Products of complementation between temperature-sensitive mutants of simian virus 40.

Copyright01975 AmericanSocietyforMicrobiology PrintedVol.in15,U.SAA.No.

Products of

Complementation Between

Temperature-Sensitive

Mutants of Simian Virus 40

National InstituteofArthritis, Metabolism,andDigestiveDiseases, Bethesda, Maryland 20014 Receivedforpublication 27 June 1974

Temperature-sensitive mutants of the D complementation group ofsimian virus 40 exhibit delayed complementation. Analysis ofthe thermal stability,

kinetic profilesintemperatureshiftexperiments, and progeny of complementa-tion have led to the hypothesis that delayed complementation is not true

complementation, but the result of a very low level ofleakiness, followed by

phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants atthe restrictivetemperature.

From complementation analyses, tempera-ture-sensitive mutants of simian virus 40

(SV40) have beendivided into classesA, B, C,

BC, and D (3).

MutantsoftheB, C and BCclasses havebeen

designatedas"late" becauseoftheircapacityto

synthesize viral DNA under restrictive

condi-tions (2, 10). Recent studies suggest that these mutants may map within a single cistron (6;

C.-J. Laiand D. Nathans, Cold

Spring

Symp. Quant. Biol., in press). Therefore, the

complementation between B and C mutants

may represent intra- rather than intercistronic

complementation. One of several possible

ex-planations offered for the existence of the BC

classwasthat the virions of suchmutants were

particularly sensitive to heat inactivation (3).

Data presented in this paper exclude that

possibility.

Mutants of the A and D classes have been

designated as "early" on the basis of their

failuretosynthesizeviral DNA uponincubation at the restrictive temperature (2). The A func-tion isrequiredtoinitiate thereplicationofviral DNA molecules (2, 10). DNA synthesis ceases when cultures infected with mutants of the A group are shifted from the permissive to re-strictive temperature. D function, on the other hand, isapparently required priorto viral DNA replication. When cultures infectedwith D mu-tants are incubated in depleted medium at the permissive temperature, andthenshiftedtothe restrictive temperature, viral DNA synthesis continues unabated, provided the incubation has beenallowedtoproceedfor 10to 20hatthe permissivetemperature (2).Inthe course ofthis investigation, itwasobserved thatthelengthof timerequiredatthe permissive temperatureto

overcomethe D virion's defectwasconsiderably reduced when fresh rather than depleted me-dium was used. Various interpretationsof this observation are discussed.

It has been proposed that D mutants are unableto "uncoat" attherestrictive tempera-ture(9), i.e., thatsomevirioncomponent hasto be removed before expression of the viral ge-nome, and that this component remains associ-ated with the genome when D mutant virions infect monkey cells at40 C. This proposal was based on the observations that (i) D101 DNA was infectiousfor oneround ofreplicationatthe restrictive temperature yielding temperature-sensitivevirions; (ii) D101 virions were noncom-plementing when tested after 3 days at 40 C; and (iii) D101 virions failed to exhibit any known early functions at 40 C although they adsorbed to, and penetrated monkey cells

nor-mally (9). The fact that 10 to 20 h of incubation

indepleted medium was required to overcome. the defect exhibited by D mutants (2) neither supported nor negated the uncoating hypothe-sis.

Newdata suggestthat the D mutants mapin that portionofthegenomewhosetranscription

only commences late, i.e., after viral DNA

synthesis has started (6, C.-J. Lai and D.

Nathans, Cold Spring Harbor Symp. Quant. Biol.,inpress; T. E. Shenk, C. Rhodes, P. W. J.

Rigby,andP. Berg, (Cold Spring Harbor Symp.

Quant. Biol.,inpress). These resultsimplythat

(i) the D product may be synthesized late in infection; (ii)therefore, any defect exhibited by D virionsearlyininfectionprobably results from the introduction ofthedefective D product by thevirionsandnot fromtheearly synthesis of a defective Dprotein(in supportoftheuncoating 127

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CHOU AND MARTIN

proposal); and (iii) D mutants should be referred to as late mutants whose phenotype is ex-pressed early.

An apparent contradiction to the proposal that D mutants are defective inuncoating arose from complementation experiments (3). Al-though D mutants failto exhibit complementa-tion after 3days of co-infection with representa-tives of the other mutant classes (3, 9), com-plementation is observed after 2 weeks of co-infection-"delayed complementation" (3). This paper primarily concerns an investigation oftheprogeny obtained after co-infection with D mutants and temperature-sensitive mutants of the other classes. An hypothesis to account for delayedcomplementation is presented.

