Vol. 35, No. 1JOURNAL OF VIROLOGY, July 1980, p. 141-1490022-538X/80/07-0141/09$02.00/0

Preferential Replication of a Class of Host-SubstitutedDefective Simian Virus 40 Variants at Low Temperature

MAXINE F. SINGER* AND RONALD E. THAYERLaboratory ofBiochemistry, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland

20205

The host-substituted variant termed CVP8/1/P2 (EcoRI res) was first isolatedseveral years ago after serial passage of simian virus 40 strain 777 on BSC-1 cellsat 370C. When BSC-1 are coinfected with wild-type simian virus 40 strain 777and variant CVP8/1/P2 (EcoRI res), the variant rapidly becomes the dominantspecies produced, often representing as much as 80% of the total DNA I synthe-sized after infection. We present evidence that the replicative advantage of thevariant was increased when the infection was carried out at 33 rather than 370C.Also described are nine new and independent serial passage experiments carriedout at 330C with several purified wild-type virus stocks, including strain 776, andboth BSC-1 and primary African green monkey kidney cells. In each seriesvariants related to CVP8/1/P2 (EcoRI res) were detected in the progeny viralgenomes after four serial passages. Hybridization data suggest that at least someof these variant DNA I molecules contain simian virus 40 DNA sequences,monkey a-component DNA sequences (highly repetitive), and the infrequentlyreiterated monkey DNA sequences found in CVP8/1/P2 (EcoRI res), all cova-lently linked as in CVP8/1/P2 (EcoRI res). It appears that this type of variantemerges with some frequency during infection and is then preferentially replicatedat 330C, thereby becoming readily detectable in passaged stocks. A variety ofcontrol experiments indicated that the repeated emergence of similar, if notidentical, variants is unlikely to be the result of inadvertent cross-contaminationor the presence of detectable amounts of the variant in the plaque-purified viralstocks.

A variety of defective variants of simian virus40 (SV40) are formed upon serial passage ofplaque-purified virus at a high multiplicity ofinfection (3, 12). The genomes of one group ofvariants, which are characterized by the pres-ence of covalently linked segments of viral DNAsequences and monkey DNA sequences, havebeen studied in some detail, and recently nucleo-tide sequence determinations for several suchvariants have been published (6, 7, 20, 27, 34).Typically, these variants consist of several (from3 to about 10) tandem repetitions of basic repeatunits comprising viral and monkey DNA seg-ments. The basic repeat units are different indifferent variants, but always contain the regionof the wild-type SV40 genome identified as theorigin of replication. The tandem repeats arearranged in a closed circular duplex DNA which,at least in packaged variants, approxiimates thesize of a wild-type SV40 genome.One such variant, termed CVP8/1/P2 (EcoRI

res) (abbreviated CVP8/1/P2 below), consists offour tandem repeats of a DNA segment 1,210base pairs (bp) long (24, 34). Figure 1 shows aschematic drawing of this repeat unit; the entire

141

nucleotide sequence is known (34). One in everythree to four repeat units differs from the otherunits by the insertion of a 223-bp repetition of aportion of the sequence. The 1,210-bp repeatunit contains monkey sequences of two types;one is homologous to the highly repeated mon-key DNA fraction termed a-component (18, 28),and the other is homologous to sequences thatare repeated only a few times in the monkeygenome (22,34; T. McCutchan and M. F. Singer,unpublished data). Separately, a similar varianttermed F161F was isolated from the same seri-ally passaged viral stocks, as was CVP8/1/P2(22).One of the striking observations concerning

variants of the CVP8/1/P2 type is that theyarose after only a very few passages of a plaque-purified stock of SV40 strain 777 (14, 22, 23, 29).They were detectable after only two passagesand by the fourth serial passage represented asubstantial portion of the total viral DNA Iisolated from infected cells (14, 29). Thus, thevariant was replicated efficiently relative to thereplication of wild-type genomes. In this paperwe demonstrate that the relative efficiency of

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142 SINGER AND THAYER

eAr ~I II XI3 i. 8 g- 'R _ Q

~~~~~~-4

I100 200 3D 400 MD 700 800 900 1000 1100 1200

I 1.

'43

Bam Hl Hinc II

FIG. 1. Schematic diagram ofrepeatingDNA segments in defective SV40 variant CVP8/11/P2. Structure ofthe major repeatingDNA segment (1,210 bp) ofthe host-substituted SV40 variant designated CVP8/11/P2. Theentire nucleotide sequence is known (34J. Four head-to-tail tandem repeats of this segment constitute thewhole genome. The solid bars represent SV40 sequences; the open bars represent monkey sequences that occurinfirequently in the monkey genome; and the cross-hatched bar represents sequences homologous to the highlyrepetitive a-component DNA of the mon:key genome. One of every three to four repeating units contains areiteration of residues 345 to 567 inserted after residue 567, as shown on the bottom of the diagram. Thesubunit structure of this variant is very similar to that of the F161F variant (22), as judged by restrictionendonuclease mapping and cross-hybridization experimnents. CVP8/11P2 and F161F have a common historyin the sense that both are derived firom the same serialpassage ofSV40 strain 777.

