Characterization of a temperature-sensitive mutant of human adenovirus type 7.

(1)

Vol. 21, No. 3 Printed in U.S.A.

Characterization of

a

Temperature-Sensitive

Mutant

of

Human

Adenovirus Type 7

MARY K. ESTES AND JANET S. BUTEL*

Department of Virologyand Epidemiology, Baylor College of Medicine, Houston, Texas 77030

Received for publication 19 November 1976

Theproperties ofanaturally occurring temperature-sensitive (ts) mutant of

human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40

(SV40)-Ad7hybrid (PARA). Growth of the mutant was restricted at40.50C, and

the ratios ofvirusyieldsin KB cells at 40.5 and 330C were 10-2 to 10-3. Viral

DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid,

hexon, and penton antigens) appeared normal at the restrictive temperature.

The assembly of virus particles was aberrant, as determined by thin-section of

infected cells. Theinfectivity of mutant virions was heat labile at 50'C,

suggest-ing a tsdefect in a structural component of the virion. Analysis by

polyacryl-amide gel electrophoresis of [35S]methionine-labeledpolypeptides synthesized in

mutant-infected cells suggestedthat at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein

synthesis lateinmutantinfections,ascomparedwithwild-type (WT) infections atboth thepermissive andnonpermissive temperatures, madequantitation of

infected-cell polypeptides difficult. Analysis of theassemblyofcapsomeres from

cytoplasmicextracts of infectedcells on sucrose gradients and by

non-dissociat-ingpolyacrylamide gel electrophoresis suggested that hexon capsomeres were

made at 40.5°C. The hexon capsomeres made by the mutant at either 33 or

40.5°C displayed a decreased migration in the non-dissociating gels compared

with the WThexon capsomeres. The molecular weights of the mutantandWT hexon polypeptides were identical. These results suggest that the ts lesion of this

group B human Ad7 mutant may bereflected in altered hexons. The mutant

Ad7 interferedwith the replication of adenovirus types 2 and 21 at the elevated temperature.

Conditional-lethalmutantsofbacteriophages and animal viruses have been valuable tools for analysis of the viral genes expression inboth theproductive and transforming cycles ofvirus

infection(10). The currentisolation of viral and

host cell mutants is allowing a more precise

analysis of the molecular mechanisms that

reg-ulateviral synthesis and cellular alterations in

eukaryotic cells. Temperature-sensitive (ts)

mutantsofadenovirustypes 2, 5,and12,

repre-senting groups A and C human adenoviruses,

and of avian orphan (CELO) adenovirus were

reported previously (9, 11, 14, 16, 20, 31-36,

39-41). The biochemical andgenetic

characteriza-tionsof these mutants haveprovided

informa-tion on the organization ofthe adenovirus

ge-nomeand have permitted identification of the

biological functions of some of the viral gene

products. Thisreport characterizes a naturally

occurringts mutantof adenovirustype7

(Ad7),

agroup B adenovirus.

MATERIALS AND METHODS

Cells. Human embryonic kidney (HEK) cells were obtained from either HEM Research, Rock-ville, Md., or Flow Laboratories, Inc., Rockville,

Md. The cellswere grown in Melnick lactalbumin

hydrolysate medium containing 10% fetal bovine

serum,0.075% sodiumbicarbonate, 100Uof penicil-lin, and 100 ,ugofstreptomycinper ml. Theywere maintainedinthesamemediumsupplementedwith 2%fetal bovineserum. HEKcellswereutilized up to passage three in tissue culture. KB cells were growninEagle medium supplementedasdescribed above.

Viruses. The prototype strain of Ad7wasobtained from the American Type Culture Collection. The strain waspassed nine times in KB cells. An Ad7 isolated from a fatal case ofpneumonia (3) was designatedAd7 (Hu) and waspassed fivetimes in HEK cells.The titerof the Ad7 (Hu) stock used for allexperiments described herewas6 x 107PFU/ml.