MATERIALS AND METHODS

Virus and cells. The virusstocks, mutants, and CV-1 cells used have been described(3).Theprimary AGMK cells were supplied by Flow Laboratories, Inc., Rockville, Md., and the BSC1 cellswerekindly provided by D. LeBlanc, and tested by him to verify thatnoinduction of host DNA synthesiswasobserved afterSV40 infection.

Media, plaque, and slant agar assays. The modi-fied nutrient mix F-12 and modified Eagle media (3E), as well as the plaque assay and slant-agar technique forcomplementation, have been described (3, 8). Depleted medium was the 3E5 (5% serum) medium removed after 4 to 5 days from confluent CV-1 cells.

Thermal inactivation of virus. Stocks of the mutant virions were diluted 50-fold with phos-phate-bufferedsaline, (8), andincubatedat50C.

Kinetics of temperature shift. Confluent mono-layers of CV-1 cells in the 24-well (1.6-cm diameter each) plates of Linbro Chemical Co., New Haven, Conn., wereinfected with themutants at multiplici-ties ofinfection(MOIs) of2 to5,after removal ofthe old medium. The viruswasallowedtoadsorb for2h at 33C, and 1 ml of fresh 3E5 mediumor depleted medium per well was added. At appropriate times, cultures were shifted to 40C. In addition, acontrol was prepared in which the adsorption aswell asthe incubation was carried outat 40C. At 96 h postinfec-tion, all of the cultures were frozen, thawed and titeredinFalcon25cm2flasksat 33C.

Products of complementation. Confluent CV-1 monolayers in the 24-well Linbro plates were pre-pared. The medium was removed, and thetwo mu-tants or mutantand wild-typevirions suspendedin 0.2to 0.3mlof3Ewereallowedtoadsorbat 40C for2 to 3 h. Fresh 3E5 (1 ml/well) was added, and the plateswereincubatedfor 96 h at 40Cwhen wildtype was co-infected with the mutants. The monolayers werealmostfully lysedby thistime.Theplateswere incubatedfor 13daysat 40C whencomplementation between twoSV40mutants wereinvestigated. Com-plete lysis was seen by 5 to 6 days in all but the

co-infected cultures containing Dmutants(whichwere not fully lysed even by 13 days).The cultureswere frozen,thawed and titeredat33C. Individualplaques were picked with asterile Pasteurpipette. The agar plugs weresuspended in 2 ml ofphosphate-buffered saline inscrew-cap tubes and storedat 4C. Totest the plaque isolates when wild-type virions were in-volved in theexperiment, 0.2 ml of each suspension wasaddedtofresh confluentCV-1 monolayers in the 24-well plates. After 2 to 3 hof adsorption at 40 C, medium in agar was added, and the cultures were incubated 13days at 40C with feeding at 3days and7 dayspostinfection. After staining, plaque containing wellswerescored aswild type.

The plaque isolates fromthe crosses between two temperature-sensitivemutants werepickedasabove, andstocksweregrowninthe 24-wellLinbroplatesat 33 C. These stockswerethentested for progeny type by the agar-slant complementation technique (3). This technique measures delayedcomplementation, sothatall mutants, including D mutants, complement by this assay. The progeny were tested for com-plementation in each case against both parental types. No wild-type recombinant virus were found amongthe limited number of progeny examined, as judgedbythe fact thatnoneappearedtocomplement bothmutantparental types. The results withA x B, A x C, and B x Cwerecompletelyunambiguous, but theD x A, D x B, and D x C resultswere occasion-ally difficult to interpret. (The agar-slant technique yields ambiguous results in about 10% ofall crosses

[31).

To distinguish D from B or from C progeny, the followingtechnique,basedonthe fact thatDmutants are unableto synthesizeviral DNA at 40C (2), was employed. Duplicate, confluent monolayers in the 24-wellLinbroplateswereinfected withafewdrops of the progeny virus stocks. After adsorption at the appropriate temperature for 2 to 4 h, 1 ml of 3E5 medium was addedto each well, and incubation at theappropriate temperature wasallowed toproceed for 2days. Next, the medium was removed, and 3E medium lacking serum but containing 1 gCi of

["4C]thymidine per ml wasadded, and the cultures

wereincubated attheappropriate temperature over-night. After salt precipitation of sodium dodecyl sulfate-extracted material (5), supernatant fractions wererecoveredaftercentrifugation, andsamples were precipitated with 10% trichloroacetic acid and counted in a Mark II Nuclear-Chicago scintillation counter.

