Genetic Analysis of Adenovirus Type 2 II. Preliminary Phenotypic Characterization of Temperature-Sensitive Mutants

JOURNALoFVIROLOGY,May 1975,p.1049-1056 Copyright X 1975 AmericanSocietyforMicrobiology

Vol. 15, No. 5 Printedin U.SA.

Genetic

Analysis of Adenovirus Type

2

II.

Preliminary Phenotypic Characterization

of

Temperature-Sensitive

Mutants

J. WEBER,* M. BEGIN, AND G. KHITTOO

Departement deMicrobiologie, Centre Hospitalier Universitaire, Sherbrooke, Quebec, Canada Receivedfor publication 21 October 1974

The properties oftemperature-sensitive mutants ofadenovirus type 2

repre-senting12complementationgroups werestudied. Allmutantswerenormalwith

respect to adsorption as measured by viral inclusion formation and viral DNA

syntbeFsis asshownby velocity sedimentation in alkalinesucrosegradients. One

mutant, however, formed viral inclusions of altered morphology atthe

nonper-missive temperature. Thesynthesis of themajor capsid proteins wasexamined

by immunodiffusion. On this basis, the complementation groups could be arranged asfollows: (i) onegroup wasnegative for allthree proteins; (ii) three groups failed to synthesize penton bases; (iii) eight groups were positive for

hexons, pentons, and fibers. The assembly of virus particles at 39C was

examined by equilibrium sedimentation in CsCl; three groups were found

defective, whereas two of the penton-negative groups were positive for virion

production. Tests of the thermolability of virions at 50C revealed eightgroups

labile whereas theremainderwere insensitive toheat inactivation. Noneof five

mutantsinoculated in newbornratsinducedtumors, although three ofthemwere

capableof invitrotransformation.

In a previous communication we have

re-ported the isolation of temperature-sensitive

(ts) mutants of adenovirus type 2 (2). These mutants were classed into 12 complementation groups and arranged in a linear sequence on a tentative ge-ieticmap by recombination

analy-sis. In this report we examine some of the functional characteristicsof these mutants.

(Preliminary reports of this work were pre-sented atthe Annual Meeting of the American

SocietyforMicrobiology, Chicago, Ill., 12 to 17

May 1974, and at the Canadian Society of

Microbiologists, Montreal, 18to 21 June1974.)

MATERIALS AND METHODS

Virusandcels.Humanadenovirustype 2(Ad 2) designatedasthewild type(WT)waspropagatedin KBcells and titratedbytheplaqueassayonKB cells orby the infected cell unit assayonHEp-2cells, as

described previously (2, 19). The isolation ofthets mutants and their preliminary genetic characteriza-tion have also been described previously (2). In the interest of abiding by uniform nomenclature, the mutant numbers published in the preceding paper which are greater than 99 have been revised as follows:ts241is ts1; ts 496 is ts 2; ts 235 is ts 3;ts 500 is ts4; ts 458 ists5;ts155is ts6; ts 517 is ts 7; ts 166 is ts 8; ts 186 is ts 9; ts 231 is ts 11; ts 469 is ts 12. Henceforth the mutants shall be referred to by this newnomenclature, in conformity with other

adenovi-ruslaboratories (7).The KBcellswereobtainedfrom

Flow Laboratories (Rockville, Md.) and were never

usedbeyondthesixthpassage. BothHEp-2 andKB

cellswereculturedinDulbecco-modified,Eagle mini-mal essential medium containing 10% calf serum.

Virus-infected cells were culturedinminimal

essen-tial mediumsupplementedwith 2.5% calfserum and

0.4mM arginine.

