DAR-viral DNA obtained from purified virions following four serial passages were subjected to alkaline sucrose sedimentation analysis, two narrow bands sedimenting at 16 S (full length, viral DNA strands) and 10.6 S were obtained (Fig. 4(a)). These two DNA species represent Ew RI sensitive and supersensitive molecules, respectively; under the centrifugation conditions employed, Eco RI resistant DNA molecules would pellet (53 S) and would not be present in the gradient. The absence of multiple, single-strand breaks in either the DNA III (14.5 S) or 10.4 S products is confirmed by the separate alkaline sucrose sedimentation analyses of these purified species (Fig. 4(b) and (c)). snalyzed by alkaline sucrose sedimentation. In the analysis shown in (a), ‘YLlabeled DNA II was added to indicate the positions of the 18 S (circular) and 16 S (linear) strands whereas in (b) end (c), 3H-labeled SV40 DNA II (2800 cts/min) was added es s sedimentation reference. --e--@,

32p; --A--*--,

3H.

284

G. C. FAREED,

J. C. BYRKE

AND

M. A. MARTIN

Double-stranded, linear DNA molecules which sediment at 10.4 S in neutral sucrose gradients are about 1.1 x lo6 daltons in mass (Studier, 1965) or one-third the mass of full length linear SV40 DNA molecules (3.2 x lo6 daltons). This size estimation for the 10.4 S segments has been confirmed both by electron microscopic eontour length measurements and by polyacrylamide-agarose gel electrophoresis. The electron microscopic measurements (Garon, Fareed & Martin, unpublished results) of 10.4 S DNA segments revealed their mean lengths to be 0.529 pm (kO.064 pm) as compared to linear SV40 DNA which was 1545 pm (50.064 pm). In the electrophoretic analysis illustrated in Figure 5, the purified 10.4 S DNA segments were compared to the Eco RI nuclease fragments of adenovirus type 2 DNA which were labeled with [13H]thymidine. Petterson et al. (1973) have reported the specific fragmentation of adenoviral type 2 DNA into six segments which range in size from 1.1 x lo6 to 13.6 x lo6 molecular weight. The experiment illustrated in Figure 5 shows 32P-labeled 10.4 S DNA has the same electrophoretic mobility as EGO RI fragment F of adenovirus type 2 DNA. The result of this experiment is in agreement with the previously presented sedimentation data and indicates that the 10.4 S DNA segment has a molecular weight equivalent to that of fragment F (1.1 x 106). Since the 10.4 S segments are one-third of the length of the original genomes and are generated by exposure of normal sized (in comparison to SV40 DNA) closed circular DNA to the Eco RI nuclease, three Eco RI sites must be present in the original 21 S DNA molecule. Furthermore, in order for the cleavage products to be one-third the length of SV40 DNA, the three Eco RI sites must be symmetrically located on the genome. (e) Reassociation kinetic analysis of the 10.4 S DNA segments In order to determine whether the 10.4 S DNA segments represent a specific one-third of the viral genome or contain all of the DNA sequences present in viral DNA which have been cleaved to 1.1 x lo6 dalton fragments by virtue of a triplication of the Eco RI site, we examined the reassociation kinetics of 10.4 S DNA. Britten t Kohne (1968) have previously shown that the rate of reassociation of denatured DNA is inversely related to the physical complexity of the DNA under study. Thus, if the 10.4 S DNA segments contain a speci$c one-third of the viral genome, it should reassociate three times more rapidly than the parental DAR-viral DNA. On the other hand, if the DNA sequences present in the 10.4 S cleavage product are representative of the entire viral genome, the rate of reassociation should be similar to that observed for DAR-viral DNA. In the experiment shown in Figure 6, the kinetics of denatured 10.4 S DNA reassociation have been compared to those of denatured, sheared, DNA III, also produced by Eco RI cleavage of DAR-heavy viral DNA. The 10.4 S DNA segments reassociated 2.8 times more rapidly than the DNA III (Fig. 6) indicating that their physical complexity was about one-third that of parental DAR-viral DNA. When 32P-labeled 10.4 S DNA was allowed to reassociate in the presence of a 5.9 x 106-fold excess of fragmented monkey DNA, no acceleration of reassociation was observed (Fig. 6). Under these conditions, the presence of 0.6 copy of DNA with a physical complexity of 1 x lo6 daltons would have accelerated the reassociation twofold. These experiments allow us to conclude that following serial passage of DAR-heavy virus, an ever increasing fraction (greater than 70%) of DNA I molecules evolves consisting of a specific triplication of one-third of the viral genome in the form of repeating 10.4 S segments.