Distinction between the A and D mutants was based upon the fact that Amutant-infected cultures incubatedat 33C for 2 to 3days and then shiftedto 40C, rapidly lose their ability to synthesize viral DNA (2). Therefore, to distinguish the progeny, duplicate cultures were infected with a fewdropsof progeny virus asabove, but both cultures were incu-batedfor 3 daysatthepermissive temperature. At 3 days, one of the cultures was radioactively labeled

with ["4C]thymidineasabove, the otherwasshiftedto

40Cfor 5 h prior to the addition ofthe radioactive thymidine. Incubation was again allowedtoproceed

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129

overnight at the appropriate temperature and o o o o

counted as above.

RESULTS

Tempeature sensitivity of viral particles.

We previously postulated that D mutants are o

defectiveinuncoatingatthe restrictive temper- x x x x

ature (9). This hypothesis suggests that D virions harbor a defective virionprotein which

in turnmight destabilizethevirions at elevated *

temperatures. Representative strains ofeach of o o o

the classes of viral mutants were therefore x x x x

tested for their ability to withstand thermal m C4

inactivation for 3 hours at 50C in buffered

solution (see above). However, D mutant

vi-rions, like wild-type virions and A mutant | x x

virions, were notinactivated underthesecondi- e gD ue

tions (Table 1). m _ c'

-Virions of the late mutant classes were

in-cluded in this study todeterminewhetherany

correlation might exist between the thermal x x x x

lability of the mutants and their ability to LO c4

complement, i.e., to determine whether BC Q 4 _

mutants were

particularly

After 3 h at 50C, B201 was not inactivated,

ii

whereas the titers ofB204 and B8dropped35- x x x x

and 257-fold, respectively; C219 did not de- X X X eee

crease in titer, whereas C244 exhibited a four- o @

fold loss of infectious units; and BC230 de-

ie

creased10-foldintiter, whereasBC245dropped e o

1,000-fold. Thus, no simple correlation can be x x x x

drawn between the heat

sensitivity

,-4

mutant virionsand theirabilitytocomplement. Virions of BC230are inactivated only 10-fold

under conditions where B204 and B8 (both of

which can complement C mutants) are inacti- x x x x

vated35- to250-fold. _

Theseresults areentirelyconsistent with the

notion that the Band Ccistronsencodeone or oo o o

moreviralcapsidproteins and suggest that the 2 _ _ _

failureofBCmutants tocomplementwith Bor . . .x

C mutants is not

simply

hypersensitivity toheat inactivation. The

fail-ure to observe heat inactivation of A and D

mutant virionsdoes not, of course, exclude the x x x x

possibility that the proteins encoded by these e

cistrons arealso virion components.

Kinetics of virusyieldintemperatureshift o o o o

experiments. It has previously been argued | x x

bothon the basis of virion inactivationbyheat a X X 00

(as above) and the kinetics of virus yield in __

temperatureshift experiments, that Bmutants

are defective in a capsid-protein component X o o0

(10). To conduct asimilar analysis with the D

3___

mutants, we have performed experiments

(based on those of Tegtmeyer [10]), involving 8 _ cqc

incubation of mutant-virion-infected cultures

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CHOU AND MARTIN

for varying lengths of time at the permissive temperature, followed by incubation at the restrictive temperature. After 4 days (total incubation time), the cultures were frozen, thawed and titered for virus at the permissive temperature. The profiles for early mutants would be expected to risebefore the profiles of late mutants, assuming, of course, that the shifting ofan infected culturetothe restrictive temperature immediately shuts off further vi-rionsynthesisand/or assembly and thatvirions formed at the permissive temperature remain viable - assumptions which for many reasons

neednotbe valid. Nonetheless, Tegtmeyerand Ozer (11) and Tegtmeyer (10) found that B4 and Bll virionyields increasedinparallel wvith, but were delayed by approximately 12 hfrom, theyields of three A group mutants.

The results oftemperature-shift kinetic ex-periments with the mutants isolated in our laboratory are not entirely consistent with the previous reports (10, 11). All oftheA mutants with the exception of A276 display similar profiles in these experiments (Fig. 1). We

can-notexplain the morerapidappearance of A276 virions except to assume either that A276 is somewhat "leakier" than the otherA mutants and hence is not entirely "shutoff' when the culturesarechangedtothe restrictive tempera-ture, orthat the otherA mutants aresomewhat defective even at the permissive temperature. All of our previous data would tend to argue against the former explanation; first, because thedifferenceinplaque-formingunits (PFU) of A276atthe permissive and restrictive tempera-tures is of the order of 106 (3), and secondly, because DNA synthesisappears to ceasewithin 1 h at the restrictive temperature with this mutant (2). We therefore assume that the A mutants, with the exception ofA276, are par-tially defective evenat 33C.

