Application of Arrhenius kinetic theory to viral eclipse: selection of bacteriophage phi X174 mutants.

0022-538X/81/080510-09$02.00/0

Application

of Arrhenius Kinetic

Theory

to Viral

Eclipse:

Selection of

Bacteriophage

4X174 Mutants

N. L.INCARDONA

DepartmentofMicrobiology&Immunology, University ofTennessee Centerforthe Health Sciences, Memphis, Tennessee 38163

Received17February 1981/Accepted15April 1981

Analysis of the

bacteriophage

4X174

eclipse period

in terms of Arrhenius

kinetic theorysuggeststhefollowing hypothesis: mutantsshould exist with two

concomitant

physiological

characteristicsastheir

phenotype.

Theseare aneclipse

rate lower than that of the wild type at

permissive

temperatures for plaque

formation and an

eclipse

rate toolow atlower temperatures to

permit

plaque

development. Thus, enrichment ofa

mutagenized

virus

population

formutants

thatfailto

eclipse during

ashort

period

at

permissive

temperatures shouldyield

eclipse mutants with the cold-sensitive

(cs;

nonpermissive

temperature, 25°C),

andnotthetemperature-sensitive (ts; nonpermissivetemperature,

420C),

plaque

phenotype.Inseveral

trials,

thefrequency of thecs

phenotype

inthe

population

increased from less than 0.2%to between2 and 4% after the enrichment step,

whereasthe

frequency

of thets

phenotype

remained

unchanged (less

than0.2%).

Moreover,80% of thesecs mutantshave

eclipse

ratesthatare3-to40-fold lower

than that ofthe wildtype atboth

370C

and

250C.

Thesuccessful

application

of

the Arrhenius theory to 4X

eclipse

may

provide insights

into the molecular

mechanismwhereby the

4X174

genomeisdelivered into the hostcell. Since the

eclipse

kinetics of other nonenveloped viruses are similar to those of

4X174,

kinetictheorymaybe

broadly applicable

intheselection and characterization of

viraleclipsemutants.

Since viral

nucleocapsids

areconstructed from

protein subunits and nucleic acid molecules,

noncovalent interactions makeasignificant

con-tribution to the structural stability of viruses

and

play

an

important

role in their

replication.

The

importance

of noncovalent bonds in virus

assembly has been

extensively

documented (3),

and the principles that govern their formation

arebeginningtoemerge (21). However, the

dis-ruption of these interactions must also play a

part invirus

replication

atthepoint where viral

genomesgainentryintohostcells.Yetevidence

for theprecise role played by noncovalent

inter-actions

inthis

delivery

of viralgenomesremains

very sketchy

(1, 6, 12-15, 18, 27, 29, 30). This is

partlydue to the dearth ofadequate means for

interrupting theprocess todetermine the

num-berandnatureof theinternediate steps. Advan-tage has been taken of the fact that, at low temperatures, many viruses adsorb but fail to enterthe

eclipse

phase (17, 18, 22, 24) and that some viruses require

metabolically

active cells

forcompleteentryintothe cytoplasm (4, 16, 19).

However, there isa need for selectiontechniques

specificforconditionallylethal mutants that are

defectiveinthe steps subsequent to adsorption

and associated with the entry into the eclipse

phase of the replication cycle. Since the viral

genomeis dissociated fromatleastsomeof the capsid proteins during eclipse, it is likely that

mutants with altered noncovalent interactions

will be generated bysuch procedures.

Structural and kinetic studies onthe eclipse

ofbacteriophage

4X174

provide a background

for thedesign of selection procedures that are

specific for eclipsemutants. One can single out

for study thesteps associatedwith the loss in

infectivity that occurs at the beginning of the

eclipse period bysynchronizing the infection in

starvation buffer at

150C

and shifting to a higher temperature toinitiate theentry into theeclipse

phase.Moreover, since EDTA partially disrupts

the outer wall of the cell, viral nucleocapsid structures canberecovered from infected cells

between the attachment and complete DNA

injection steps, inclusively. Use of these

tech-niqueshasrevealed that at the low temperature,

4X174

adsorbs to starved

cells

but does not

begintoeject itsDNA. Therefore, the recovered

virusremainsinfective. Shifting thesevirus-cell

complexesto temperatures above

200C

in

star-vation bufferresults in the partial ejection of the

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VOL. 39, 1981

DNA with the concomitant loss ofinfectivity

that defines thebeginning of theeclipseperiod.

Addition of nutrientstothe starvation buffer is

required for complete injection of the viral ge-nome (22).