TRIPLICATION

OF A UNIQUE

DNA

SEGMENT

285

FIG. 6. The reassociation kinetics of 32P-labeled 10.4 S DNA segments. The 32P-labeled 10.4 S and 14.5 S products of Ew RI digestion of DAR-viral DNA I from the fourth serial passage were purified as described in the legend to Fig. 4. The pursed 32P-labeled 10.4 S DNA was heat denatured and allowed to reassociate in the presence of unlabeled fragmented salmon sperm ( 0) or monkey liver (0) DNAs (6.8 mg/ml) in 0.6 nr-sodium phosphate, pH 6.8, at a concentration of sheared (Gelb et 9.9 x 10V4 pg/ml. A sample of the 32P-labeled DNA III (0) was mechanically al., 1971), heat denatured, and allowed to reassociate at a similar concentration in the presence of unlabeled fragmented salmon sperm DNA (6.8 mg/ml). Both reactions were incubated at 68°C; samples were removed at the indicated times and the percentage of DNA reassociated was determined by hydroxyapatite chromatography.

(f) Eflects of subsequent serial passages on DAR-viral

genomes

Progeny DAR-heavy virions have been serially passaged eleven times thus far. Following the eighth serial passage, a new species of viral DNA appeared that was accompanied by a marked reduction in the proportion of both Eco RI sensitive and Eco RI supersensitive DNA molecules. The newly evolved viral DNA was supercoiled, somewhat shorter in molecular length and noticeably heterogeneous in size in comparison to SV40 DNA (Fig. 7(a)) prior to cleavage with Eco RI nuclease. Following enzymatic cleavage, a more homogeneous band of Ew RI resistant, shortened DNA molecules was obtained (Fig. 7(b)). In addition, the 10.4 S DNA. fragments, while reduced in amount, exhibited considerable size heterogeneity as judged by sedimentation in neutral sucrose (Fig. 7(b)) and also confirmed by alkaline sucrose sedimentation (data not shown). After the subsequent passages (ten and eleven), the major viral DNA species appears to be shortened (20 S), Eco RI resistant, superhelical DNA molecules ; DNA molecules containing three Ew RI sites that were the predominant species in passages five through eight, were now barely detectable (Fig. 7(d) and (f)).

4. Discussion Serial passaging of DAR-heavy virions at high input multiplicities in monkey kidney cells has resulted in the rapid evolution of a new species of viral DNA. Once they appear, the new viral genomes either have a selective advantage over other forms of viral DNA and rapidly accumulate or (less likely) predominate as a consequence of a process of continuous creation. The specific genetic reassortment that

286

G. C. FAREED,

J. C. BYRNE 500

H9 Control

i

400

400

AND

600 5J 480

300

M. A. MARTIN -

-600

400

-480

600

-

450

-300

2

_

F.

-360

-200, 3 - 240

300

200

500

‘f

100

-100

150

10

0

0 0 1000

IO I

,

20 I

I

30 I

I

1

I

I

I

IO

20

30

40

-0

50

500

1

H IO B Control

I

(cl

0

50

40

-120

500

150

f %

400

120

300

90

200

60

100

30

0

0 0

IO

20

30

40

50

0

60

IQ

20

30

40

50

0 60

Fraction no.

DNA from progeny virions of the Pm. 7. Sedimentation analysis of 3ZP-labeled DAR-viral ninth, tenth and eleventh serial passages. The 3aPWlabeled viral DNA I preptarations were purified from progeny virions. and analyzed by neutral sucrose gradient centrifugation after incubation nuclertse. The nuclease with ((b), (d) and (f)) or without ((a-), ( o ) and (e)) the EGO RI restriction reactions and sedimentation snalyses were performed as described i,n the legends to Figs 1 and 2. The locations of SV40 DNA I and II in these gradients was assessed by adding i4C-labeled DNA I and II to the sa.mples at the termination of the reactions. (a), (b), (c). (d), (e) and (f) are, respectively, sedimentation profiles for uncleaved and cleaved DNA from the ninth, tenth and elevenht serial passages. -o-e--, 32P; --o--O--, 3H; --A--A--, r4C.

gives rise to triplicated genomes involves the deletion of two-thirds of the original viral genome and a threefold amplification of remaining viral DNA sequences. This triplication of a specific DNA segment in DAR-viral genames has been demonstrated by characterizing these molecules, after cleavage with the EGO RI‘ restriction endonuclease. We have used the Eco RI cleavage site as. a physical marker on the viral