The kinetic studies of virus yield with the late mutants generate afamilyof curvesrather than auniqueprofile(Fig.1,middle panel). Thiswas not unexpected since suchmutantsmight have different thermal stabilities even at 40C,orbe

differently affected in the synthesis, folding,

turnover, or assembly of capsid protein.

Mu-A mutants

I

20 40 60 80

Time

(hours)

FIG. 1. Kineticsof virus appearanceon temperatureshifttotherestrictive temperatureinfreshmedium. After2hofabsorptionat33C the unadsorbed viruswereremovedbyaspiration,andfresh3E5 mediumwas

addedtoeach culture. Thecultureswereshiftedto40Catthetimesindicated,and all cultureswerefrozenand thawedat96h. The titersarepresentedas apercentageofthe titerobtainedafter96hofincubationat33C. Symbols, left-handpanel:A,A207; V,A209; x,A239;0, A241; T,A255andA,A276.Middlepanel: 0, B201; V,B204;A,BC230; V, BC245; x, C219 andA,C240.Right-hand panel:0,D101;A, D202;V,D222;A,D238; V,D236 and x, D270.

100

L. 10

a)

0I

0 c

(00)

't0.I

D mutants

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tants C219 and B201 reach 10% of their final yield by approximately 42 h atthe permissive temperature prior to the temperature shift, whereas mutants BC245, BC230, and C240 do not attain this percentage of their final yield untilapproximately55h. B204 requires67hat the permissive temperaturebeforetemperature shift toreach 10% ofthe yield foundat 96h at the permissive temperature. Whatever the cause(s) forthe family of curves generated by the latemutantsinthese kinetic experiments, it isclear thatvirions of theB, C, and BCmutants

always appear at, or after the A mutants

(compare left-hand and middle panels,Fig.1).

Similar kinetic experiments performed with

the Dmutantsyieldastrikingly differentprofile (Fig. 1). When the virionsareadsorbed (asinall ofthese experiments) for 2h at the permissive temperature and immediately shifted to the restrictive temperature, the yields after 4 days ofincubation are already over 10%of thefinal yields found when the entire incubation is carriedout atthepermissivetemperature! Sim-ilar results were obtained whether the experi-ment was performedatMOIs of2 to5PFU/cell

oratMOIsaslowas0.1PFU/cell.Yet, the titers

ofseveral ofthese mutantsdifferbymorethan 105 at 33 C versus 40 C by plaque assay (3). Controls inwhich theadsorption aswell asthe incubation was carried out at 40 C gave titers that were 10-fold less than those observed at time 0. By contrast, when the same stocks of viruswereusedtomeasurethe induction of host and viral DNAsynthesis,noviral DNA synthe-sis was observed by 24 h at the restrictive temperature,and theinduction of the host DNA

synthesis wasreduced (2).

One notable difference between the experi-ments ofFig. 1 and those involving the induc-tion ofviral DNA synthesis (2) was that after

adsorption, fresh medium was added in these

experiments, whereas serum-depleted medium was used for the latter. We therefore reex-amined the kinetics ofvirion appearance with

depletedmedium.

The kinetic profiles of the D mutants are

markedly affected by carrying out the

experi-ment in depleted medium (Fig. 2). Indepleted

medium, the titer obtained for the sample

shifted to the restrictive temperature

immedi-ately afteradsorptionwasthesame asthe titer

obtained from thecontrol in which the adsorp-tionwas carried out atthe restrictive tempera-ture, andthetiters ofbothsampleswere atleast 100-fold less than when the D mutants were allowed toincubatefor 4daysatthe permissive temperature. Furthermore, itnow took 14 h at

L..

*-1-C 0

L..

0

(0

0)

'l

_1o-10

I0

0.I

0.01

0

20

40 60

80

Time

(hours)

100

FIG. 2. Kinetics ofvirus appearance on tempera-ture shift to the restrictive temperature in depleted medium. The detailsoftheexperimentare asinFig. 1, with the exception that depleted medium was employed.Symbols: 0,D202; x,D270;V, A209 and A,B204.

thepermissivetemperaturepriortothe shift for D202 to reach 10% of the yield observed when the entireincubation wasallowedtoproceedat thepermissivetemperature.The10%markwas only slightly delayed with the other mutants (compareFig. 1).These results with the virions corroborateourpreviousobservation thatby20 h of incubationat the permissive temperature indepleted medium, D mutantinfections have reached a stage such that subsequent incuba-tion attherestrictivetemperature allows DNA replicationtoproceed unabated (2).