The kinetics ofthe lossin infectivity

associ-ated withthe partial ejection process (for

con-venience, termed the eclipse reaction) can be

followed by diluting virus-cell complexesmade

at

150C

into starvation bufferat higher

temper-aturesand then stopping the reactionbydiluting

into EDTAat

00C

(23). Therateof thiseclipse

reaction for wild-type

OX

and a cold-sensitive

(cs) eclipse mutant, cs7O, has been examined

over awide temperature range. At the

nonper-missive temperature

(2500)

usedtoselect thecs

mutants of

OX,

cs7O hasaneclipse ratethatis

essentiallyzero.This is thedefectivestep that is

responsible for itscsplaque phenotypesince its

eclipse and latent periods, as well as its burst

size,arethesameasthose of thewildtypewhen

virus-cell complexesmade at

370C

in starvation

bufferareshifted tonutrient broth at

250C

to

complete the infection (28). However, the

un-usualpropertyof thismutantis thelowereclipse

rate at all temperaturesbetween 30 and

400C,

the permissive range (8). Analysis of these

ki-netic properties in terms of Arrhenius theory

providesatheoretical basis for the lowereclipse

rate of cs7O at permissive temperatures.

Fur-thermore, since the

theory requires

that such

eclipse

mutantshave the cs

plaque

phenotype,

specific procedures

have beendevised for

select-ing thesemutants.

The

cs7O mutation is believed

tobe ingeneF(26).

MATERIALS AND

METHODS

Viral and bacterial strains. Wild-type 4X174

originallycamefrom thelaboratoryof R. L.

Sinshei-merandwassubsequentlyplaque purified.High-titer stocksareroutinelygrownbyinfecting25-mlcultures ofEscherichiacoliC/1atacelldensityof

108/ml

with randomlyselectedsingleplaques. Lysisisinhibitedby the addition of3mlofsterile2MMgSO4atthetime of infection (8). After3hof incubation at370C,the cellsarecollectedbycentrifugation,suspendedin5ml ofpH9.5saturated sodium boratecontaining0.05M EDTA, and storedat2°Covernightforlysis.Stocks preparedin thismanneralways give plaquesat420C. E. coli C/1 and C/+X have been previously de-scribed(11).

Plaque assay. Theprocedure is thesame asthat previouslydescribed(10),withmodificationstoreduce the size of theplaquesforcountingwithaNew Bruns-wick BiotranIIAutomatic ColonyCounter. For the wild type, the top agar concentrationis1.1%;1.0mlof alate-log-phaseculture of E. coliC/1is used perplate,

and theplatesareincubated for1hat370Cand then overnightat230C.With thecsmutants,only0.2ml of

SELECTION OF ECLIPSE MUTANTS 511

bacteria is used with 0.7% top agar. The plates are incubated at 37°C until plaques are faintly visible (between 2 and 4 h) and then shifted to 23°C for overnight incubation.

Chemical mutagenesis. Several high-titer stocks ofwild-type4X174were diluted to1010PFU/ml and mutagenized with hydroxylamine as outlined by Tess-man (31). After incubation at370Cwith 0.10 M hy-droxylamine for7 min,the survivors (20%) were di-luted at least 100-fold in ice-cold medium to quench the chemical mutagenesis. Then an appropriate vol-umeof the diluted survivors was added to a culture of E. coliC/1 at a multiplicity of less than 0.01. The conditions of the infection and lysis were identical to those described above for the high-titer stocks. The low multiplicity was used to reduce the probability of multiply infected cells, and an additional 50-fold dilu-tion ofthe mutagen was also accomplished at this step.

Selection ofmutants. The above-described ly-satesof the survivors' progeny(10'0to 10'lPFU/ml) werediluted with starvation buffer (23) to 109 PFU/ ml and incubated at370Cfor 10 min. At thattime, a 0.1-ml samplewasadded to 0.9 ml of E. coli C/1 in starvation buffer at 150C and stirred for a 30-min adsorptionperiod. The cellsweregrown to2 x108/ml inKC broth (23), washed once with starvation buffer, and concentratedto109/ml.Atthe endof the adsorp-tion period, the reaction flask was transferred to a waterbathshaker at370Cfor a 10-min eclipse period, after which the reactionwasquenched by a 100-fold dilution into ice-cold EDTA elutionbuffer (22). After 10min, the cells were removed by centrifugation at 12,000 xgfor 10 min, and the supernatant solution wascarefullyremovedand stored at20C.At a conven-ienttime, the solution wasplatedonE. coli C/1 at

370C, and individual plaques were transferred with

sterile toothpicksto platesto testfor the cs (2500)

and ts (4200) phenotypes. Eachmutant was plaque

purified several times and retested for thetwo phe-notypes.