replication of the variant is even greater at 33than at 370C. This finding was used to determinewhether variants like CVP8/1/P2 arise repeat-edly in infected cells. Whatever the mode offormation of such variants, passaging of virus at33 rather than 370C would favor accumulationof the CVP8/1/P2 type of variant and therebyaid in its detection. As reported here, newlyinitiated serial passages (at a high multiplicityof infection [MOI]) of several different strains ofplaque-purified wild-type SV40 on differenttypes of cells consistently gave rise to viral pop-ulations containing defectives similar to theCVP8/1/P2 type when the passaging was carriedout at 330C. These findings are consistent withearlier indications that variants of this typemight recur independently after SV40 infections(21; E. Winocour, M. F. Singer, and E. L. Kuff,Cold Spring Harbor Symp. Quant. Biol., inpress).

MATERIALS AND METHODSCells. The BSC-1 (10) monkey kidney cells used in

most experiments were progeny of a clone isolated in1977 in this laboratory from cells originally obtainedfrom E. Winocour. For some experiments (see Table3) BSC-1 cells derived from the same Winocour stockand maintained independently for about 5 years in thelaboratory of Norman Salzman without interveningcloning were used. BSC-1 cells were grown in Dulbeccomodified Eagle medium containing glutamine and sup-plemented with penicillin, streptomycin, and 10% fetalcalf serum. Primary African green monkey (AGM)kidney cells were grown in Eagle no. 2 medium sup-plemented with streptomycin, penicillin, achromycin,mycostatin, glutamine, and 10% fetal calf serum.

Virus. Viral stocks were assayed for plaque-formingunits by standard procedures (15). The medium forviral infections was as described above except that 2%fetal calf serum was frequently used. SV40 strain 776was obtained from D. Nathans. A plaque derived fromthree sequential plaque isolations was used to preparea stock (CV776) by infection of BSC-1 cells at between10-3 and 10' PFU/cell at 370C. A stock of plaque-purified strain 777 SV40 (CVB) was originally obtainedfrom E. Winocour (15); the strain 777 stocks used inthese experiments were prepared as described aboveby infection with a thrice-purified plaque isolated fromthe CVB stock. The two stocks were identified asstrains 777 and 776 by the characteristic products ofdigestion of the corresponding DNA I progeny by endoR.HincII and endo R.HindIII (11); the digests gaveno indication of the presence of detectable variantgenomes. The stock of variant CVP8/1/P2 (23, 24)contains wild-type strain 777 helper. The CV371 andCV372 stocks were prepared as described above fromseparate thrice-purified plaques. DNA I made withthese stocks gave restriction endonuclease productstypical of strain 777 (11), with no indication of thepresence of detectable variant DNA.

Serial undiluted passaging. Passaging was car-

ried out as described previously (14, 15, 29). For allinfections, virus was adsorbed to cells at 370C for 1.5to 2 h, medium was added, and the cells were incubatedat either 33 or 37°C, as indicated below. Stocks pre-pared from plaque-purified virus were used undilutedto initiate each passage series; the MOI was generallybetween 10 and 20. After full cytopathic effects were

observed, the medium was collected, and the virus wasdispersed by three cycles of freezing and thawing. Theresulting material was called passage 1. Between 2 and4 ml of passage 1 lysate was used to infect fresh cells,and after full cytopathic effects were observed, themedium was collected, treated as described above, and

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REPLICATION OF DEFECTIVE SV40 VARIANTS 143

designated passage 2. Passages 3 and 4 were preparedin analogous fashion. Lysates are designated by atripartite nomenclature, which was introduced by Laviand Winocour (15). First, the plaque-purified virusused to initiate the passages is named, then the num-ber of the particular passage series is given, and finallythe passage number is named. For example, CV776/1/P1 is the lysate obtained after the first passage (P1) inthe first independent passage series (series 1) initiatedwith the CV776 stock; CV776/3/P4 is the lysate ob-tained after the fourth serial passage (P4) in the thirdindependent passage series (series 3) initiated with thesame stock.