DNAsynthesis.Confluent monolayersof KB cells grown in 50-mm diameter Falcon petri dishes were

inoculatedwith ts mutants at a multiplicity of input of 5 PFU/cell. After a 50-min adsorption period at 33 C, the cells were washed three times with

Tris-buffered saline (pH 7.4). Fresh medium was then added and the infected cultures wereincubatedat33 and 39C, then labeled with5

,Ci

of ['Hlthymidine

per ml(40 to 60Ci/mM) 24 and 16 h later,

respec-tively. At 40 hpostinfection, the mediumof the cul-tures incubated at 39 C was removed, and the cells were washed with Tris-buffered saline, scraped off

with a rubber policeman, and resuspended in colQ

Tris-buffered saline. The cultures incubated at 33 C

received the same treatment 65 h postinfection. In someexperimentsthecellswere labeled for 1honly andeitherlysedimmediately or further incubated in medium without ['H]thymidine. Linear, 5 to 20% sucrose gradients (in 1.0 M NaCl, 0.01 M EDTA, and 0.3 M NaOH, pH 12.9) were prepared in poly-allomer centrifuge tubes essentially as described by

Doerfler (5). A lysing layer (0.20 ml) consisting of 0.5 MNaOH and 0.01 M EDTA was carefully applied ontop of thepreformed gradients. Samples of about 1049

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WEBER, BEGIN,ANDKHITTOO

10'

cells (contained in 0.05 ml) were mixed with

[l'C

lthymidine-labeled marker virus and gently pipetted onto the lysing layer. The cells wereallowed to lyse for 1 h at room temperature or overnight at 4 C. The preparations were then centrifuged in the B60 International ultracentrifuge using the SB-405 rotor, at 60,000 rpm for 84min at 4 C. At the end of the run the gradients were collected dropwise on Whatman (GF-81) fiber glass filter disks from the bottom of the tubes. The filters were washed with ice cold 5% trichloroacetic acid solutionfollowed by ethanol, and then dried and counted in a toluene

basedscintillation fluid.

Immunodiffusion. Double diffusion in agar gels was carried outby a modification of the microtech-nique ofCrowle (4)and alsobythestandard ouchter-lony macrotechnique using the Gelman (Gelman

Instruments, AnnArbor, Mich.)apparatus(14).After

developmentfor1to2days,the slidesweresoakedin 0.15 MNaClfor 1 to 3days,then dried atadistance of 25 cm under a 250-W infrared lamp. To prevent

shrinkage during drying,the wells of the macrotech-niquegels were filled withwater.Theprecipitinlines werestained with amido black (1 g of stain in450ml of 1M acetic acid, 450ml of 0.1 M sodium acetate, and 100 ml ofglycerol).

Antisera. Whole virion antiserum waspreparedby

immunizingrabbitswith virions whichwerepurified by two extractions with fluorocarbon (Freon 113,

DupontCo.) followedbytwocyclesofcentrifugations

toequilibriuminCsCl. Hexon,penton,andfiberwere

purified by chromatography ofthe top components from CsCl gradients on DEAE-cellulose (9). The purity of these proteins were verified by sodium

dodecylsulfate-polyacrylamide gelelectrophoresis

(1)

and by immunodiffusion against whole virion anti-serum. Antisera against theseproteins was also

pre-pared by immunizing rabbits. The rabbit antisera wererecheckedby immunodiffusion.

Virusassembly.KB cell monolayers wereinfected with virus, incubated at 33C, andlabeledat 24hor

incubatedat 39C andlabeledat 18hafter infection with 1MCiof3H-labeledaminoacids mixture per ml

(International Chemical and Nuclear Corporation,

Irvine,Calif.).Thelabel was left on the cells until the cells were harvested inTris-buffered saline 2 and 3

daysafterinfectionat39and 33C, respectively, lysed,

and extracted with trichlorotrifluoroethane (Freon

113, Dupont of Canada, Ltd.). This material was gentlypipetted on top of a CsCl solution (1.40g/cm3)

and centrifuged to equilibrium overnight. The tubes were photographed; then fractions were collected through the bottom. The density of the CsCl was determinedbyrefractometry whereas the radioactiv-ity was determined as 5% cold trichloroacetic acid-precipitable counts in toluene based scintillation fluid. Thenegatives of the photographs were scanned at 540 nm in a Gilfordspectrophotometer equipped

with a linearscanning device.