' F 2 .' Q z

TRIPLICATION

OF A UNIQUE

DNA

SEGMENT

287

genomes and have identified three classes of molecules arising after serial passaging of the DAR virus in monkey cells. The first two classes, which have been identified previously for standard XV40 after serial undiluted passages tMulder I%Delius, 1972 ; M&&et al., 1973), are molecules resistant to EGORI cleavage and molecules sensitive to EGO RI cleavage at a single site. The third class of genomes which becomes the predominant viral DNA species between serial pa&ages five to eight are Eco RI supersensitive molecules having three, symmetrically placed Eco RI cleavage sites. In a separate series of experiments the original DAR virus stock, which contained no detectable EcoRI supersensitive genomes, was serially passaged as has been described previously. EGORI supersensitive DNA molecules again appeared following the fourth serial passage. Since the original DAR virus stock was not prepared from plaque-purified virus and the evolution of viral DNA containing a ‘specific triplication occurred during serial passage at high input multiplicities, we are unable to determine, at present, whether supersensitive DNA was present as a “contaminant” in the original isolate or resulted by recombination after the fourth passage. The appearance of a new species of DAR-viral DNA with an even simpler structure (Pareed, Martin, Lee & Nathans, unpublished observation) following the eighth serial’passage (Fig. 7) suggests to us that DAR virus readily undergoes specific reassortment of viral DNA sequences during its growth in monkey kidney cells ; the sudden emergence of this new class of viral DNA at the ninth passage from altered molecules pre-existing in the original stock therefore seems unlikely. Serial passage of SV40 results in the incorporation of monkey cellular DNA into the supercoiled form of viral DNA (Lavi I%Winocour, 1972; Lavi et al., 1973). Duplication of certain DNA sequences and an overall simplification of the resultant DNA accompany this process (Brockman et al., 1973; Rozenblatt et al., 1973; Martin et al., 1973). The alterations and rearrangements of DAR-viral DNA which occur following serial passage, on the other hand, appear to be limited to viral DNA sequences. A 5.9 x 10s-fold excess of green monkey DNA failed to accelerate the reassociation of fragmented 10.4 S DNA (Fig. 6). Furthermore, we have subsequently shown by heteroduplex mapping as well as DNA-DNA hybridization with specific Hin SV40 DNA fragments, that sequences from two separate regions of the SV40 genome have recombined to form the 10.4 S segment (Khoury et al., 1974). Subsequent amplification (triplication) of these sequences without detectable introduction of host DNA sequences has resulted in the accumulation of Eco RI supersensitive DNA molecules. The evolution of a new species of supercoiled molecules following the eighth serial passage (Fig. 7) suggests that DAR virus readily undergoes further rearrangements of its DNA. REFERENCES

B&ten, R. J. & Kohne, D. E. (1968). Science, 161, 529-540. Brockman, W. W., Lee, T. N. H. & Nathans, D. (1973). Vvirology, 54, 384-397. Eagle, H. (1959). Science, 130, 432-437. Fareed, 0. C., Sebring, E. D. & Salzman, N. P. (1972). J. Biol. Chem. 247, 5872-5879. Fareed, G. C., Takemoto, K. K., & Martin, M. A. (1973). Fed. Proc. 32, 461. Gelb, L. D., Kohne, D. E. & Martin, M. A. (1971). J. Mol. Biol. 57, 129-145. Hirt,

B. (1967). J. Mol.

BioZ. 26, 365-369.

Khoury, G., Fareed, G. C., Berry, K., Martin, M. A., Lee, T. N. H., & PiTathans,D. (1974). J. Mol.

Biol.

87, 289-301.

Koch, M. A., Eggers, H. J., Anderer, F. A., Sohlumbcrger, H. D. & Frank, H. (1967). Virology, 32, 503-510.

288

G. C. FAREED,

J. C. BYRNE

AND

M. A. MARTIN

Lavi, S. & Winocour, E. (1972). J. Viral. 9, 309-316. Lavi, S., Rozenblatt, S., Singer, M. & Winocour, E. (1973). J. V$roZ. 12, 492-500. Levine, A. J., Kang, H, S. & Billheimer, F. E. (1970). J. Mol. Biol. 50, 549-568. Martin, M. A., Gelb, L. D., Fareed, G. C. & Milstien, J. B. (1973). J. Vivirol. 12, 748-757. Martin, M. A., Gelb, L. D., Garon, C., Takemoto, K. K., Lee, T. N. H., Sack, G. H., Jr & Nathans, D. (1974). VGoZogy, 59, 179-189. Morrow, J. & Berg, P. (1972). Proc. Nat. Acad. Sci., ‘U.S.A. 69, 3365-3369. Mulder, C. & Delius, H. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 32153219. Pettersson, U., Mulder, C., Delius, H. & Sharp, P. A, (1973). Proc. Nat. Acad. Sci., U.S.A.

70, 200-204. Radloff, R., Bauer, W. & Vinograd, J. (1967). Proc. Nat. Acad. Sk., U.S.A. 57, 1514 1521. Rozenblatt, S., Lavi, S., Singer, M. & Winocour, E. (1973). J. ViroZ. 12, 501-510. Sack, G. H., Jr, Narayan, O., Danna, K. J., Weiner, L. P. & Nathans, D. (1973). Virology,

51,345-350. Sebring, E. D., Kelly,

T. J., Jr, Thoren, M. M. & Salzman,

N. P. (1971). J. ViroZ.

8,

478-490. Smith, H. 0. & Nathans, D. (1973). J. Mol. BioZ. 81, 419-423. Studier, F. W. (1965). J. Mol. BbZ. 11, 373-390. Tai, H. T., Smith, C. A., Sharp, P. A. & Vinograd, J. (1972). J. ViroZ. 9, 317-325. Uchida, S., Watanabe, S. & Kato, M. (1966). Virology, 28, 135-141. Westphal, H. & Dulbecco, R. (1968). Proc. Nat. Ad. Sci., U.S.A. 59, 1158-1165. Weiner, L. P., Herndon, R. M., Narayan, 0. & Johnson, R. T. (1972). J. ViroZ. 10, 147

149. Yoshiike,

K. (1968). I’irology,

34, 391-401.