Invitro transformation reaction.Virus stocks with titers between5x109 and1x 1010PFU/mlwere diluted 100-fold into starvation buffer maintained at 37.00 + 0.010Ctoinitiate the transformation to the form which adsorbsat low temperatures. At various timeintervals, sampleswere withdrawn and diluted 100-fold into tubescontaining E. coliC/1 in starvation bufferateither2 or150Cataconcentration of2to3

x109/ml.Aftera15-minadsorptionperiod,each

sam-plewascentrifugedfor 1 min inanEppendorf3200 centrifuge. The supernatant solutions were main-tained in ice until the concentration of unadsorbed viruswas measuredbyplaque assay.The zerotime pointwasobtained bydilutingthestock into buffer maintainedinice. Asamplefrom the zero timeflask and thereaction flask(afterthe lasttimepointwas

taken)wasalso diluted intostarvation buffer without cellsas a measureof thermal inactivation of the virus. Nosignificantinactivationwasdetected in anymutant

examined,sothesetwovalueswereaveragedandused tocalculate theunadsorbed fraction.

Invivoeclipsekinetics. Theprocedureof New-bold and Sinsheimer (23) wasused withtwo

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cations. First,thevirus-cell complexes werenot

col-lected after theadsorptionstep.However,thelevelof

unadsorbed viruswasalwayscheckedby

centrifuga-tion of asample atthe endof theadsorption period

and maintainedbelow25%byanappropriate choice

of thepreincubation timeat37°Candadsorptiontime at15°C.Second,theeclipsereactionwasinitiatedby

either a 10- or 100-fold dilution of the adsorption

mixture. The corrected rate constant for the faster component of theeclipserate curve wasobtainedas previously described(8).

RESULTS

Theoretical

analysis.

Historically,

Arrhen-ius kinetic theory was the first successful

at-tempt to account for the fact that chemical

reactionratesincrease with

increasing

temper-ature. The

relationship

between the rate

con-stant, k, and the absolute temperature, T,

(ex-pressed inits

logarithmic form)

is:

Ink =InA_-E ()

R\T

Here

E.,

the activation energy,isobtained from

theslope of the linear

plot

of

log

kversus

1/T,

and the preexponential

factor, A,

comes from

the

intercept

with theyaxisatinfinite temper-ature (R is the ideal gas constant).

Although

recenttheories haveattemptedtorelate thetwo

fundamental parameters,EaandA,tothe

struc-tural properties of atoms and

molecules,

the

above form will be sufficient for

application

to

viral

eclipse.

One needsonlytorealizethat

nu-mericalvalues for thesetwoparameters are

de-terminedby the molecularstructureofthe

reac-tantsandthe activated

complex,

aswellas the

mechanism ofthereaction. TheArrhenius

equa-tion, therefore, reveals the contributions that

thesestructuralproperties maketothereaction

rate.

Inthe caseof

4XX174,

the kinetics associated with the

eclipse

period of

wild-type

virus and the

eclipse

mutant,

cs7O,

can be analyzed in

termsof theArrhenius equation.Concentrating

firston theviral

eclipse

step, the

parallel

solid

lines in Fig. 1 are the Arrhenius plots for the

wild-type andcs7O eclipse reactions,whose rate

constantshave beenmeasured between 30 and

400C (8). For the purpose ofthis analysis, the

experimental

lines

have beenextended over a

widertemperature rangetoinclude the nonper-missive temperatures of the ts and cs plaque mutants of

OX.

IntermsoftheArrhenius

equa-tion, the cs7O mutant eclipse reaction has the

sameactivation energy (slope) as does the wild

type,but it has athreefold-lower preexponential

factor (y intercept) (8). This means that the

threefold-lower eclipse rate ofthe mutant

ob-served in the permissive temperature range

should also

apply

at

250C,

the

nonpermissive

temperaturefor

plaque

formation(Fig. 1).

Also

important

totheanalysisarethe

similar-ities between cs7O and wild-type intracellular

development after the eclipse step. Since the

mutantand the wildtypehave thesameeclipse

and latent

periods

at

400C,

and at

250C

whena

shift from the permissive temperature occurs after the eclipse reaction (28), no intracellular

stepsaftercomplete DNAinjectionare

signifi-cantly

impairedin the mutant. Thus,the

rate-limiting

stepintheintracellulardevelopmentof the mutant is the same as that for the wildtype

at all temperatures between 40 and250C, and

only

oneArrheniusplotisrequiredtodefine the

temperaturedependenceofits rate(dashedline

in Fig. 1). A comparison of the rates for the

intracellular and eclipse reactions is necessary

tounderstand why the csplaquephenotype is

theoretically linkedtothelowereclipserate at

permissive temperatures. Theactivation energy

for thisintracellularreaction wasestimatedfrom the three- to fourfold increase in thelength of

the eclipse period between 40 and 250C

(28).