Theaboveresults indicate that theexpression of normal D function is required during the initialstages of infection.They donothowever,

distinguish between the possibilitiesthat (i) D

encodesadiffusible proteinsynthesizedearlyin infection;(ii) Dencodesanondiffusibleprotein *131

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CHOUAND MARTIN

synthesized earlyininfection; and(iii)late in in-fection D is translated into a structural com-*ponent of thevirion andthattherelease of this protein fromviral DNAuponsubsequent

infec-tion isrequired for the expression of viral func-tions. Possibility (i), seemingly eliminated by the observation that D mutants did not

com-plement after3 days of co-infection with other temperature-sensitive mutants, must again be entertained because Dmutantsdocomplement

onprolonged incubation. Inthiscase, normal D

product shouldact in trans. In the latter cases

(ii) and (iii), complementation experiments at the restrictive temperature between wild type and the D mutantsshould indicate that D

mu-tants are cis acting, i.e., the progeny should

be predominantly of the wildtype.

Products of complementation with

wild-type andtemperature-sensitive mutants. To

determine if Dproductwerecis acting, cultures

were co-infected with wild-type and mutant virions. Theprogenyofsuch co-infections were

plated at the permissive temperature, and plaqueswere pickedatrandom,suspended in2 ml ofphosphate-buffered saline and then tested fortheir ability to grow at the restrictive tem-perature.

Dmutantsarecis acting (Table 2). Compari-sonofthe ratios ofmutant towild-type particles

inthe inputversusprogenyviruspools (Table2,

lastcolumn) indicatedapreferential replication

ofwild-type particlesoverthe Dmutant parti-clesuponco-infectionattherestrictive temper-ature. Inthecontrol experiments withA,B, BC and C mutants, wild-type progeny

approx-imated the input ratio ranging from50% (B204

x wild type) to a sevenfold increase (A209 x

wild type). However, the same ratio was

in-creased 17- and 83-fold when D mutants were

employed. The statistical significance of the

precise increase is less with the Dmutantsthan with the controls (i.e., the ratio might possibly be much higher), since 25 to 50%o of all the

progenyinthe A, B, BC, and C mutant experi-ments were mutant type, whereas only 3of 95

D202 x wild-type progeny and2 of75D270 x

wild-type progeny were of the mutant type. Furthermore, the final yields of the Dmutants

among the progeny (approximately 3% of the

total PFU, or 6 x 106 PFU) were very closeto the inputs for these mutants. Thus, the data suggest that Dmutant replication proceeds, if atall, ata reducedrate upon co-infection with

wild type. Other classes of mutants behave normally upon co-infection with wild type at therestrictive temperature, i.e., they are

com-plemented by wild type and appear at nearly theinput ratio.

Products ofdelayed complementation. The aboveresultssuggestthat Dfunctioncannotbe supplied in trans by a co-infecting wild-type

virus particle. Consequently, one would again

expect D mutants tobe noncomplementing as

they are in the usual assay with CV-1 cells

whereco-infectionis allowedtoproceed for only 3days (3). To determineifthe hostcell hadany

effect on the complementation, we repeated

these experiments with primary AGMK cells (Table 3) andBSC-1 cellswhich donot exhibit stimulation of host DNA synthesis upon

infec-tionby SV40(Table 4),andagain observed that Dmutantsfailedtocomplementothermutants

upon co-infection for 3 days at the restrictive temperature.

Wewerepreviously unabletoaccountfor the complementation observedwith the Dmutants when co-infection wasallowed to proceed for 1 to 2 weeks. Asexplained more fully below,the

observation that Dmutants are cisactingupon

co-infection with wild-type virions led to the

TABLE 2. Productsofcomplementationbetweentemperature-sensitivemutantsofSV40and wild typeatthe restrictive temperature

ParentalVirionsa Progenyvirions

Mutant strain Input(I) No. Progeny (P) RatioI/ratioP

ratioc examined ratioc

A209 2.8x105 1.8 1.2x 107 47 0.27 6.7

A276 2 x105 1.3 1.4x 107 48 0.55 2.4

B204 105 0.63 107 40 0.5 1.3

BC230 3 x10' 1.9 107 29 0.93 2.0

C244 10' 0.63 107 46 0.24 2.6

D202 8x104 0.5 2.4x107 95 0.03 17.

D270 4x 105 2.5 2.2x 107 74 0.03 83.

aEach culture contained approximately 105 cells and received1.6 x

10'

cMutant virions towildtype.