A second series of KB cell cultures were infected with virus and incubated at 33, 39, and 40.5C, then labeled with 2 uCi of [8H]thymidine per ml (60Ci/

nM, NewEngland Nuclear) at 6, 4, and3h after in-fection, respectively. The labelwas left on the cells

until the end of the infection period at 3 days (33 C) and 2 days (39 and 40.5C) postinfection. The virus was extracted with Freon as above andtreated with 50

Ag

of pancreatic DNase I per ml (Calbiochem) at 37Cfor 30min,and then 0.1 ml was layered on top of a preformed CsCl gradient (1.2 to 1.5 g/cm3) and centrifuged at 60,000 rpm for 1 h in rotorSB-405 of an International B60 ultracentrifuge. Thefractions were treated as described above.

RESULTS

Adsorption and inclusion body formation. Theefficiency of adsorption of the mutants was tested by the formation of inclusion bodies (15, 19, 20) at the permissive and nonpermissive temperatures. Single cover slips (1-cm diame-ter) with a confluent monolayer of HEp-2 cells wereplaced in separate petri dishes and prein-cubated at 33 or 39 C, and then 25

Al

of prewarmedvirus at several dilutions was added to corresponding cover slips and allowed to adsorbat 33 or 39C.After 1 h,unadsorbedvirus wasrinsed off, mediumprewarmed to 33 C was added, and all cover slips were incubated at 33 C for 3days. At this time they were fixed and stained,and the number ofvirus-induced inclu-sionbodieswascountedasdescribedpreviously (19). Since several dilutions of virus were tested, it was possible to choose a dilution which produced between 0.1 to 1% inclusion bodies (19), andtoread the cover slipscorresponding to this dilution at the two adsorption tempera-tures. The ratio of inclusion bodies at 33 C to inclusion bodies at 39C did not significantly deviate from unity for the 15 mutants tested (belonging to the 12complementation groups). As all the mutantsappeared normalfor adsorp-tion, the actual dataare not shown.

In a second experiment we wished to deter-mine whether the mutants were capable of forming inclusion bodies at the nonpermissive temperature. The experiment was essentially conducted asabove, exceptthatadsorptionwas at 33C and incubation at both33and39 C.

As in the adsorption experiment, the ratio of inclusion bodies at 33 C to inclusion bodies at 39 C was notsignificantly differentfromunity. With the exception of ts 3, the morphologyof theinclusionswasalsoindependentof tempera-ture.Although at 33Cthe ts3-infected cellsare indistinguishable from the WT, incubation at 39C causes the cells to appearcrenated.

It was concludedthat these ts mutants were not defective in adsorption and inclusion body formation.

Viral DNA synthesis. The ability of the mutants to synthesize DNA at the nonpermis-sive temperature was determined by velocity

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VOL.15.1975 PHENOTYPIC CHARACTERIZATION OF TS AD 2 MUTANTS

sedimentation of ['H

jthymidine-labeled

in-fected cell DNA in alkaline sucrose gradients.

Under the conditions employed, viral DNA

from infected cells cosedimented with purified Ad 2 marker DNA, whereas cellular DNA was pelleted. Figure 1 shows typical profiles ob-tained for ts 53,74, 6, 8, 9, 11, 3, 1, 5, 2, 4, 7,and WT. Similar results were obtained when using the following modifications of the basic proce-dure: (i) thelabeling periodwith [8H

]thymidine

was reduced to 1 h and the cells were lysed

immediately or after a 24-h chase; (ii) the alkali lysis period was extended to overnightat 4 C;

(iii) the temperature ofincubationwasraisedto 40.5 C. In all cases there wasalways somewhat more DNA synthesized at the nonpermissive than at the permissive temperature. Reversion

can be ruled out since we are dealing with a single growth cycle and the rate of reversion was never greater than 10-'(2). Although leak-iness cannot be ruled out, it appears highly

improbable that DNA synthesis during a 1-h labeling period should be appreciably affected by it. From this result it may be concluded that these mutants are not defective in viral DNA synthesis.