However,sincethereis not anabsolute valuefor

the rateof this reactionat even onetemperature,

one mustpostulateavaluefor the

preexponen-tial factor. This will determine where the plot

interceptstheyaxis and theposition oftheplot

in

Fig.

1relative to the twoplotsfor the

eclipse

reactions.

The value for A can beestablished bymaking

onesimplepostulate: the intracellularreaction

is the

rate-determining

step for plaque

forma-tion. If the eclipse rateisgreater thantherate

oftheintracellular reaction, plaques will form.

If it isless, theuninfected cells surrounding the

infectedcell canestablishsurviving colonies

be-fore the virus burst, and no plaques will be

visible. Therefore, the rateof the intracellular

reaction must be less than both the cs7O and

wild-type eclipse rate at 370C since both give

plaquesatthis temperature. However,at

250C,

therateof theintracellular reaction must beless

than only the wild-type eclipse ratesince only

the wildtype givesplaquesatthisnonpermissive

temperature. Thisreasoningthenrequiresthat

the Arrheniusplotfortheintracellular reaction

inFig. 1must crossbelowbotheclipse reaction

lines atthe

370C

ordinatebutbetweenthe two

eclipse linesatthe250C ordinate,thus

establish-inganapproximate value forA.

Tosummarizethe experimental basis of Fig.

1, the twoArrheniusplots forthecs7Oand

wild-typeeclipse reactionswereestablishedfrom

ex-perimentally measuredrateconstantssothat no

assumptionsarerequiredtoestablish the values

for

E.

and A (8). Anestimateforthe activation

energyof therate-limitingreaction for the

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SELECTION OF ECLIPSE MUTANTS 513 Temperature (OC)

k

(minf')

I/T

FIG. 1. Theoretical basis for the cs plaque phenotype of4X174 eclipse mutants. The solid lines are extrapolations of the experimentallydeterminedArrhenius plots for the eclipse reactions of the wild type and thecs7O eclipsemutant(8).The dashed line is a postulated Arrhenius plot for the rate-limiting intracellular reaction based on the measured eclipse periods at 40 and 25°C (28). Since thecs70 eclipse rate is greater than therateof the hypothetical intracellular reaction at 37°C, plaques areformedat this temperature. However, at25°C,theeclipse reaction ofcs7Obecomes rate limiting, and the uninfected cells outgrow the virus so that theplaquesare notvisible. wt, Wild type.

cellulardevelopment of bothmutantand

wild-typevirus wasobtainedfromexperimental data

at twotemperatures (28).Only the

preexponen-tial factor for this intracellular reaction has a

postulatedvalue, based onthe hypothesis that

therateofintracellular

development

of the virus

is therate-determiningstepin

plaque

formation.

Test for

predicted

plaque

phenotype.

Since thevalidity of

applying

Arrheniustheory

to 4X

eclipse

rests on a

simple,

but

crucial,

assumption,one must find logicalconsequences

that can be tested by experimental data. The firstprediction can beillustrated byan exami-nationofFig. 1in lightof the abovepostulate.

Mutantsshouldexist witheclipseratesthatare

lessthanwild-type valuesatpermissive

temper-atures, such as

370C.

For this to occur, the mutation mustresultinachangeinEa orA or

bothsuch that theArrheniusplot forthemutant

eclipse reactioncrossesthe

370C

ordinate below

thewild-type

eclipse

line. Those thatcrossboth

the 37 and

250C

ordinateabove the plot for the

intracellular reaction will give plaques at

all

temperaturesbetween25and

420C

andwillbe

indistinguishable fromthewild type in the

typ-icalscreenfor thecsandtsplaque phenotypes. However,thosethat crossabove the

intracellu-lar lineat

370C

butbelowit at

250C

mustalso

crossthe

420C

ordinate above theintracellular

reaction

plot.

Thus, these latter mutants will

yield

plaques

at

370C

and have the cs,butnot

thets,

plaque phenotype.

Those thatcrossboth

the37and

250C

ordinate below theintracellular

line will be lost if 37°C is used to obtain the

plaquesforscreeningattheothertwo

tempera-tures.

A testofthispredictionisa

procedure

which enriches a mutagenized

population

of 4X for

eclipsemutantsthat formplaquesat

37°C,

fol-lowedbyascreenforboth thecsandts

pheno-types. If the

prediction

is

valid,

thereshouldbe a

larger

increase in the

frequency

of the cs

VOL. 39, 1981

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phenotype in the population. Twotrials of the following schemewereperformed.After

chemi-cal mutagenesis, the survivors weregrownata

lowmultiplicitytoexpressthemutantgenotype in the capsid proteins. Anenrichment for

mu-tantswithalowereclipseratewasaccomplished

byadsorbing the mixed population ofprogeny

onto sensitive cells at 150C and shifting the

reaction flaskto37°Cfora10-mineclipseperiod.