Mutant Ad7(19),E46-,wasderived from the paren-tal PARA-Ad7 stockby three successiveplaque pu-rificationsinHEK cells andwasthenpassed eight

1159

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

1160 ESTES AND BUTEL

additionaltimes inHEKcells. Theplaque-purified progeny nolonger induced simian virus 40(SV40) antigens (4)and couldnotreplicate in green monkey kidney cells inthe absence ofSV40 (5). The titer of the Ad7 (19) stockused for allexperiments described in this report was 7 x 108 PFU/ml. Other Ad7 strains were kindly provided by H. Ginsberg and Wyeth Laboratories, Philadelphia, Pa., and were designated as such. Adenovirus type21 (Ad21) was obtained from W. Parks and was used after one passagein KBcells andtwopassagesinHEK cells. Adenovirus type 2 (Ad2) was obtained from M. Ben-yesh-Melnick and had undergone six passagesinKB cellsand one passage in HEK cells.

The adenoviruses were assayed by the plaque techniquein HEKcells as previously described (7). Virus assays wereperformed at370C. The relative plaquing efficiency of the mutant at370C/330Cwas

0.95to1.0,andat40.50C/330Cit was10-2 to10-3.

Growth curves. Growth studies wereperformed withMagniwhirl waterbaths to assure precise tem-perature control. HEK andKB cell monolayers in tubeswereinfected withAd7 (19) or Ad7 (Hu) at a multiplicity of infection (MOI) of 2 to 5 PFU/cell. After adsorption at370C, monolayers were washed with Tris-buffered saline (TBS), maintenance me-diumwasadded, and replicate tubes were incubated

at33 or40.50C. Replicate tube cultures were

har-vested at various intervals postinfection (p.i.), and theclarified lysates were assayed for virus yields as described above.

Antisera. Antisera to purified Ad2, Ad7, and Ad2M were prepared in rabbits as previously de-scribed (8). Adl2 tumor antibody was obtained from tumor-bearing hamsters. Antisera to Ad7 hexon and penton antigens, kindlydonated by G. Dreesman, were prepared in rabbits against purified hexons and pentons isolated from infected cells by gel filtra-tion onSepharose 2B, followed by sucrose gradient centrifugation (manuscript in preparation).

Immunofluorescence techniques. Monolayers of HEK cells on cover slips in Leighton tubes were infected with Ad7 (19) orAd7(Hu) and incubated in waterbaths at the permissive (33°C) or

nonpermis-sive(40.5°C)temperature.At24and48h, the cover

slips were harvested, fixed for 3 or 10 min with

acetone, and stained for tumor, capsid, hexon, or penton antigen, respectively, with the sera de-scribedabove. Thisprocedurewasfollowed by add-ing anti-rabbit globulin baboon globulin labeled with fluorescein isothiocyanate as previously de-scribed (27).

Co-infection studies. Tube cultures ofKB cells wereinoculatedsimultaneously in mixed infections with3 to 5 PFUper cell ofeach of the two viruses to be tested. Singly infected control cultures received the same multiplicity ofeach virus. After adsorp-tion, the cells were washed three times with TBS, incubatedinwater baths at33°C for 96 h or at 40.5°C for 72 h, and harvested by three cycles of freeze-thawing. The cell debris was removed by low-speed centrifugation; the supernatant fluids were pooled, treatedwiththe appropriate antiserum at37°C for 1 h, and analyzed for viral content at 37°C in HEK cells.

Viral DNA. Cultures ofHEK cells were mock

infected with TBSorinfected with virus atanMOI of 10 PFU/cell. After a 90-min adsorption period, depleted medium (12)wasadded and cultures were incubated at 33 or 40.5°C. Since initialexperiments with 2-h pulses showed that peak labeling of viral DNAoccurred atapproximately 32hp.i. at40.5°C and at 36 h p.i. at 330C, 5 ,uCi of [3H]thymidine (TdR) per ml was addedtothe cultures from 24 to48 h p.i. At48h p.i., DNAwasextractedby the Hirt procedure (13), and DNA in the supernatant fraction was purified by treatment with RNase, Pronase, andphenol. DNA wasalso extracted frompurified virionsby sequentialtreatmentwithsodium dode-cyl sulfate (SDS), RNase, and Pronase, followed by extractions withphenol. Samples containing 1 ,ug of DNAwerecentrifuged for72hat35,000 rpmat200C toequilibrium in gradients ofCsCl with an average density of 1.70 g/ml by usingaTi5O rotor. Gradients were fractionated by bottom puncture of the tube; refractive index of each fraction was determined with a Bausch and Lomb refractometer. Each frac-tion was precipitated with cold trichloroacetic acid onglass-fiber filters, dnd theradioactivity was de-terminedin aBeckman LS250liquid spectrometer. Electronmicroscopy.Cultures of HEK cellswere mock infectedorinfected with Ad7 (19)orAd7(Hu) at anMOI of10PFU/cell. After incubationat33or