Synthesis of viral capsid antigens. Using the standard macroimmunodiffusion tech-nique, three distinct precipitin lines were

ob-tained with infected cell lysates and antiserum prepared againsthighlypurified completevirus

12

-10

x

*-0

6

FRACTION

FIG. 1. Viral DNAsynthesis. Alkalinesucrose

gra-dient velocity sedimentation of

[3H]thymidine-labeledDNA from KBcellsinfected withts mutants.

This profile isrepresentative of all thets mutantsand WT tested. The arrow indicates the position of ["C]thymidine-labeled Ad2markerDNA. Symbols: A, ['H]thymidine counts/min labeled at 33C; A,

['H]thymidinecounts/min labeledat39C.

(Fig. 2). A fourth faint line was also present sometimes. By means of the microtechnique, two tothree additional, fainter precipitin lines canalsobedetected. The three majorprecipitin lines were identified by means of monospecific antisera to hexons and pentons (penton base andfiber). At the present time we do not know the nature of the extra precipitin lines. The ability of the ts mutants to synthesize immuno-logically cross-reactive material of the three major capsid antigens (hexon, penton, and fiber) was tested underpermissive and nonper-missive conditions by both these techniques. The results are summarized in Table 1. Itcan be seen that ts3 fails to synthesize any of the capsid antigens; ts 11, ts 4, and ts 7 representing three distinct complementation groups pro-duced normal precipitin lines correspondingto hexon and fiber antigens but reducedprecipitin

lines corresponding to pentons, whereas the remaining mutants representing eight com-plementation groups appear to synthesize nor-mal levels of theantigens.

Virus assembly. The immunodiffusion ex-periments described above have demonstrated that some ts mutants were defective in the synthesis of one ormorecapsid antigens at the nonpermissive temperature, whereas the major-ity had apparently synthesized a pattern of antigens indistinguishable from the WT virus. Since the synthesis of cross-reactive material is noguarantee of the functionalintegrity of the antigen, northeabsence of cross-reactive mate-rial fortheabsenceofvirions, theproblemof the assembly of virus particles was examined by the following experiments. KB cell cultures weresingly infected with mutants or WTat 33 and 39 C and labeled with 'H-labeled amino acids, and the infected cell extracts were cen-trifuged to equilibrium in CsCl. The virus bands were photographed in the tubes, and then fractions were collected and measured for den-sity andradioactivity. Figure3shows the radio-activity profilesandthe densitometric tracings

of the photographs of three mutants and the WT. The profiles show thesharpvirus band in the 1.34 density region and a broader band of material which includes empty virions, virus

capsids (top components), and probably some cellularproteinsaround the 1.30density region (3, 8, 17). The disproportionate virus band in the densitometric tracings is due to the strong diffractionofvisible lightbyvirions. Of the mu-tants tested, ts 53, 74, 6, 8, 9, and 1 had pro-files identical toWT; ts 2, 7, and 11produced a normal amount ofvirus but a reduced quan-tity of top components; ts 4 and 5 produced 1051

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WEBER, B1GIN,ANDKH1TTOO

FIG. 2. Doublediffusion inagar. Centerwell containsrabbit anti,erum againstAd2andperipheralwell's

extractsfromcellsinfectedwithvirus aisfollows:1and4,ts at39C; 2, WTat39C;3and6,ts at33C; 5,

at33C.

no virus but an apparently normal amount of

top components;andts3producedneither virus

nortop components. Since ts3was apparently

defectiveintheproductionofcapsid

cross-reac-tivematerial, it issatisfyingto notethe drastic reduction of both virionsandtopcomponents at

thenonpermissive temperature.Although ts4, 11, and 7 were defective in the production of

pentons,only ts4appearedalsotobedefective in virionproduction. The defects ints5 and 2

may involve a minor virion component or a

nonstructural virus function. The defects in

mutantswhichwereindistinguishable from the

WT will need to be investigated by other methods.