The complexeswere then diluted into ice-cold

EDTA-borate elution buffer to dissociate the unecipsed virus fromthe cells. Over 99% of the wild-type virus, and those mutants with an

eclipse ratethe same asthat of the wild type, areinactivated in thistime interval at370C (8), butasignificant fractionof thepotentialeclipse

mutant population is still infectious due to a

lower rate of eclipse. After centrifugation to

remove all cells that may contain completely

injected

4X

DNA, the unecipsed population

wasthen screened for thecsandtsphenotypes by platingat370Candtesting individual plaques at25 and420C.The results(trialsAlandA2 in Table 1)clearly showatleasta10-foldincrease

in only the cs phenotype. In a third trial, the

enrichment stepwas a45-mineclipseperiodat

250C (thereasonforselecting thistemperature willbe discussedbelow), followed bya screenof plaques obtainedat370Cfortheplaque pheno-types. Since the desired mutantshave alower

eclipseratethan the wildtypeat250C (Fig. 1), theycanbe enriched in thepopulation by

inac-tivating the wild type during an eclipse period

at250C, as well asat370C. Ofcourse, theycan

be recovered by plating the survivors at370C

(Fig. 1). Trial B in Table 1 shows that the results

are again a 10-fold enrichment ofonly the cs

phenotype. Thus, the prediction concerning the plaque phenotype is confirmed.

Test for predicted classes ofeclipse

mu-tants.Asecondpredictionarises whenone

con-siders all of the possible ways of altering the values of Ea and Atoobtain the Arrheniusplots forpossibleeclipsemutants.InFig.2,four ofthe eight possibilities are imustrated and represent the caseswhere onlyone of theArrhenius

pa-rameters is changed from the wild-type value. Since cs7O is an example ofa single mutation

altering onlyoneof the two parameters, there is

precedent forinitially considering only the sim-plestcasesinFig. 2. For thetwo classes above

the wild-type line,one candesignanenrichment

step that takes advantage of their increased eclipserateoverthat of the wild type; but

nei-ther class is expectedtohave either thetsorcs

plaque phenotype for screening the enriched population (compare Fig. 1 and 2). As for the two classes with the eclipse rates slower than that of the wildtype,theenrichment and

screen-ing procedures usedtoobtain thecsmutantsin Table 1 should select for both classes. Thus, examples of both the "higher

E."

and"lower A" mutantsshould bepresentineach population of

csmutants (compare Fig.1and2).

Totestthisprediction,apreliminary

classifi-cation was obtained by measuring the eclipse

Temperature (°C)

/T

FIG. 2. Theoretical basis for the existence of dif-ferent classes of4XM74eclipsemutants.Thesolid line isanextrapolationoftheexperimentally determined Arrheniusplot for the eclipse reaction ofthe wild-typevirus (8). Thedashed linesareexamples ofsome

postulated classes of eclipse mutants with altered valuesofeithertheArrheniusactivationenergy,Ea, orpreexponentialfactor,A.Wt, Wildtype.

TABLE 1. Effectiveness of eclipse mutant screen

Procedure Frequency of phenotype No. of eclipse

Growth No.tested mutants/no.of

temp(°C)

Enrichment

cs

(2500)

ts(420C) tested

Control 37 None 550 0.002 <0.002 NDa

Al 37 Uneclipsed at370C 100 0.02 <0.01 1/2

A2 37 Uneclipsed at

370C

600 0.037 <0.002 4/6

B 37 Uneclipsed at 25°C 450 0.040 <0.002 6/6

C 37 Unadsorbed at

150C

400 0.032 0.002 ND

aND, Not

determined.

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VOL. 39, 1981

rate at 37and 250C forarepresentative group

from each of the screening procedures. Six of the

csmutantsfrom the370Cenrichment (trial A2 inTable 1) arecompared with the wildtypein Table 2. The kineticmeasurementswere made

with high-titer crude lysatessinceapreliminary

experiment with wild-type 4X174 (data not

shown) gave an Arrhenius plot with only a

slightly higher A but withan

E.

identicaltothat obtainedwith purified virus. For themost part,

asinglekineticrun wasmadeateach tempera-ture so thatmoremutants could be examined. This necessitates a conservative approach in

comparingmutant and wild-type eclipse rates,

sinceuncontrolled variations in cell properties occasionallyleadtolarge differences inratesfor thesamevirus sample. Althougha

reproducibil-ity of 10to 20%canbe obtained from multiple

determinations (8), tentative classification can

be madebyconsidering only ratios ofwild-type to mutant rates that are greater than two as

significantly different. By this criterion,atleast two-thirds of the cs mutants in Table 2 are

defective intheeclipsereaction. Moreover,

ex-amples of both classesarepresent. C33-25and -30probably belongtothehigher Eagroup,since

their ratiosat250Caresignificantlygreaterthan thoseobservedat370C.Onthe otherhand,

C33-27and -28 probablyrepresentthe lower A class in that their ratios at both temperatures are

withinafactor of 2. InTable 3, six of themutants generated by the enrichment at250C are

com-pared. All mutants of this group had a lower

eclipseratethan did thewildtype atboth tem-peratures, andagain, both the higher

E.