40.50C for 96 or 72 h, respectively, the cells were

washed withTBS,trypsinized, pelleted by low-speed centrifugation, and fixedat 40C in3% glutaralde-hydeinMillonigsphosphate buffer forembeddingin Spurr low-viscosity media. The embedded cultures werestained with saturateduranyl acetatein50% ethanol andReynoldslead citrate, sectioned on an MT2-B ultramicrotome, and examinedon an RCA EMU 3 microscope operated at 100 kV. Duplicate cultureswereharvestedby freezingandthawingto monitor virus yields.

Heat inactivation. Virus stocks werediluted

10-fold in TBS andsonically treated for30 s. Aftera

further 100-fold dilutionincoldTBS,0.2-mlportions were dispensed into replicate tubes that were im-mersedina500Cwaterbath. Atspecified intervals, atubewasremoved, and thecontents werediluted withcold TBSandassayed for surviving infectivity by the plaquetechnique.

Analysis of viral polypeptides and capsomeres.

Polypeptides synthesized in infected cells were

la-beled byadding [35S]methionine (Amersham/Searle Corp., ArlingtonHeights, Ill.)at afinal concentra-tionof 5,Ci/mlto a culture mediumcontaining0.1 the normal concentration of methionine. Infected cultures wereexposedtothe radiolabeled precursor forvaryinglengths oftime.

Infected-cell extractswereprepared from100-mm petri dishes. The cellswereremovedby scrapingat

40C andweresedimentedat1,000 xg for5min; the cell pack was washedtwicewith ice-cold TBS. The cell pellet was suspended again in 1 ml ofTBS containing300,ugofphenylmethyl sulfonyl fluoride per ml and frozenat -20°C untilanalysis. Atthat time, the cellpelletsweredisruptedinsample buffer

(0.0625MTris,2%SDS, 5%2-mercaptoethanol,30%

sucrose, and 0.005% bromophenol blue, pH 6.8), heated to 1000C for 2 min, and analyzed by the discontinuousSDS-polyacrylamide gel

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

ts MUTANT OF ADENOVIRUS TYPE 7 1161 sis (SDS-PAGE) system of Laemmli (15). Samples

were subjected to electrophoresis on slab gels 1.5-mmthickfor4h at 100 V.Separating gels contained 12%acrylamide, 0.12% bisacrylamide, 0.1% SDS, 0.5 M urea, and 0.375 M Tris-hydrochloride (pH 8.8); stacking gels contained4%acrylamide, 1% bisacryl-amide, 0.1% SDS, 0.5 M urea, and 0.05 M Tris-hydrochloride (pH 6.8).

Intact viral capsomeres were analyzed either on non-dissociating slab gels containing 5% acrylamide and 0.13% bisacrylamideinTris-glycine buffer (pH 8.3)with a spacerpreparedby the method of Maizel (21), or bysedimentation of [35S]methionine-labeled cytoplasmic extracts in 5 to 20% neutral sucrose gradients inan SW41 rotor at 39,000 rpm for 16 h (22). Cytoplasmic extracts were prepared from washed cells suspended againin 0.14 MNaCl-0.01 MTris-hydrochloride (pH 7.2)-1.5 x 10-3 MMgCl2, andlysedby the addition of NonidetP-40(NP-40)to afinalconcentrationof0.5%.After5min at40C,the nuclei were removed by centrifugation at 200xg for 5 min (6). The cytoplasmic and nuclear fractions weredialyzed against the electrophoresis buffer con-tainingphenylmethylsulfonyl fluoride priorto elec-trophoresis. Insomecases, thecytoplasmicextracts werecentrifuged at45,000 rpm at4°C for 2h in a Ti5Orotor to remove aggregated material (24), but this wasfoundtobeunnecessary. After

electropho-resis, the gels were fixed with 5% methanol-7.5% aceticacid, driedonfilterpaper, andexposedto

X-TABLE 1. Relativeyields ofseveral Ad7 isolatesfrom KBcells atthe permissive(33°C) and nonpermissive

(40.5°C) temperatures

Virus yield(PFU/ml)a from Virusyield Ad7isolate cultures incubated at: at

40.5°C/vi-rusyieldat

33°C 40.5°C 33°C

Prototype 4.0 x 107 4.0 x 107 1.0 Wyeth 4.0 x 106 8.0 x 106 2.0 Ginsberg 4.5 x 107 6.9 x 107 1.5

Hu 1.4 x 107 3.4 x 107 2.4

19 2.0 x 107 6.0 x 104 3.0 x 10-3

aVirusyields from infectedKBcellcultures

incu-bated at 33 or 40.5°C for 96 or 72 h, respectively,

weredetermined by plaque assay in HEK cells at 37°Casdescribedinthetext.

rayfilm. The developedautoradiograms were

ana-lyzedwithaCanalco model G densitometer.