To test someof thepropositionsenumerated

above, the experimentwasrepeated withsome

modifications. Although the above experiment dealt with changes detected on the basis of

following the proteins, the present one was

designed tostudytheassemblyof nucleic acid-containing particles. Virus was labeled with [3H1]thymidine at33, 39, and 40.5 C and puri-fied asdescribed above. The radioactivity

pro-filesareshowninFig.4.Underthese conditions

ts1, 2, 6, 8, 9, 53, and74 wereidenticaltoWTas expected; ts 3 and 4 remained

unchanged

whereas ts 5, 11, and 7 (panel B) showed a

considerabledrop invirion

assembly

or mainte-nance at 40.5C. This result appears to be

consistent with the notion ofleakiness ints 11

and7.

Heatinactivation. Figure 5gives the results of experiments testing the

thermolability

of mutant and WT virus particles at 50C. The

slopeoftheinactivationcurvevaried

depending

onthe stateofpurification andthesuspension

medium. To have uniform conditions, crude virussampleswerediluted with thesamebuffer and all viruses were tested at the same time. The mutants appeared to cluster in three groups: those indistinguishable from WT (ts 2,

4, 7, 8); those inactivated 10- to50-fold faster than WT (ts 1, 3, 5, 6, 9, 11); and those inactivated 1,000-fold faster than WT (ts 53,

74). To check if the survivors contained revert-ants, some plaques from the 32-min exposure were picked and replaqued at 33 and 39C; all were shown to be ts mutants. This result suggests that mutants of eight

complementa-V:i.

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VOL. 15, 1975 PHENOTYPIC CHARACTERIZATION OF TS AD 2 MUTANTS

TAau 1. Immunodiffusionoflysates of cells infected

with ts mutants at 39Ca

Complementa- Mutant Virus capsid

antigens

tion group Hexon Fiber Penton

J ts 3

E tal11

F ts1 (+) (+) (4)

C ts 7~

B ts53

H ts 74

H ts 26

G ts 6

L ts 8

Ats 9 (+ (+ ()

D tal1

D tsl12

I tas

K ts2

Unassigned ts 10

aThisrepresents aqualitativesummaryofseveral

experiments using both the macro- and microim-munodiffusion methods with complete virus anti-serum and monospecific antiserum to hexon and pentonantigens.

tion groups

(A, B, D,

E,

G, H, I, J)

mayhave defects in genes for structural components of the virion.

Tumorigenesis. WT Ad 2 has been shown tobeoncogenic

indirectly by

thetransplantation

of in vitro transformed cells(6). To our

knowl-edge there exists no recent report to induce tumors by direct inoculation ofthe virus into

newborn rodents. To fill this gap, and more importantly to test the oncogenicity of ts mu-tants,newbornrats(Sprague-Dawley, Madison, Wis.) were inoculated with WT and 5 ts mu-tants. At about6 to 8 weeksof age, and also 1

week later, each rat received intramuscular injections of 2.5 mg of cortisone acetate as an immunosuppressive. The rats were palpated weekly for the presence of tumors up until a maximum of 14 weeks of age. Although the

rat's body temperature of 38.2 C closely

ap-proaches the nonpermissive temperature, in

this experiment neither the WT nor the ts mutants induced tumors at the site of inocula-tion (Table 2). Preliminary results of trans-formation experiments using primary cultures ofrat embryo cells indicate that WT andts 1, 3, and4arecapable oftransformation ateither 33or39 C. Thereasonforthe failure ofinvivo transformation remains unclearatpresent.