Preparation of 'H-labeled viral DNA I frominfected cells. Confluent monolayers were infectedas described above. At specified times [methyl-3H]-thymidine (50 Ci/mmol; New England Nuclear Corp.)was added to a concentration of 10 uCi/ml, and incu-bation was continued for 24 h unless indicated other-wise. Viral DNA was extracted by the method of Hirt(9) and deproteinized by repeated extractions withphenol and a mixture of CHC13 and isoamyl alcohol(24:1). For the experiment reported in Table 1, DNAI was purified further by banding to equilibrium indensity gradients of CsCl in the presence of ethidiumbromide and then dialyzed (final dialysis against 1.5mM NaCl/0.15 mM sodium citrate). For the experi-ment reported in Table 2, the deproteinized prepara-tion was treated with 50 ,ug of heated (80°C, 10 min)pancreatic RNase per ml and then with 50 ug of self-digested proteinase K per ml, each for 10 min at 37°C.DNA was recovered after precipitation from 0.3 Msodium acetate with 2.5 volumes of ethanol. DNA Iwas then purified by a modification (K. Chowdhury,National Institutes of Health, personal communica-tion) of the acid-phenol technique (35). For the exper-iments reported in Tables 3 through 6 the DNA I waspurified by centrifugation through alkaline sucrosegradients, as described below.Determination of the proportion of CVP8/1/

P2 in DNA I isolated from infected cells. Theproportion of the variant in the mixtures of CVP8/1/P2 DNA I and wild-type DNA I isolated from cellscoinfected with the variant and its wild-type helper(strain 777) was determined by measuring the per-centage of 3H-labeled DNA I resistant to treatmentwith endo R.EcoRI. CVP8/1/P2 DNA does not con-tain a cleavage site for endo R-EcoRI (23, 34), whereaswild-type SV40 DNA contains one such site. At least90% of the EcoRI-resistant viral DNA molecules incells infected with stocks of this variant are CVP8/1/P2 DNA (23, 24). The data were not corrected for thesmall difference in the sizes of variant DNA I andwild-type DNA 1 (5,060 bp [34] and 5,226 bp [4, 25],respectively). 3H-labeled DNA I (approximately 0.1Lg; about 1.4 x 105 cpm/gg) was incubated with andwithout endo R.EcoRI for 2 h at 37°C in a solution(total volume, 50 pl) containing 0.1 M Tris-hydrochlo-ride (pH 7.5), 0.01 M MgCl2, 0.1 M NaCl, and 8 U ofendo R.EcoRI (Boehringer). The reactions werestopped by making the mixtures 0.05 M in NaOH and0.05M in EDTA (final volume, 0.1 ml), and incubationwas continued for 10 min at 37°C. Then the materialwas layered on top of a 4.2-ml gradient (5 to 20%sucrose; 0.1 to 0.4 N NaOH) above a 0.2-mil cushion of

60% sucrose; all sucrose solutions were prepared in 0.5M NaCl-2.5 mM EDTA. The tubes were centrifugedin an SW50.1 rotor with a model L5-65B Beckmancentrifuge at 4°C for 90 min at 49,000 rpm, and ap-proximately 20 fractions were collected from the bot-toms of the tubes into scintillation vials. The alkaliwas neutralized with a drop of glacial acetic acid, andthe samples were counted in 10 ml of Ultrafluor (Na-tional Diagnostics) containing 10% water. The radio-activity in the fastest-sedimenting peak (DNA I) wasmeasured.

Filter hybridization. AGM liver DNA (2) andSV40DNA (23) were prepared by previously pub-lished procedures and fixed to nitrocellulose filters(Millipore Corp. or Schleicher & Schuell), and hybrid-ization was carried out as described previously (15).The filters were washed by a slight modification (30)of the original procedure. DNA was eluted from thefilters as described previously (15).

Preparation of 32P-labeled fragment C. A frag-ment (termed fragment C) representing the monkeyDNA segment from residue 364 through residue 679in the repeating unit of CVP8/1/P2 DNA (Fig. 1) wasobtained from purified CVP8/1/P2 DNA by cleavagewith endo R.BamHI and endo R.HindIII, as de-scribed previously (34; Winocour et al., in press). Frag-ment C is 315 bp long and contains 282 bp of low-reiteration-frequency monkey DNA, as well as 33 bpof highly reiterated a-component DNA. This 33 bp didnot hybridize significantly to a-component sequencesunder the hybridization conditions used in the presentexperiments (see Tables 5 and 6). Purified fragment Cwas labeled with 3P in vitro by nick translation, usingthe method of Rigby and co-workers (26). The specificradioactivities ranged between 107 and 108 cpm/yg ofDNA.Assay for variants containing fragment C se-

quences in infected cells by in situ plaque hy-bridization. This assay was as described by Winocourand co-workers (Winocour et al., in press) and screensfor infectious centers that hybridize with the fiagmentC sequence. In brief, growing BSC-1 cells were infectedwith plaque-purified viral stocks, and the cells weretrypsinized 2 h after the adsorption period was com-plete. Infected cells were mixed with a 20-fold excessof uninfected cells, about 106 cells were placed in eachof several 60-mm dishes, and monolayers were allowedto form and then overlaid with agar. At varying timesthereafter (6 to 10 days) the agar overlays were re-moved, and the cell monolayers were transferred tonitrocellulose filters (32). The filters were treated withalkali, hybridized with 3P-labeled fragment C probe,and autoradiographed. Cells infected with CVP8/1/P2itself served as the positive control. Rounded, well-localized dark spots indicated single infectious centersproducing variants containing sequences homologousto fragment C. This procedure readily permits screen-ing of 1 x 106 to 2 x 106 infected cells; between 30 and50% of the infected cells can be visualized, as indicatedby reconstruction experiments (Winocour et al., inpress).