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TAm 3. Vinuyields after co-infection ofAGMK cells for 3 days at40 C

PFU/ml Mutants

A209 B201 C219 D202

A209 32 2.4x10 4x 104 200 (185)a (17) (1.8) B201 1.3 x 10' 8x104 1.6 x10'

(22) (1.2)

C219 2.4x10' 1.6x103

(0.7)

D202 80

aNumbers in parenthesis represent the titer di-vided by the sum of the titers of the two parental strains alone.

TABz 4.VirusYields after co-infection ofBSCI cells for3daysat 40C

PFU/ml Mutants

A209 B201 C219 D202

A209 40 7.4x 10' 10' 2x102

(460)a (17) (0.8) B201 1.6x 10' 1.4x10' 3.6x 10'

(18) (2)

C219 6x10' 7x103

(1.1)

D202 2.4x102

aNumbersinparenthesisasinTable 3.

hypothesis that delayed complementation was

the resultofa rareproductiveinfection followed

by phenotypic mixingofthe progeny particles.

A prediction of this hypothesis is that the

progeny ofdelayed complementationbetweena D mutant and any other co-infecting mutant virion should consist predominantly ofD

mu-tant particles - the inverse ofthe result

ob-tained when D mutants co-infect wild-type

in-fectedcultures.

Cultures were co-infected with variousA, B,

orCmutants atMOIsof1to2and D202at an

MOI of approximately 0.2 to 0.5.

(This

necessary since at higher MOIs ofD202, only

D202 progeny wereobserved.) Afterincubation

for 13 days at the restrictive temperature, the

cultureswere frozen, thawed and titeredatthe

permissive temperature. Individual plaques

were picked, and stocks were prepared from

thematthepermissivetemperature. The

prog-eny viral types were then distinguished as

follows; all were testedbythe agar-slant

com-plementation technique previously described

(3). In addition, the D mutants were distin-guishedfrom B and C mutantsbytheirinability

tosynthesizeviral DNAafterincubation atthe

restrictive temperature. The data on 20 of 90 such analyses are presented in Table 5. D mutants were distinguished from A mutants by their ability to synthesize viral DNA upon shifting to the restrictive temperature after 3

days ofincubation at the permissive

tempera-ture (data from the first 10 of 43 progeny

examined in Table6).

The progeny resulting from delayed com-plementation between D mutants and other mutant types are predominantlyof the D class

(Table 7). When combinations ofA, B, and C

mutants were used toco-infect a monolayer of

CV-1 cells at the restrictive temperature, the progeny mutant yields reflected the parental input. This isdemonstrated bythe fact that the ratios ofparentaltoprogeny mutant types was

approximately unity(Table 7). This ratio,

how-ever, varied between3.5and 22inexperiments involving D202, indicating a preponderance of D mutants among the progeny.

DISCUSSION

Examination of the thermal lability of the virions of various temperature-sensitive SV40 mutants revealed that members of the late groups,B, C, and BC,areunstable. No

correla-tion between thermallability and

complemen-tation behavior was observed among the late mutants. The BC mutants do not therefore

TABLE 5. Analysisof representative plaque isolates from the D202 x C219 or D202 x B204

complementationexperiments D202 xC219' D202 x B204a Plaque

Counts/min

Counts/min

isolate Mtn

type type

40C 33C inferred 40C 33c inferred 1 0' 520 D202 2,150 780 B204 2 400 700 C219 2,120 720 B204 3 0 410 D202 2,550 910 B204 4 29 430 D202 1,720 420 B204

5 0 540 D202 45 300 D202

6 0 175 D202 2,050 750 B204

7 250 820 C219 81 400 D202

8 0 390 D202 1,880 640 B204 9 41 460 D202 2,310 760 B204

10 0 310 D202 63 210 D202

aDataonthe first10of46progeny exami-ned in the D202 x C219 cross and the first 10 of 44 progeny examined in the D202 x B204 cross.

bNumbers represent the counts per minute in the supernatantfractions after extraction and salt precip-itation by the method of Hirt (5). The counts/min of mock-infected controls have been subtracted. They were192count/min at 40 C and 144count/min at 33 C.