DISCUSSION

The present study showed that Ad 2 ts mutants representing 12 complementation

[image:5.505.51.245.79.302.2]

groups were all defective in late functions.

Table 3presentsasummaryof the properties of the mutants studied. All of the mutants ad-sorbed normally, synthesized viral DNA, and produced a cytologically recognizable intranu-clear inclusion body. The morphology of the inclusion ofts3 atthenonpermissive tempera-ture was atypical. This was also the only mu-tantwhich failedtoproduce hexon,penton,and fiber antigens as evidenced by immunodiffu-sion. Working with Ad 31tsmutants, Suzukiet al. (18) have observed similar alterations in inclusion body morphology, accompanied by a reduction in the accumulation of the capsid

antigens. The drastically altered phenotype of ts 3 however is not likely due to a multiple mutational event as evidenced by the com-plementation, recombination, and reversion tests (2).

Immunodiffusionanalysis also revealed three complementation groups which showed reduced accumulation of the penton antigen, whereas

E

Pv"I

u

; 3

I I v

3

frocton number

FIG. 3. Radioactivity and absorbance profiles of cesium chloride equilibrium gradients ofextracts of

cells infected in the presence of 'H-labeled amino acids with WT and ts mutants at the permissive and

nonpermissivetemperatures.AandC, WT, ts 53, 74, 6, 8, 9, 1; B andD,ts4and5; E andG,ts11, 2, 7; F andH,ts 3. - , 33C; ---, 39 C.

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BgGIN,

ANDKHITTOO

E

I.-,E

.

, U

froaion number

FIG. 4. Radioactivity profiles of cesium chloride equilibrium gradients ofextracts ofcellsinfectedin

the presence of [3H]thymidine with WT and ts

mutants atthepermissiveandnonpermissive temper-atures.Panel A:ts1, 2, 6, 8, 9, 53, 74,and WT.Panel B:ts5, 7, 11. Panel C:ts4. Panel D:ts3.Symbols: *, 33C; *, 39 C; A,40.5 C.

normal levels of hexon and fiber antigen were

present. The precipitin line corresponding to

the penton cross-reacts with that of the fiber

and has been showntocontaintheentirepenton capsid, consisting of penton base and fiber(9). Since the fiberantigen ispresentineach ofthe three mutants, and current data suggests that the penton base may consist ofmore than one copy of a single molecular weight class of

polypeptide (10), the existence of three

com-plementationgroupsappearstobeanintriguing

finding. Two of these complementation groups mayinfact be involved withregulatoryaspects

affecting the synthesis, processing, or

metabo-lism ofpenton bases.Alternatively, wemaybe dealing with intracistroniccomplementation,or

multiple-step mutants. The first possibility is

unlikely as the complementation indices were

greaterthan10for thecombinationsts 11x ts4 orts11 x ts7and, though that betweents4 x

ts7wasonly2,thesemutantsmapped distantly byrecombination.The genetic evidence and the highrateof reversion (2) alsoargueagainstthe

second possibility. Russell et al. (12, 13) have demonstrated six complementation groups

which affect hexon synthesis, and three

com-plementation groups which affect fiber

synthe-sis, whereas only one group affected penton

synthesis.

Of the three mutants defective in penton

synthesis, onlythe leastleakyone, ts4,wasalso

10~~~~~~~~~N

8 3

U~~~ ~lUE AT 50°

10

ts4

ts7

86316s32

FIG. 5. Heat inactivation of virions of ts mutants.

Crudesamples of virus grown at 33 C were sonically

treated to disperse possible aggregates, and diluted

1:10 in Thris-buffered saline, pH 7.4. Samples were

exposed to 50 C for the indicated times, then cooled

rapidlyin an ice bath. Survival was assayed by plaque

formation at 33 C.