RESULTSReplication of variant CVP8/1/P2 DNA

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is more efficient at 33 than at 37°C relativeto the replication ofwild-type DNA. Variousobservations made over the course of severalyears suggested that the DNA of variant CVP8/1/P2 was efficiently replicated in cells infectedwith stocks containing both variant and helperviruses (8, 13, 14, 23, 29). In all cases, as well as

in the present experiments, the helper virus wasstrain 777; variant CVP8/1/P2 was isolated afterpassaging a viral stock prepared from a purifiedplaque (termed CVB) of strain 777 (14, 15, 23).Table 1 confinns the earlier observations. BSC-1 cells were infected with mixtures of wild-typevirus (strain 777) and the CVP8/1/P2 variant,and the percentage of labeled variant DNA I intotal pulse-labeled viral DNA I was estimated.Identical experiments were carried out simulta-neously at 33 and 370C. At both temperaturesand at all relative MOIs with the variant, a

substantial percentage of the total labeled DNAI was variant DNA.Although the differences were small, a com-

parison of the results at 33 and 370C (Table 1)at each MOI with CVP8/1/P2 suggested thatreplication of the variant might be relativelygreater at 33 than at 370C. Therefore, a similarexperiment was carried out in which the per-centages of variant DNA I synthesized at vary-ing times after infection at 33 and 370C were

determined (Table 2). The relative MOI withCVP8/1/P2 was 0.1 on the scale indicated inTable 1. At all times studied, the intracellularvariantDNA I was synthesized at a substantiallygreater rate at 33 than at 370C relative to wild-type DNA; the difference was most striking earlyin infection.

In other similar experiments the percentageof DNA I that was variant was measured afterlong periods of labeling in order to estimate therelative accumulation ofthe two DNAs. At 370C,labeling was started 24 h after infection, andCVP8/1/P2 DNA was 27 and 22% of the totalDNA I isolated at 48 and 72 h, respectively, afterinfection. At 330C, labeling was started 36 hafter infection; 50 and 54% of the DNA I wasvariant DNA after 72 and 96 h, respectively.Presence of defective substituted var-

iants in viral stocks passaged at 330C. Asdescribed above, the results presented thus farindicate that passaging viral stocks at 330Cmight favor the accumulation of defective var-

iants which, like CVP8/1/P2, are replicated rel-atively efficiently at the lower temperature. In-deed, it has been noted that stocks of tempera-ture-sensitive mutants of SV40 that are rou-

tinely prepared at 330C often contain significantamounts of defective variants (33; E. Winocour,personal communication).Table 3 summarizes the results obtained when

TABLE 1. Relative rates of synthesis of CVP8/1/P2DNA and wild-type DNA late in mixed infections at

33 and 37°CRelative MOI of Temp % CVP8/l/P2 inCVP8/1/P2b (OC) DNA Ic

1 33 8537 81

0.2 33 5637 45

0.05 33 3237 28

0.01 33 1137 7

0.00 33 4a Plates (60 mm) containing confluent monolayers

of BSC-1 cells (approximately 106 cells) were infectedin duplicate with 1 ml of a mixture containing 107 PFUof wild-type SV40 strain 777 and varying amounts ofCVP8/1/P2 variant virus. The plaque-forming unitsof wild-type SV40 strain 777 were composed of virusderived from the purified stock described in the textand virus present as helper in the stock of the CVP8/1/P2 variant. After adsorption, one of each duplicateset of plates was placed at 330C and the other wasplaced at 370C. After 48 h at 370C or 72 h at 330C,[3H]thymidine was added, and incubation was contin-ued at the same temperature for 2 h, after which viralDNA was extracted and DNA I was purified by band-ing in cesium chloride-ethidium bromide. Mock-in-fected controls had no DNA I band.

b The concentration of CVP8/1/P2 virions in thestock was not known. The actual volumes of CVP8/1/P2 stock used in each infection were 0.2, 0.04, 0.01,0.002, and 0.000 ml, giving relative MOIs of 1, 0.2, 0.05,0.01, and 0.00, respectively. The CVP8/1/P2 stockcontained 5 x 107 PFU of helper strain 777 virus perml.

'Equal portions of each DNA I (about 104 cpm)were incubated under identical conditions with andwithout an excess of endo R-EcoRI, and the amountof DNA I was estimated as described in the text. Thepercentage of CVP8/1/P2 DNA formned is the ratio ofthe counts per minute in DNA I after EcoRI treatmentto the counts per minute in DNA I before EcoRItreatment, multiplied by 100. The total radioactivitiesrecovered in the samples of each pair of gradients wereessentially identical.