RESULTS

Isolation and growth characteristicsof

mu-tant virus. Ad7 (19) was isolated by plaque

purification asthe nonhybrid adenovirus

com-ponent from the defective SV40-Ad7 hybrid

(PARA) population (4, 5). It has been used in

many subsequent studies of the defective

hy-bridvirus (26). Thets natureof thisisolatewas

investigated after the observation that ats

le-sion exists inthe adenovirus regionofthe

hy-brid PARAgenome thataffects the replication

of PARA (M. K. Estes, M. J. Guentzel, and J.

S. Butel, Abstr. Annu. Meet. Am. Soc.

Micro-biol. 1973, V36, p. 200).

Comparisons of virus yields from the

permis-sive (33°C) and restrictive (40.5°C)

tempera-tures withseveral strains of Ad7 are shown in

Table 1. All the wild-type (WT) Ad7 isolates

tested (i.e., prototype, Wyeth, Ginsberg, and

Hu)exhibited similar virus yieldsatboth

tem-peratures. In contrast, the Ad7 (19) isolate

showedatleasta300-fold reduction in yieldat

40.5°Cascompared with thatat33°C,

illustrat-ingthetsphenotype of this virus. An analysis

of thekinetics of growth of Ad7 (19)inKBcells

confirmed themarkedinhibition of virus

multi-plication at40.5°C (Table 2). The growth cycle

was essentially complete by 48 h p.i. at both

temperatures. Alsoconfirmed in Table2is the

fact that Ad7 (Hu), the WT virus used in all

furtherexperiments, didnotrevealany

restric-tion ofgrowthat40.5°C.

The Ad7 (19) mutant typically exhibits a

ra-tio ofyieldsin KBcells at40.5°C/33°Cof 10-2to

10-3. The plaquing efficiency of the uncloned

adenovirus component in theparental

PARA-Ad7 population at 40.5°C/33°C is 10-3 or less.

The cloned Ad7 (19) mutant displays greater

leak and/or reversion during growth in HEK

cells, such that the relative yield at 40.5°C to

that at33°C occasionally approaches 10-1.

Be-cause ofthis, experiments performed in HEK

TABLE 2. Kineticsofgrowth ofmutantAd7inKBcellsa Incubation Virus yield (PFU/ml) at hours p.i.:

Virustemp (~C) 6 24 48 72 96

Ad7 (19) 33 5.0 x 103 5.0 x 105 1.5 x 107 2.2 x 107 2.4 x 107

40.5 2.8 x 103 1.2 x 103 1.1 X 105 6.4 x 104 6.2 x 104

Ad7(Hu) 33 NDb ND ND ND 6.0 X 106

40.5 ND ND ND ND 6.0 x 106

aVirusyieldsfrom infected KB cultures incubated at 33 or40.5°Cforindicatedtimesweredeterminedby

plaque assayin HEKcellsat37°C, asdescribedinthe text.

bND, Not done. VOL. 21, 1977

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.501.57.246.395.487.2] [image:3.501.60.452.555.635.2]

(4)

cells always included duplicate cultures

har-vestedtomonitor theamountof leak and

rever-sion; no results were considered valid if the

ratio of virus yield at 40.5°C/33°C wasgreater

than 10-2.

Induction of viral DNA synthesis. The abil-ityoftsmutantAd7 (19)toinduce thesynthesis

ofviral DNA in HEK cells under

nonpermis-sive conditions was examined by using a 2-h

pulse of [3H]TdRatvarious timesp.i. (Table 3).