(C32-11 and -13) and lower A (C32-12, -14, and -18)

arerepresented. Thus,thesecondpredictionof

theArrheniustheory is also fulfilled.

Ifone considers the possibility that asingle

mutation might simultaneously alter both Ar-rhenius parameters,twoother classesshould be presentinthecspopulations. Mutants with both alower

E.

andalowerAwouldbeselectedby enrichment at either 37 or 250C, since an

Ar-rheniusplot with thesameyintercept but with asmallerslopethan the lower A mutant inFig. 2would fall between thewild-type and lowerA lines. Moredetailed kinetic dataatseveral tem-peratures will be required to distinguish this class. However, certain mutants with both a

higher

E.

andahigherAcould be revealed by this data. For example, an Arrheniusplotwith

ahigheryinterceptandasteeperslopethanthe

wild-type line in Fig. 2 could cross the 370C

ordinateatthesamepointasthewild-typeline

but below the wild-type line atthe 250C ordi-nate. Since this mutant would have the same

[image:6.503.257.456.55.469.2]

eclipse rate as does the wild type at

370C,

it

TABLE 2. Kinetic characterization of eclipse mutants'

In vivo eclipse (single compo-nent)

k(wild Tentative Mutant k

(min-f)

type)/k(mu- class

tant) 37°C 25°C 370C 25°C Wild type 3.4 0.42b

C33-15 3.5 0.20-0.25 1 2 Wild type C33-29 2.8 0.23-0.29 1 2 Wild type C33-25 1.0c 0.030 3 14 HigherEa C33-30 0.60c 0.02 6 20 HigherE. 033-27 0.66c 0.040 5 10 Lower A C33-28 0.69 0.041 5 10 LowerA

a

Trial

A2of Table1.Virus was grownand enrichedat37°C. bBiphasic first-orderplots;the rate constant isfor the cor-rected faster component.

'These showed bothabiphasicplotandasinglecomponent plotonrepeat kineticruns.However,the correctedrate con-stant for thefastercomponentwaswithinexperimentalerror of the rate constant obtained from the single-component

curve.

TABLE 3. Kineticcharacterizationofeclipse mutantsa

Invivoeclipse(single

compo-nent)

Mutant_Muat k (min~l)_k

_(min-')

k(wild_(mutant)type)/k Tentative_class

370C 250C

370C

250C

Wild type 3.4 0.42

C32-11 0.69 0.03 5 14 HigherEa C32-13 0.46 0.01 7 42 HigherEa C32-14 0.35 0.02 10 21 LowerA

C32-16 0.20 -b 17 -b

C32-12 0.23 0.02 15 21 Lower A C32-18 0.39 0.03 9 14 Lower A

a

Trial

Bof Table 1. Virus was grown at

370C

and enriched

at250C.

"Thefirst-orderplotshowedaloss in PFUsatearlytimes

butaregaining of PFUs at later times.

would,therefore, be lost during the enrichment

step at

370C

butnot at

250C.

Yet thismutant

doesnot seem toberepresented in Table3,since

none of the six examined have arate at

370C

that is closetothat of thewildtype.

Transformation of

OX174

from inactive

to active form. In the course of this

study,

a

certain featureof the

-OX

adsorption properties

wasobservedtobealtered in these

eclipse

mu-tants. To insure that thischangeinadsorption

characteristicsdidnotintroduceanartifact into

themeasurement of the eclipseratesexhibited

by these mutants, a

preliminary

study of the

wild-type

and eclipse mutant

adsorption

was

made. Since the measurement of the

eclipse

reaction rate

requires

synchronized

infections,

the

adsorption

step had to be carried out at

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[image:6.503.259.457.270.466.2]

15°C. When care was taken to maintain the virus samples in ice from the time they were

removed from the refrigerator until the

begin-ning of the adsorption period, only 10% of the

PFUsattachedtosensitivecells.If,however,the

sampleswereinadvertentlyleftat room

temper-ature for a short period of time, the level of

adsorbed viruswasmuchhigher.This variation

was never observed in adsorption studies at

370C,suggestingthat

OX

canexist in an inactive

formduring storage at low temperatures and is

transformedinto theactive format

higher

tem-peratures. Ifthis is the case, theproblemwould

onlybeencounteredin studiesrequiring

adsorp-tionatlow temperatures.