several different stocks of wild-type SV40 wereserially passaged at 330C at a high multiplicityon either BSC-1 or AGM kidney cells. Eachpassage series involved four serial passages; onlythe results for passages 1 and 4 are given. ViralDNA I was purified from BSC-1 cells infectedwith the passaged virus and screened for thepresence of defective substituted variants byhybridization to AGM liver DNA. This proce-dure detects only variants substituted with the

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REPLICATION OF DEFECTIVE SV40 VARIANTS 145

TABLE 2. Comparison of the relative rates ofCVP8/1/P2 and wild-type SV40 DNA synthesis at

33 and 37°C at varying times after infection'Relative radio-

Temp Time post-in- activity (cpm) % CVP8/1/P2(0C) fection (h) in total viral in DNA Ic

DNAb

33 48 3.5 5333 72 27 6133 96 37 6533 120 40 6333d 120 32 0

37 24 1.0 1437 36 3.0 1437 48 21 1937 59 17 2937 72 35 50

a Plates (100 mm) containing confluent monolayersof BSC-1 cells (approximately 3 x 106 cells) were eachinfected with 1 ml of a mixture containing 22.5 jA of apurified stock of wild-type SV40 strain 777 (1.2 x 109PFU/ml) and 60 pl of the CVP8/1/P2 stock (5 x 107PFU of wild type per ml). After adsorption one-half ofthe plates were placed at 330C, and one-half wereplaced at 37°C. At the indicated times after infection[3H]thymidine was added, and incubation was contin-ued at the same temperature for 2 h, after which viralDNA was extracted and purified by using the acid-phenol procedure.

b Relative amount oflabeled viral DNA synthesizedin each plate as measured by the counts per minuterecovered after acid-phenol purification. The data arenormalized to the counts per minute incorporated 24h after infection at 37°C and thus indicate the relativerates of total viral DNA synthesis in the various plates.

'See Table 1, footnote c.d Control in which infection was with the purified

strain 777 stock alone (MOI, 10).

highly repetitive a-component fraction of mon-key DNA since only this DNA is present in asufficient amount on the filters (15). Previouswork has indicated that many, but not all, sub-stituted variants contain sequences homologousto a-component (7, 19-21, 27). Each of the DNApreparations was also hybridized under identicalconditions to filters containing wild-type SV40DNA as a positive control. In addition, DNA Isynthesized after infection with the initiatingstock virus itself was tested (Table 3).DNA I isolated from cells infected with the

purified wild-type stocks or with lysates ob-tained after the first high-multiplicity passage ineach series (passage 1) hybridized to an insignif-icant extent to AGM DNA (Table 3). Thus,within the level of sensitivity afforded by thisassay, the viral stocks used to initiate the pas-sages did not contain variants substituted witha-component. This is consistent with the obser-vation (see above) that such DNA I contained

TABLE 3. Detection ofSV40 variants substitutedwith monkey a-component sequence in stocks

passaged at 33°C% of Viral DNA I hybridized

to filtersdPassage Cell Tempseries' typeb (°C) Passage 1 Passage 4

AGMe SV40' AGM' SV40'

CV776/1 BSC-1 37 0.3 22 0.4 33CV776/2 BSC-1 33 0.2 13 0.7 16CV776/3 BSC-1i 33 0.1 24 6.4 37CV776/4 AGMKf 33 0.1 25 3.3 44

CV371/1 BSC-1 33 0.0 21 7.8 26CV371/2 BSC-1i 33 0.0 19 2.2 41CV371/3 AGMKf 33 0.0 34 2.9 39

CV372/1 BSC-1 33 0.2 26 3.8 18CV372/2 BSC-i 33 0.7 31CV372/3 AGMKf 33 0.0 25 4.4 42

CV776' 37 0.2 21CV776' 33 0.3 15CV371 33 0.0 21CV372' 33 0.1 26

'The passage series are designated by the name of thecloned viral stock (see text) used to initiate the passage seriesand a numeral indicating the particular series of passagesinitiated with that stock. Thus, CV776/1 is the first series ofpassages initiated with the stock of strain 776.

b The type of cells used for the passage series.'Temperature used for serial passage and for preparation

of progeny DNA I for hybridization.d The preparations of DNA I analyzed in these experiments

were made by infecting BSC-1 cells with the designated pas-saged stock at the same temperature that was used for theserial passage. Approximately 5 x 103 to 10 x 103 cpm of[3H]DNA I was used for each hybridization. Blank filterscontaining no DNA absorbed 0.1% or less of the radioactivityin almost every case. The blank values have not been sub-tracted.

e The filters contained either 2.5 pg of SV40 DNA or 5 ug ofAGM liver DNA for the passage series designated 1 and 2 andfor the stock DNAs (see footnote g) or 5 pg of SV40 DNA or15 pg of monkey DNA for the passage series designated 3 and4.

f These passage series were carried out in the laboratory ofMalcolm Martin. The BSC-1 cells were those maintained byNorman Salzman (see text). AGMK, AGM kidney.