The incorporation of [3H]TdR by mock-infected

control HEK cells was greatly decreased at

40.5°C compared with that at 33°C; markedly

enhanced incorporation of label did occur at

40.5°C, however, when the cellswere virus

in-fected. These datasuggested that the majority

ofthe DNA being synthesized at40.5°C by 34h

p.i. was probably virus specific. However, to

confirm that the DNA synthesized at 40.5°C

was viral in nature, samples from infected

HEK cells wereanalyzed by equilibrium

centi-fugation. The DNA in Hirt supernatant

frac-tions obtained from infectedHEKcells, which

had been labeled with [3H]TdR from24 to 48 h

p.i., was characterized. A similar,

homogene-ous peak of labeled material that bandedat a

density of 1.710 g/ml was obtained from

mu-tant-infected cells incubated under either

per-missive or nonpermissive conditions (Fig. 1).

DNA with a similar buoyant density was

ex-tracted from WT-infected cells and from

puri-fied Ad7 (Hu) virions (datanot shown).

Induction of virus-specific antigens.

Mu-tant-infected cells weremonitoredby

immuno-fluorescence for the production of virus-specific

antigens. HEKcells infectedwith Ad7 (Hu) or

Ad7(19)atanMOI of 2PFU/cellwerefixed and

examined for the presence of tumor, capsid,

hexon, andpentonantigens.As shown inTable

4, all these virus-specificantigenswere

synthe-TABLE 3. Incorporationof[3H]TdRinto trichloroaceticacid-precipitablematerialbyHEK

cells infectedwith Ad7(19)at33or40.5oCa

cpm/,g of DNA Time of

pulse a- 33°C 40.5°C

bel(h

p.i.) Mock in- Ad7 (19) Mockin Ad7 (19)

fected fected

0-2 3,460 3,500 1,966 900

24-26 6,871 6,666 49 4,200

32-34 5,640 10,750 50 11,549 36-38 4,465 21,989 50 4,223

48-50 1,708 7,404 100 787

aControlorvirus-infected HEK cellswerelabeled

with 5

ACi

of[3H]TdRpermlfor the indicated time

intervals p.i. Cellextracts wereprepared and

pre-cipitatedwithtrichloroacetic acidatthe endof the pulse timeasdescribedinthetext.

4

C-)

x

I

3

2

1

1.80

1.70 gm/ml

1.60

FRACTION NUMBER

FIG. 1. Densitygradient analysis of DNA

[image:4.501.261.452.61.256.2]

synthe-sized in Ad7 (19)-infected HEK cells at 33 and

40.50C. Cultures of HEK cells were infected withan

MOI of 10 PFUlcell and labeled with 5puCi of [3H]TdR per ml from24to 48h p.i.Viral DNA was extractedby the Hirt procedure,purified,and centri-fugedtoequilibriuminCsCl for72hat35,000 rpm at200Cin a TiSO rotor. Symbols: *, DNA synthe-sizedat330C; 0,DNAsynthesizedat40.50C. Arrow indicates positionatwhich marker-cell DNA bands. TABLE 4. SynthesisofvirusantigensinHEKcells

at40.50Ca

Synthesis of virus-specific antigen: Virus

Tumor Capsid Hexon +

fiber

Ad7 (19)b + + + +

Ad7(Hu) + + + +

a

HEK

cells growingon coverslipswereinfected

withAd7 (19) orAd7 (Hu) at anMOI of2PFU/cell. At48 hp.i., the cellswerefixed withacetoneand stainedby the indirect immunofluorescence test as describedinthetext.

Relative yield of mutant

(40.50C/330C)

= 1 x 10-2.

e +,Presence ofantigen.Approximately40 to 50% of the cellsin a culture were positive for a given antigen. Allfluorescentreactions were observedin the nucleus.

sized at 40.50C, with approximately the same

percentage of cells showing positive

fluores-cence at40.50C as at 330Cfor eachgiven

anti-gen. All the virusantigens detectedby

immu-nofluorescence werelocated in the nucleus.

Production ofvirus particles. Electron

mi-croscopic examination of thin sections of

in-fected HEK cells wasperformed. The nuclei of

WT Ad7 (Hu)-infectedcellsrevealed large

crys-talline arrays of virus at both temperatures.

Typical WT-infected cells at 33 and40.5°C are

showninFig.2Aand B. Thecytoplasmic

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.501.262.452.378.440.2]

(5)

MA

An'~~~~~~~~~~~~~~r

.. ;.- ~W 'P O

FIG. 2. Electronmicrographs of thin sections ofHEK cells infected with Ad7 (Hu) or Ad7 (19) for 72 or 96 hat33 or40.5 C,respectively. (A) Ad7 (Hu)-infected HEK cell at 330C. x4655. (B) Ad7 (Hu)-infected HEK cellat40.50C. x7130. (C) Ad7 (19)-infected HEK cell at 330C. x8060. (D) Ad7 (19)-infected HEK cell at 40.50C. x6080.