Inapreviousreport(2), atransformation from

a thermally stable form of

OX,

incapable of

adsorbingtocells at low temperatures,to amore

labile form that could adsorb at 00C was

ob-served when the viruswas incubated inpH 7

phosphatebufferat370C.Todetermine whether

this or a similar transformation could account for the variable levels ofadsorptionencountered

atthebeginningof thisstudy, experimentswere

designedto testforsuchatransformation in the

starvation buffer used for the

eclipse

reaction.

Anticipating apossible rapidrate of this

trans-formationat370C,the reactionwasinitiatedby

a100-folddilutionof ice-coldvirusstocks intoa

reaction flask containing starvation buffer. At

various time intervals, samples were removed

and diluted 100-fold into tubes containing sen-sitive cells for a 15-min adsorption period at either 2 or

150C.

Toinsure thatadsorptionwas

rapid andcomplete inthis timeperiod, the cell

concentration was above109cellspermlandthe

multiplicitywasbelow0.01.Aftercentrifugation,

theconcentration of unadsorbedPFUswas

de-termined in each supernatant solution. Since

control experiments showed that incubation

alone did not leadto virus inactivation, these

titers represent the amount of virus that has

failed to attach to the cells. Over 95% of the

wild-typeandcs7O stockswereconverted within

10 min at 37°C to an active form capable of adsorbing to cellsatlowtemperatures (Fig. 3). However, the oneeclipse mutant from trial Al inTable 1 showed asignificantlylower rate for

this transformation, and a 90-min incubation

wasneeded to convert 90%of thismutant to an adsorbableform.When the 12 csmutants, whose

eclipseratesare shown inTables 2 and 3, were

examinedby thisprocedure, a possible correla-tion emerged. All of the mutants with lower

eclipseratesthan that ofthe wildtype also had

lower rates for thistransformation reaction. The differences in the ratesamong the mutants var-ied from 25- to 200-foldlower than that of the wild type. The two cs mutants whose eclipse

0.01 k(mirrl)

wt o-o 2.0 C 33-5 0.43

cs70W 2.0

0.001

0 10 20 30 40 50 60 70

[image:7.503.258.446.71.328.2]

Time(min)at37°C

FIG. 3. Comparison of the in vitrotransformation reactionforwild-type4X174 and its eclipsemutants. Virusstocks maintained continuously at 2°Cwere diluted 100-fold into starvation buffer at 370C. At each timepoint, sample was diluted 100-fold into starvationbuffercontainingE.coliC/1 cellsat2°C. Theconditionsof adsorptionforthisexperimentare 15min at a cell density of3 x 109/ml and at a multiplicityofinfection ofless than0.001. Thezero timepoint wasobtainedbydiluting thevirus stock into starvation bufferat20C, andafter10min, the sample was diluted into buffer containing cells at

2°C.wt, Wild type.

rates are notsignificantlydifferent fromthat of the wild type (C33-150 and -29 inTable2) also

had transformation rates that were similar to

that of the wild type.

This suggests that a slower transformation

rate may be a property of eclipsemutants

se-lectedbytheproceduresdescribed in this report

and could be the reason for their enrichment

during the eclipse step at 37°C. If this is the

case, these same mutants should beselectedby

screeningtheunadsorbed mutagenized

popula-tion of

OX.

Trial C in Table1shows theexpected enrichment ofonlythe csphenotype. Thus, the cs mutants selected in this study survived the

eclipse step because they have either a slower

eclipserateafterattachment at150Cora slower

transformation rate to the adsorbableform.

The reversibilityofthis

transformation

reac-tion was tested during the initial experiment

withwild-type

4X.

Asampleofvirus was diluted

at the end of the

370C

incubation period in

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39, 1981

starvation buffer at 2°C for a 60-min period

before cellswere added. The level of free virus

in thiscase was the same as that at the 8-min

timne

pointinFig. 3. This means that no

signifi-cant transformation back to the inactive form

occurred in 1 h at low temperatures, and the

data in Fig. 1 represent the concentration of

active virus present at

370C

and during the

adsorptionperiod at lowertemperatures.

Although the molecularbasisforthe

intercon-version betweenthese forms is not

understood,

the above resultsweretakenintoaccountinthe

design of the selection proceduresandthe

char-acterization of themutantsthatweregenerated

by them. For example, the transformation

reac-tionwasexaminedwitheach mutant so that an

appropriate preincubationperiod at

370C

could

be used before the adsorptionstep toinsure that

morethan 90% of the viruswasattachedtocells

at the beginning of the eclipse kinetic

experi-ments. Moreover, the level of free virus was

usuallymeasured atthe end ofthe adsorption

period. As for the selection procedures, the

mu-tagenized

wild-type

stock was always

preincu-bated at

370C

for15minbefore the additionof

cells. These precautionswerenecessary toinsure

that the majority of the infectious virus were

attachedto

cells

during the enrichmentstep.