' The [3H]DNA I used in the hybridization was from theprogeny isolated after infecting BSC-1 cells with the purifiedstock used to initiate the various passage series. Infection wasat an MOI of 20 PFU/cell.

no detectable variant DNA when characterizedby restriction endonuclease digestion. However,in each passage series tested after four serialpassages at 33°C, a significant increase in theextent of hybridization to AGM DNA occurred,suggesting the presence of defective SV40 var-iants containing sequences homologous to a-component DNA. Because of the possibility thatthe consistent detection of variants containingsequences homologous to the a-componentDNA of the monkey genome resulted from in-advertent contamination of the newly made pas-

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146 SINGER AND THAYER

sages with defective variants already present inthe laboratory (e. g., CVP8/l/P2), several of theseries of passages were carried out in anotherlaboratory in another building at the NationalInstitutes of Health, using cells and mediummaintained there (Table 3).Table 3 includes an analysis of one passage

series (CV776/1) carried out at 370C. In thisinstance little or no variant containing the a-component sequence was detected, althoughmany reports (3, 12, 15, 19, 21, 29) indicate thatsuch variants are occasionally detectable afterfour passages at 370C. Thus, the result withCV776/1 (Table 3) may be considered a fortui-tous negative.With passage series CV776/1, CV776/2,

CV371/1, and CV372/1, DNA I was also isolatedfrom cells infected with stocks corresponding topassages 2 and 3 (e. g., CV776/1/P2 and CV776/1/P3) and analyzed by hybridization, as de-scribed above for the experiments reported inTable 3. In most cases, hybridization to filterscontaining AGM DNA was detected even in theDNA I produced after infection with passage 2or passage 3 stocks.Table 4 indicates that the a-component DNA

found in the passaged material was covalently

TABLE 4. Covalent linkage ofSV40 and monkeyDNA sequences in CV371/1/P4 and CV372/1/P4

% HybridizedInput DNA to filters con-

Hybrid- taining:[3H]DNAa ization

step Radioac- SV40 AGMType tivity DNA DNA

(cpm)CV371/1/P4 ib DNA I 59,500 42 16

2c SV40 14,000 26 3AGM 5,900 27 8

CV372/1/P4 lb DNA I 13,169 30 72C SV40 2,000 21 1

AGM 600 31 9

[3H]DNA I was prepared from BSC-1 cells infected withstocks of CV371/1/P4 or CV372/1/P4 (see Table 3).

bHybridization step 1 was the initial hybridization of thenicked, denatured DNA I to filters containing either 5 utg ofwild-type SV40 DNA or 10 ,ug of AGM liver DNA. Step 1hybridizations were carried out in quadruplicate, and theresults shown are the averages of the four determinationsuncorrected for hybridization to filters containing no DNA;the blank filters generally contained 0.5% or less of the inputradioactivity.

e The radioactivity on the filters after step 1 was determinedin a toluene-based scintillation fluid, and the filters were rinsedthree times with 10-mi volumes of fresh toluene and driedovernight in air. The DNA on each filter was then eluted byincubating each filter with 1 ml of elution buffer (15) at 37°Cfor 3 h with shaking. The eluates from the sets ofquadruplicatefilters were pooled and used as inputs for the step 2 hybridi-zation. The input DNAs labeled SV40 and AGM were thoseeluted from the step 1 filters containing SV40 DNA and AGMDNA, respectively.

linked to SV40 DNA sequences. The methodused has been described previously (15). [3H]-DNA I isolated from cells infected with CV371/1/P4 or CV372/1/P4 was first hybridized tofilters containing either SV40 DNA or AGMDNA, as described above. The hybridized DNAwas eluted from each type of filter and rehybrid-ized in a second round of hybridization to bothtypes of filters. In each case [3H]DNA that hadbeen eluted from filters containing SV40 DNA(designated SV40) hybridized to SV40 andAGMDNA (the results with CV372/1/P4 are justsignificant). Similarly, [3H]DNA eluted from fil-ters containing AGM DNA (designated AGM)hybridized to both SV40 and monkey DNA.Thus, significant amounts of variants containingcovalently linked SV40 and a-component se-quences occurred repeatedly when viral stocksfree of detectable substituted variants beforepassaging were serially passaged at 330C.Presence of low-reiteration-frequency