1163

-31

;.". a

... ..-t.

-:. ..-v".", 1; 't

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.501.59.443.51.620.2]

(6)

1164 ESTES AND

phous material seen in mutant-infected cells at

40.5'C, and describedbelow, waslackinginthe

WT-infected cells,

although

somenuclear inclu-sionbodieswere seen(Fig. 2B). Thepercentage

of cells infected at 40.50C with Ad7 (Hu) was

approximatelyhalf that observed at330C, even

though the WT virus produced 108 PFU/ml at 40.50C.

Cells infected with Ad7 (19) revealed that

intact virusparticleswereapparentlynotmade

as efficiently at 40.50C as at 330C. In cells

in-fected at330C, 80 to 90% of the nuclei contained

vast crystalline arrays of virus (Fig. 2C). In

contrast, virus particles were observed inless

than50%of thecells maintainedat40.50C,and very few crystalline arrays were detected.

In-stead, large areas of nuclear inclusion bodies

were seen, and amorphous substances were

prominent in the cytoplasm of cultures from

40.50C (Fig. 2D). TypeIandtype IIinclusions,

asdescribed for Ad12 infections (23), were sel-dom seen at 330C but were common atthe

re-strictivetemperature. Nuclear membrane

frag-mentation occurred late after infection (not

shown) at

330C,

but itwas seldomobserved at

40.50C

in mutant-infected cells. The relative

yield of the ts mutant in these experiments

(40.50C/330C)

was7.5 x 10-3.

Heatstability ofmutant virions. That Ad7

(19) particle assembly appeared altered at the

nonpermissive temperatureraised the possibil-ity that a structural component of the virion might beaberrant. Therefore,the effect of

heat-ing at500Contheinfectivityof boththe mutant

Ad7 (19) and the WT Ad7 (Hu) virus grown at

37°C was examined (Fig. 3). The infectivity of

Ad7 (19) was inactivated more rapidly than

that of theWT virus; only 1%of theinfectious

mutant virus survived after 9 min at 500C,

whereas about 26min ofheatingwasrequired

toreduce theWTinfectivitytoasimilar level. The heatlability of the mutant virus suggests

that thetsdefectmay lie in astructural compo-nent of thevirion.

Synthesis of polypeptides in infected cells.

The technique of PAGE is a powerful tool for

probing events in infected cells at the

poly-peptide level. The synthesis of polypeptides

in WT-and Ad7 (19)-infectedcellslabeledwith

[35S]methionine wasmonitored todetermineif

any structural polypeptides were lacking that

might account for the aberrant virion

assem-bly. Analysis by SDS-PAGE of cells labeled

from 18to 42 h p.i. at 40.5°C or from 24 to 48 h

p.i. at 33°C revealed that at least the major

structural polypeptides were synthesized in

both mutant- and WT-infected cells at both

temperatures (Fig. 4). Polypeptides II, III, IV,

V, andVI, asdesignatedby Maizel (22), can be

100

0

r

(L-C., a

-n

(f)

I--a

LLI

Q~

10

10 20 30

MINUTES AT 50 C

FIG. 3. Heat inactivation of Ad7 (Hu) and Ad7 (19)at50°C.Portionsofsonically treated virus stocks were immersed ina waterbath at50°C forvarying intervalsoftime and thenassayedfor surviving

in-fectivity, asdescribed in thetext.

detected, and the patterns in mutant- and

WT-infectedcells were similar when compared at 33

or40.5°C. Becausethese experiments withlong

labeling periods did not reveal differences in the polypeptide patterns between the Wt- and

mutant-infected cells at the nonpermissive

temperature,noattempts were made to

investi-gate the processing ofpolypeptides.

Oneinteresting observationwasthat the

mu-tant virions did not appear to shut off host

proteinsynthesis aseffectivelyasWTvirus,as

evidencedbythehigher levels of background in

the mutant-infected cells at both thepermissive

and nonpermissive temperatures. All the

ex-tracts for polypeptide analysis were prepared

from cells infected with 5 PFU of virusper cell,

and 100,000 counts of radioactivity was

ana-lyzed with eachsample. Experiments designed

tofurthercharacterizethe lack of inhibition of

host protein synthesis were hampered by the

leakiness exhibited by this mutant at higher multiplicities of infection. The lack of

inhibi-tion ofsynthesis of host proteins late in

infec-tionobscures thequantitation of the

virus-spec-ified proteinsin the mutant-infected cells.