TABLE 2. Attempts to inducetumors in rats with WT

and tsmutantst

Virus dose No. ofbaby Number of Virus (PFU/rat) rats injected free

I017_10 25 13-14

ts 1 107 27 9

ts 6 107 8 8

ts 4 106 8 11

ts 3 107 5 10

ts74 106 15 10

aNewborn litters were injected subcutaneously

with0.1ml of virus within 24hofbirth and checked for tumorsweekly. At about6 to8weeks of age and1 weeklater, eachratreceivedtwointramuscular injec-tions of 2.5mg of cortisone.

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VOL.15, 1975 PHENOTYPIC CHARACTERIZATION OF TS AD 2 MUTANTS TABLE 3. Properties ofts mutantsoftype2adenovirusa

DNA Inclusion Antigensproduced (39C) Virion Thermo- Invivo Invitro

Mutant synthesis assembly sensitivity tumor for-

-(3 (39C)

C

)

Hexon Fiber Penton 39 or (50C) mation

(m3a9tC)

H2tsJ 3 1 + + + +

H2tsE 1 1 + + + + 4 4 + NT NT

H2tsF 4 + + + + 4 _- _ +

H2tsC 7 + + + + 1 - NT NT

H2tsB 53 + + + + + + + NT NT

H2tsH 74 + + + + + + + _ NT

H2tsH 26 NT + + + + NT NT NT NT

H2tsG 6 + + + + + + + _ NT

H2tsL 8 + + + + + + _ NT NT

H2tsA 9 + + + + + + + NT NT

H2tsD 1 + + + + + + + _ +

H2tsD 12 NT + + + + NT NT NT NT

H2tsI 5 + + + + + 4 + NT NT

H2tsK 2 + + + + + + _ NT NT

H2ts 10 NT + + + + NT NT NT NT

WT + + + + + +

_

_

+

aAbbreviations: NT, nottested;H2, human type 2adenovirus; J,E,F, etc., denoted the complementation

group.

defective in virion assembly at 39C. The two

leaky mutants, ts 11 and ts 7,

only

showed

reduced assembly at40.5 C.

The assembly of virions in the absence of

adequate numbersofpentons isnot as surpris-ing in view ofthe experiments of Prage et al. (11) which showed that virions lacking pentons and even peripentonal hexons arestableinCsCl

gradientsand canbe visualizedbymeansofthe electron microscope. Further phenotypic sepa-ration of these mutants was obtained by the

demonstration of thethermolabilityofonly one ofthese three mutants. Increased thermolabil-ity of the mutant virions has been taken to suggest thedefectivenessofastructural compo-nent or aprotein implicated in the determina-tion of the structure of the virion. Unaltered

lability however, fails to indicate if the ts lesion is in astructural or a nonstructuralprotein.By this criterion, ts 3 and 5 are twofurther com-plementation groups which fail to produce vi-rions andareprobably defective inastructural protein. Intotal, eight complementation groups

showed increased thermosensitivity, thus sug-gestingeightcistronsspecifyingproteins

impli-cated in the architecture of the virion. The remaining four complementation groups failed to show increased thermolability. According to currentdata(1), Ad 2maysynthesize upto 14 molecular weight classes of structural proteins and perhaps seven nonstructural proteins. As-sumingthattheseproteinsareindeed codedfor

byAd2, the total molecularweight of 920,000of these21proteinsaccountsfor80% ofthecoding

capacity of the virus. The 12 complementation groups discussed in this paper may represent

about half of the total genetic complement of Ad 2.

ACKNOWLEDGMENTS

The technical assistance ofKen Harrison and in part Diane Duranleauare gratefully acknowledged.

J.W. is ResearchScholarofthe Medical Research Council of Canada. M.B. is recipient of a studentship from the Conseil de RecherchesM6dicales duQu6bec.Thiswork was supported bygrants from the National Cancer Instituteof Canada and MA-4164 from the MedicalResearch Councilof Canada.

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