monkey DNA found in CVP8/1/P2 in sub-stituted variants accumulated at 330C. Thepassage 4 DNA I populations described in Table3 were also tested for the presence of sequenceshomologous to a monkey DNA segment of lowreiteration frequency (fragment C [see above])that is present in variant CVP8/1/P2. In oneapproach, 32P-labeled fragment C, either aloneor mixed with [3H]DNA I, was incubated withfilters containing SV40 or AGM DNA (Table 5).Significant amounts of 32P hybridized to bothtypes of filters only when the passaged DNAwas present. Thus, these data suggest that se-quences homologous to fragment C are presentand covalently linked to both SV40 and a-com-ponent DNA. In other experiments, 32P-labeledfragment C was mixed with [3H]DNA that hadbeen selected by prior hybridization to filterscontaining either SV40 or AGM DNA as de-scribed in Table 4 and then hybridized to freshfilters containing unlabeled SV40 orAGM DNA.The results for CV371/1/P4 are shown in Table6. 32P-labeled fragment C by itself bound little,if at all, to filters containing SV40 DNA; someradioactivity (about 1%) was bound to filterscontaining AGM DNA. However, when 3H-la-beled variant DNA preselected by hybridizationto either SV40 or AGM DNA was included inthe reaction, 32P-labeled fragment C bound toboth types of filters. These results indicate thatvariant viral DNAs containing both a-compo-nent DNA and sequences homologous to frag-ment C are present and covalently linked toSV40 DNA in CV371/1/P4.As noted above, DNA I preparations made

upon infection with the viral stocks used toinitiate these passages were free from detectablevariants, as judged by restriction endonuclease

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REPLICATION OF DEFECTIVE SV40 VARIANTS 147

TABLE 5. Presence of sequences homologous tofragment C in CV776/4/P4, CV371/3/P4, and

CV372/3/P4a

Radioactive probesRadioactivity (cpm)

bound to filters contain-ing-

[3H]DNA I nP-la SV40 DNA AGM DNA

beledRadio- frgType activity rag-b 3H n2p 3H 32p

(cpm) ment C

CV776/4/P4 11,266 + 3,618 379 1,045 329None + 88 38

CV371/3/P4 12,266 + 2,475 332 1,905 413None + 27 49

CV372/3/P4 14,731 + 3,209 212 2,283 295None + 40 36

a Hybridization reactions were set up as described for step1 in Table 4, footnote b, except that 3P-labeled fragment Cprobe was included.

'The fragment C used was contained within a pBR322 (1)plasmid vector, fragment C was ligated into pBR322 by meansof its endo R.HindIlI and endo R-BamHI termnini and thecorresponding sites on pBR322 (T. McCutchan and R. Thayer,unpublished data). Each reaction contained 26,000 cpm of 3P2labeled plasmid, of which approximately 7% (315 of 4,302 bp)or 1,800 cpm represented fragment C.

TABLE 6. Presence of low-frequency monkey DNAsequence characteristic of CVP8/1/P2 in CV371/1/

P4 DNA a

% Of 32P-labeled probe hybridized to

[3H]DNA filters containing:

SV40 DNA AGM DNA

None 0.1 1.0CV371/1/P4 SV40 7.0 4.8CV371,V1/P4 AGM 3.6 4.4

a Filter hybridizations were set up exactly as de-scribed for hybridization step 2 in Table 4, footnote c,except that 37,500 cpm of32P-labeled fragment C probe(see text) was included in the input to the hybridiza-tion. The filters incubated with the 3P-labeled probewere counted under conditions allowing determinationof both 3H and 32P bound to the filters. The 3H datawere the same as those reported in Table 4 for CV371/1/P4, step 2 (the experiments were carried out simul-taneously).

digestion and by hybridization to AGM DNA.In view of the data reported above, it becameimportant to test the initiating stocks in a muchmore sensitive manner for possible contamina-tion by variants containing the fragment C se-quence. Consequently, CV776, CV371, andCV372 were tested for the presence of suchvariants by an in situ plaque hybridization pro-cedure (Winocour et al., in press), as describedabove. A total of 106 cells infected with CV776(MOI, 1 or 6), 1.3 x 106 cells infected with CV371(MOI, 10), and 9.5 x 105 cells infected with

CV372 (MOI, 10) were screened for infectiouscenters synthesizing detectable amounts of var-iants that might hybridize with fragment C.CV371 and CV372 showed no hybridizing infec-tious centers. The experiments with CV776 suf-fered some from problems with backgrounds,but no clearly positive centers were observed.