DISCUSSION

The

preliminary

characterization of the

eclipse kinetics for thesemutants

clearly

estab-lishes the

selectivity

of the combined

enrich-mentandscreening procedures.Inall fourtrials,

therewas atleasta10-fold increase in the

fre-quencyofthepredictedcs

phenotype. Moreover,

intwo

trials,

the eclipse kinetics of sixmutants

were examinedat two temperatures, and over

two-thirds of the mutants had the

predicted

lower

eclipse

rate. In

measuring

the

eclipse

ki-netics,

one

begins

the reaction with a 100-fold

dilution of virus-cell

complexes

fornedat

150C.

This design excludes any effect ofaltered

ad-sorption

kineticssince sufficienttime is allowed for

adsorption

tobe

completed

before the

eclipse

reaction is

begun.

Thus,

the observed lower

eclipse rate in the mutants is not a result of

altered

adsorption

properties.

Additional

exper-imentsare

required

todeterminewhethersome

of the mutations may be

pleiotropic

and may

impart

altered

adsorption

kinetics as well. Ifa

reversible

adsorption

step

precedes

the

irrevers-ible

eclipse reaction,

more detailed studies are

requiredtodetermine what

contribution,

ifany,

anincrease inthe detachmentratemakestothe

observed decrease in the rate at which these

mutantsenterthe

eclipse phase.

Becauseof this

possibility,

the

adsorption

kineticsatlow tem-peraturesmustbe

carefully

examinedto

estab-MUTANTS 517

lishwhether the firststepleadingto

OX

infection

isreversible. However, it is clearthat the

pro-cedures describedin this report arehighly

selec-tive for mutants with defects in steps thatare

subsequenttoadsorption.

Theselectivity of the technique and the

pre-liminarycharacterizationofthe mutantssuggest

that the invivoeclipsereactionof4X174may,

indeed, be understood in terms of kinetic theory.

Two ofthe threedetectableclassespredicted by

the theoryhave been selected. As pointed out

earlier,mutants with a higherEaand a higher A

were not represented in the group of six that

were examined in detail (Table 3). However,a

single base change which results in an altered

value for both Arrhenius parameters may not

exist or may occur soinfrequently that a large

number ofmutants may have to be examined

for one to be found. The initial success of the

theory inselecting thesemutantswarrantsmore

detailedkinetic and structuralstudies, with the

hope of revealing the molecular propertiesthat

determine the values ofbothArrhenius

param-eters. Although kineticanalysis of biochemical

reactions has yielded values for the

transition-state equivalents of Ea and A, the

OX

eclipse

reaction is the firstsystem todemonstrate that

one canalter thevalues of these parametersby

genetic mutation. With the availability of the

aminoacidsequence ofthecapsidproteinsand

complete nucleotide sequence of the 4XDNA

(26),rapid sequencing methods forlocating base

changes in thesemutants will providea

begin-ningin

identiffying

thedomains in theDNA and

capsid structures that are involved in forming

the activatedintermediate for the reaction.

Asfor theapplicability of these selection

pro-cedurestootherviruses, key features of

adsorp-tion and eclipse kinetics of

OX

are shared by

many

nonenveloped

viruses. For

example,

whereas the

adsorption

rate exhibits

only

a

slight

dependence on temperature (9), the

eclipse kinetics showanalmost 100-fold decrease

between37and

150C

(8, 23). That thismay be

ageneral featureissuggested

by

the

diversity

of

theviruses in whichit hasbeen observed. These

include R17 (4), Ti

(25),

T4

(16),

lambda (20),

poliovirus (7), rhinovirus (18), and adenovirus

(17,24).Thesameholdstruefor thedissociation

ofcertaincapsid

proteins during

the

beginning

of the eclipse period. In additionto

4X174,

the

phenomenon occurswiththe phagesM13 (13),

R17(15),T4(29),lambda(27),P22

(12),

and429

(6) and the following animal viruses: human

rhinovirus (18) and adenovirus (30).

Thus,

a

geneticselection

technique

that is basedonsuch

a commonpropertyasthetemperature

depend-enceof theeclipseratecould be

applicable

toa widerange ofviruses.

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ACKNOWLEDGMENTS

This work wassupported in part by General Research Grant RR05423 fromthe National Institutes of Health and grantPCM77-15982 fromtheNationalScience Foundation.

Technical assistancewasprovided byMarshaL.Brown. Thehelpful discussion andsuggestionsof JamesKane,Robert Reeves,and David Kingsburyaregreatlyappreciated.

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