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CHOUAND MARTIN represent a class of mutants which are

exqui-sitelysensitive to thermal inactivation. A and D mutants were not inactivated underthe condi-tionsused.Whereas these results areconsistent with the late mutants corresponding to the majorcapsid protein(s),they donotexclude the possibility that the proteins encoded by the A and Dmutants are also structural components of the virions. Indeed, recent mapping data suggest that D mutants map in the lateregionof the genome (6; C.-J. Lai and D. Nathans, in TABLE 6. Analysis of representative plaqueisolates from the D202 x A209complementation experimenta

Counts/min

Plaqueisolate Mutanttype

40 C 33C inferred

1 466 180 D202

2 530 200 D202

3 650 200 D202

4 740 350 D202

5 100 300 A209

6 550 190 D202

7 980 440 D202

8 590 150 D202

9 420 150 D202

10 50 270 A209

aCounts per minute less mock-infected controlsare

presented as inTable 5.Themock-infected controls had 82counts/minat 40C and84counts/minat 33 C.

Thecountsper minute forstrainD202at40Cand33 Cwere800and360,respectively,and for strainA209 were 70and 390counts/min, respectively.

press;T. E. Shenk, C. Rhodes, P. W. J. Rigby,

and P. Berg, in press).

The kinetic experiments in which mutant-infected cultures were incubated for varying

lengths of time at the permissive temperature before shift to the restrictive temperature dis-tinguish among the early mutants, in addition todistinguishingtheearlymutantsfromlate. D function was required only during the earliest

stagesof virion infection.

Interestingly, serum stimulation seems to partially overcome the defect exhibited by D mutants. Theprofiles of the virion yields in the kineticexperimentsweredisplacedtowardlater

timeswhendepleted mediumwasused in place

of fresh medium.Possibly,someserum-induced

proteaseof the host isnecessaryforthe uncoat-ing of virions, and D mutants are altered in

their susceptibility to the protease. Whatever the mechanism by which fresh medium

over-comes the defect exhibited by D mutants, we

wishtoemphasize that the properterminology

todescribe this phenomenon is "suppression," notleakiness. Thedifference in the titer of D202 at 33C and 40C is greater than 7 x 105 (3).

This means that when a monolayer of 3 x 106

monkeycellswasinfectedatMOI = 1,lessthan

five plaques wereobserved (3), although

statis-tically 10' cells were infected with 5 or more

PFU.The fact that after4daysin fresh medium the viral yield at 40 C was 10% of that at the permissive temperature means that fresh

me-dium can suppress the D mutations.

Suppres-TABLE 7. Productsofcomplementation betweendifferent mutantstrainsof SV40

Parental virions Progenyvirions

Mutant strain 1xMutantstrain2

Progen2

Input(l)

Infecting Infecting ratio PF plaques ratiob

Strain PFUa Strain PFU examined

A209 7 x104 D202 2x104 3.5 6x 105 43 0.39 9

C219 1.25 x106 D202 2x 104 6.3 106 46 0.28 22

B204 106 D202 2x 104 5.0 3 x 106 44 1.44 3.5

A209 7 x104 B204 10 0.7 4 x 106 50 0.47 1.5

A209 7x 104 C219 1.25 x 105 0.56 4 x 105 59 0.84 0.67

B204 105 C219 1.25x 105] 0.8 6 x 106 31 2.9 0.3

aThe titers of all of theparental stocks with the exceptionofC219 were checked atthe same time.

bMutant1/mutant2.

cAnotherwayofpresenting the datainthis column would be to point out that with the A mutant as strain 1

and the BorCmutantasstrain2,theI/Pratiosare1.5and0.67(average = 1.1)ascomparedwith avalue of 9 with the Dmutant asstrain 2.Similarly,with theB mutant as strain1and the A or C mutant asstrain 2, the ratios are0.67(1/1.5)and0.3(average =0.49), respectively,ascompared with a value of 3.5 with theD mutant as strain 2. Finally, with the Cmutant as strain 1 and theA orB mutant as strain 2, the I/P ratios are 1.5 (1/0.67)and3.3(1/.3)(average = 2.4)versus aratio of 22with the D mutant as strain 2. Combining the data in thisway, the averageI/Pratio forthe combinations excluding D202is 1.3[(2.4 +1.1 + 0.5)/3],andthe average including D202is 11.5 [(9 + 22 + 3.5)13],or adifference of ninefold.

134 J. VIROL.

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sion of certain phage mutations by nutrient factors is welldocumented (1).