DISCUSSIONSince the earliest recognition of substituted

defective variants of SV40 (see above), it hasbeen assumed that the wild-type SV40 originsequence is necessary for replication of the de-fectives (12, 29). Several characterized variantscontain multiple origins (3, 6, 12, 20; Winocouret al., in press). Lee and Nathans (17) recentlydemonstrated by using a series of such variants(not including CVP8/1/P2 or F161F) that therate of variant DNA replication relative to therate of helper virus replication is an exponentialfunction of the number of origin segments pergenome. These workers concluded that a majorfactor in the evolution of SV40 variants was thereplicative advantage derived from the reitera-tion of the origin. Variant CVP8/1/P2 DNA hasfour reiterations of the origin region and, asexpected, is replicated efficiently compared withwild-type DNA. Furthermore, as shown here,CVP8/1/P2 DNA is replicated more efficientlyrelative to wild-type DNA at 33 than at 37°C.The difference between 33 and 37°C is mostmarked very early after the initiation of viralDNA synthesis in infected cells. The preferentialreplication of the variant at 33°C was evidentwhen both short labeling periods (indicating rel-ative rates ofsynthesis) and long labeling periods(indicating accumulation of the variant) wereused. These results are unexpected if the repli-cative advantage ofthe variant arises solely froma multiplicity of origins since the relative effectof a decrease in temperature should be equiva-lent for identical origin sequences, regardless oftheir number. It is possible that other featuresof the CVP8/1/P2 structure influence its repli-cation.A large number of high-multiplicity serial pas-

sages of plaque-purified wild-type SV40 havebeen carried out at 37°C in the last few years (3,12, 15, 21). Many such passage series yieldedsubstituted defective variants containing mon-key DNA segments, and it generally has beenassumed that the variants are the result of re-combination between viral and host genomes.Many of the variants contain DNA homologousto a-component DNA (14-16, 19, 21, 27, 29).This finding is not surprising, given the presenceof several million copies of the a-componentmonomer in the monkey genome (19, 30, 33). Inaddition, several different monkey DNA seg-

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148 SINGER AND THAYER

ments derived from the low-reiteration-fre-quency class of monkey DNA have been foundin different variants (3, 5, 7, 12, 17, 20, 27, 34).Interestingly, Oren and co-workers (21) reportedthat sequences homologous to the low-reitera-tion-frequency monkey segment of F161F (andtherefore to fragment C of CVP8/1/P2) weredetectable in two other, apparently independentpassaged stocks. These workers concluded thatalthough SV40 DNA could recombine with avariety of monkey DNA segments to form sub-stituted defectives, certain monkey sequencesmight be preferred.The preferential replication of CVP8/1/P2 at

330C suggested that passaging experiments car-ried out at the low temperature might favoraccumulation of CVP8/1/P2-type variants. Inthe experiments reported here, each of nine in-dependent passage series at 33°C yielded, afteronly a few passages, stocks in which variantscontaining the a-component monkey sequencewere readily detectable. Furthermore, the datasuggest that the defective variants in several ofthe passage series may contain variants similarto CVP8/1/P2 in that both a-component andfragment C DNA sequences are present andcovalently linked to SV40 DNA. This suggestionhas been confirmed and extended by purificationand characterization of defectives in stocks ofCV371/1/P4, CV372/1/P4, CV776/3/P4, andCV776/4/P4 (J. Papamatheakis, T. N. H. Lee,R. E. Thayer, and M. F. Singer, manuscript inpreparation). Thus, it appears that passage at33°C of several different strains of purified wild-type viruses in two different types ofAGM cellsleads to marked accumulation of defective var-iants related to, if not identical to, CVP8/1/P2(or F161F). We conclude that although a varietyof defective variants may arise during passaging,the CVP8/1/P2 type of variant becomes abun-dant at 330C because of its preferential replica-tion. We do not know whether the lower tem-perature also influences the rate at which CVP8/1/P2-type variants are formed. It is of courseprobable that other variants, which were notdetectable by the methods used here, may alsoconstitute a substantial portion of the popula-tion of viral genomes in the passaged materials.Although we cannot rigorously conclude that

the CVP8/1/P2-type variants emerged inde-pendently in each ofthe passage series describedhere, a number of observations support such apossibility. First, each of the initiating viralstocks was prepared by taking a thrice-purifiedviral plaque and infecting cells at a multiplicityof 10-3 to 10-4 PFU/cell. Second, the possibilitythat the viral stocks or the cells were contami-nated in the laboratory by CVP8/1/P2 itself is

unlikely because (i) the existing stocks ofCVP8/1/P2 were not used at all during the period whenthe present passages were carried out; (ii) six ofthe passage series were carried out in anotherlaboratory in another building, using cells, me-dia, and incubators belonging to that laboratory;and (iii) analysis of the initiating wild-typestocks by the in situ plaque hybridization pro-cedure suggested that such contamination, if itexists, is at a level below 1 in approximately 107PFU (106 cells infected with an MOI of about10). Finally, it is pertinent that the CVP8/1/P2(and F161F) variants were isolated after passageof strain 777 virus and had not been observedpreviously in passaged strain 776 stocks. Thus,variants of this type may indeed emerge repeat-edly and independently either during the prep-aration of viral stocks or during passaging. Iffurther work supports this view, then it will beof some interest to elucidate the mechanism offormation of the recurring variants. Among thepossibilities are specific recombinational eventsbetween the viral genome and host DNA, as wellas the possible presence inAGM cells of a crypticDNA whose replication is helped by SV40.

ACKNOWLEDGMENTSWe are very grateful to Malcolm A. Martin and Norman

Salzman for making laboratory space and materials availablefor some of the experiments reported here. Leonard Rosenthalkindly supplied wild-type SV40 DNA. This work benefitedsubstantially from the critical interest of Edward Kuff, ErnestWinocour, Cary Queen, Thomas McCutchan, and Joseph Pa-pamatheakis. We thank May Liu for her expert preparation ofthe manuscript.

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