Al-though the major virionpolypeptides appeared

tobe synthesized in mutant-infectedcells,SDS

gelsseparate polypeptides onlyonthe basis of

molecularweight, and,therefore, these

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.501.285.426.64.315.2]

(7)

ts MUTANT OF ADENOVIRUS TYPE 7 1165

4*

.4 E n

-1*0 ma

A

i

w

II

S

-_F

A_

vCi

1 _ _r~~-

11

i - 94K

-_-

-III

...A

Imokl'

--

iia

50K

--v

- 45K

-

VI

- 23.5K

_W- ..- Vil

vIi*

A.B

A

B

C

D

E

F

G

H

I

FIG. 4. SDS-PAGE autoradiogram of Ad7 (Hu)- or Ad7(19)-infected KB cells labeled from18to 42hat

40.50Corfrom24to 48h at330Cwith 5pLCiof[35S]methionineper ml.Plates (100 mm)infected withanMOI

of5PFU/cellwereprocessed as describedin the text, and approximately 100,000cpm wasappliedtoeach

samplewell. Thesampleorder isasfollows: (A) mock-infectedcellsat330C; (B)Ad7(Hu)-infectedcellsat

33C; (C) Ad7(19)-infected cells at 33 C; (D) mock-infected cells at40.50C; (E)Ad7(Hu)-infected cells at

40.50C; (F) Ad7 (19)-infected cells at40.50C; (G)purified [35S]methionine-labeled virus; (H) hexon band

elatedfrom the non-dissociating polyacrylamide gel ofAd7 (Hu)-infectedKBcell cytoplasmic extractshown inFig. 6C; and (I) hexon band elated from the non-dissociatingpolyacrylamide gel ofAd7 (19)-infected KB cell cytoplasmic extract shown in Fig. 6B. Wells (A) through (G) were subjected to electrophoresis inthe same slab gel. Roman numerals indicate the viral polypeptides (nomenclature ofMaizel[22]) and the arabic numbers indicate the location of molecular weight markers detected by Coomassie brilliant blue staining: phosphorylase A, 94,000; heavy-chain gamma globulin, 50,000; SV40 polypeptide I, 45,000; and light-chain gammaglobulin, 23,500.

ments could not determine ifassembly of the

polypeptides intocapsomereswasaberrant.

Synthesis of viralcapsomeres.Todetermine

ifthe viral polypeptides were assembled into

capsomeres, cytoplasmic extracts of WT- and

mutant-infected cells labeled with

[35S]methio-ninewereanalyzedonsucrosegradientsandon

non-dissociating polyacrylamide slab gels (pH

8.3). From the sucrose gradients, it was

ap-parent that, although the extent of

incorpora-tion of radioactivity was reduced at 40.50C,

capsomereswereinfactformed(Fig. 5).

Analy-sis of fractions from the sucrose gradient by

SDS-PAGE showed that they contained

pre-dominately the hexon andpentonpolypeptides.

This observation is in agreement with earlier

work with Ad2 (17).

An analysis of infected-cell cytoplasmic

ex-tracts (viral capsomeres) on 5%

non-dissociat-ing polyacrylamide gels (pH 8.3) revealed the

presenceofonemajorandoneminorband that

were not present in extracts ofmock-infected

cells (Fig. 6). The major band from both WT-andmutant-infected cellextracts wasidentified

asthe hexon

polypeptide by eluting

the band andanalyzingitby SDS-PAGE (Fig.4HandI). The Ad7 (19) hexon had a molecular

weight

identicaltothat ofWThexon whenanalyzedon

5or12%SDS-PAGE. The minor band could not

beanalyzedfurther due toinsufficient

incorpo-rationofradiolabelcounts. Thematerialatthe

top of thegels, seen inFig6BandC, contained

primarilythe corepolypeptidesVand VII and

specifically lacked hexon

polypeptide.

The

bands at the bottom represent free

[35S]_

methionine.