When cultures were co-infected with D mu-tants and wild type under restricting condi-tions, the progeny were predominantly wild type, suggesting that the D function cannot be complementedin trans.The cisaction ofthe D mutants is most easily explained byone of two mechanisms: either (i) the protein encoded by

the D cistron is a virion component introduced with the viral DNAupon infectionand serving to prevent the expression ofall viral functions until released from the genome; or (ii) the D cistron is transcribed and translated from the genome soon after infection, and the protein synthesized remains associated with the paren-tal DNA. Whereas bacterial models for both mechanisms exist (7; B. Hoffman and M. Le-vine, personal communication), the former seems morelikelyineukaryotic cells where itis presumed that viral transcription and transla-tion are compartmentalized inthe nucleus and cytoplasm, respectively. We have been unable to find anyother plausible explanation forthe cis behavior of the D mutants. Whether the product of the D mutant were required in stoichiometricorcatalyticamounts,the ratio of D mutant to wild-type progeny should remain the same asthe parental ratio, provided the D product were a diffusible substance. While we cannoteliminate the theoreticalpossibilitythat upon co-infection wild-typevirions exclude the

adsorptionof D virions(butnotA,B,etc., even

though D mutants adsorb normally by

them-selves [9]), orthat the DNA of the D mutants contain a temperature-sensitive site, we find these explanationsunlikely.

Since the D cistron might encode a virion component (9) and possibly even one

synthe-sized late in infection (6), we propose the

followingasthebasisfordelayed

complementa-tion: ifDmutants are veryslightly leakysothat

perhaps 1in 10'to106cells infectedat anMOI

of 2 leads to a single round of infection, a low level of progeny D mutant virions will be re-leased into the medium. We have previously demonstrated thatby2 to 3dayspostinfection at the restrictive temperature, 80% of all cells which have been infected witha Dmutantlose their ability to lead to a productive infection upon subsequent incubation at the permissive temperature (9). Thatis, virion-infected cells in which virion replication cannot occur as the result of atemperature-sensitive lesion tendto become "cured" attherestrictivetemperature. Thus, thenewD virionsreleased from the 1 in 10'to101 infected cellswill,withhigh

probabil-ity, encounter cured cells after their release. However,the probability of infection will again beonly 1 in 10'to10,and since the burst size is less than 10' virions per cell, even prolonged incubation will not lead to an extensive infec-tion. When the same event takes place during co-infection withanother mutant, however, the result will be quite different. Only in the rare cell in which the D mutantreplicates will any virus be produced. But because this cell is co-infected with a different mutant, of classX, both will replicate. However, the virions re-leased will be phenotypically mixed, i.e., some ofthe D mutantvirions will haveawild-typeD cistron protein provided bythe other tempera-ture-sensitive mutant. Since most ofthe host cells for the second round of replication are effectively cured, anumber of types of infection arepossible. Single infectionsby the normal D virion, the X virion, and the phenotypically mixed X virion, will all be unproductive. Even most double infections by both mutant types (those in which the D virion contains the defective D-encoded product) will be nonpro-ductive. However, those cells receiving a phe-notypically mixed D virion will be productive for a single round, and those cells receiving a phenotypically mixed D virion and the Xvirion will beproductive and providemore phenotypi-cally mixed D virions.

The net effect will be that "complementa-tion" will not occur until many rounds of replication have taken place.Inaddition, there will beanincreaseinthe ratio of D virionstoX virions but not to an overwhelming degree.

Thosecellsinfectedbyonlyasingle,

phenotypi-cally mixed D virion will go through a single

round of replication and will release normal D virions. But the normal D virions released will

not be capable of efficient reinfection. This

prediction was tested, and D mutants indeed

prevailindelayedcomplementation byabouta

factor of9 (Table 7).

We therefore conclude that delayed com-plementation is nottruecomplementation, but the combined effect of a very low level of leakiness and phenotypic mixing of theprogeny D mutants. Inherent in this conclusion is the assumption that the D-encoded protein is part ofthe normal virion and that the Dvirions are blockedinuncoatingattherestrictive tempera-ture.Whether theDproteinissynthesized early orlate after infection cannot be deduced from these experiments.

While theevidencesupportingourconclusion concerningthe nature ofdelayed complementa-tion is only circumstantial, the datapresented 135

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CHOU AND MARTIN

in conjunction with the otherknown properties ofD mutants (3, 4, 6,9) areentirelyconsistent

with the proposed hypothesis.

ACKNOWLEDGMENTS

We thank J. L. Rosner, M. Singer, R. Saral, and D. LeBlanc for theirhelpful criticisms of the manuscript and Sylvia Bailey for technical assistance.

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5. Hirt, B. 1967. Selective extraction of polyoma DNA from infectedmousecellcultures. J. Mol. Biol. 26:365-369. 6. Lai, C.-J., and D. Nathans. 1974. Mapping

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9. Robb, J. A., andR.G. Martin. 1972.Geneticanalysis of simianvirus 40.III.Characterization ofa

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