A comparison of the mobility of the hexon

capsomeres from extracts of WT- and

mutant-VOL. 21, 1977

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.501.53.451.74.345.2]

(8)

1166

ESTES AND BUTEL

0o

m

CD

x

z

2

LLJ

AAd7 (19)-33 C AAd 7 (19)-40.5 C oAd 7 (Hu)-33 C

*Ad7(Hu)-40.5 C

5 10 15 20 25

FRACTION NUMBER

FIG. 5. Sucrose gradientcentrifugation of capso-meresfrom cytoplasmicextractsof KB cells infected with Ad7(Hu)orAd7 (19) atanMOI of 5 PFU/cell

and labeled with [35S]methionine from 24 to 48 h (330C) or from 18 to 42 h (40.50C). Cytoplasmic

extractsweremade andanalyzedasdescribed in the

text.Thecapsomereregion(fractions 16 through 19) contained both hexon and pentonpolypeptides, as

shown bySDS-PAGE analysis. The variousextracts

areindicatedby the following symbols:0,Ad7

(Hu)-infected KB cells, 330C; 0, Ad7 (Hu)-infected KB

cells, 40.50C; A, Ad7 (19)-infected KB cells, 330C; and A,Ad7(19)-infected KB cells, 40.50C.

infected cellsinnon-dissociating gels, however,

revealed a decrease inthe mobilityof the

mu-tant hexon (Fig. 6B through E). This altered

mobility wasevident in hexons extracted from

KB or HEK cultures incubated at the

per-missive (330C) as well as the nonpermissive

(40.50C) temperatures. Two bands are evident

when themutantand WTcapsomeres are

sub-jected toco-electrophoresis (Fig. 6F).

Hexons extracted from cells infected with

Ad7prototypeorAd7 (Ginsberg) showed

identi-calmigrationtothe Ad7(Hu)WThexons(data

not shown). These resultssuggest that the

de-fective structural protein of the Ad7 (19)

mu-tantmaywell be the hexonpolypeptide.

Interference with replication of

heterolo-gous adenoviruses. Co-infection experiments

in KB cells were performed as described in

Materials and Methods to ascertain whether the mutantlesioninAd7(19)exertedanyeffect

on the replication of heterologous Ad2 and

Ad2l. In the presence ofco-infecting WT Ad7

(Hu), less thanatwofoldreductioninthe yields

of Ad2l orAd2 at either the permissiveorthe

nonpermissive temperature took place (Table

5). Similarly, in the presence of co-infecting

Ad7 (19) under permissive conditions (330C),

minimal reductions in the yields of

heterolo-gousvirusesoccurred(1.8-foldfor Ad2land

7.8-foldwith Ad2.). However, under conditions of

incubation that block the production of

infec-tious mutant viruses (40.50C), there were

marked reductions in the yields of both Ad2M

(18-fold) and Ad2 (19-fold). The basis for this

interference exerted by mutant Ad7 (19), and

notby WTAd7, is not known.Onepossibilityis

that a mutant protein bearing the Ad7 (19)

lesion is used by the co-infectingheterologous

adenoviruses andrenders them either

noninfec-tious orunabletoreplicate efficiently.

Titration of the Ad7 yields from the doubly

infected cellsrevealedthat, in some instances,

the presence ofAd2 orAd2M hadadeleterious

effecton the replication of human Ad7 (Table

6). Ad2M co-infection resulted in a marked

de-creaseinyields (112-fold)of Ad7(Hu) at40.50C

and of Ad7 (19) at 330C (84-fold). Ad2M had

A

B

C

D

E

F

G

FIG. 6. Non-dissociating PAGE autoradiogram of Ad7 (Hu) or Ad7 (19) viral capsomeres. KB cells infectedat an MOIof 5PFU/cell werelabeled with

5,uCiof[35S]methionineper ml from 24 to 48 h(330C)

or 18 to 42 h (40.50C). Cytoplasmic extracts were preparedasdescribed in the text. One hundred thou-sand counts weresubjectedtoelectrophoresis atpH 8.3 inTris-glycine buffer. The anode is at the bottom. The cytoplasmic extracts were from the following samples:(A) mock-infected KB cells,330C;(B) Ad7 (19)-infectedKB cells,330C; (C) Ad7 (Hu)-infected KB cells, 330C; (D) Ad7 (19)-infected KB cells,

40.50C;(E) Ad7(Hu)-infectedKBcells,40.50C; (F) mixture ofequal amounts ofsamples (D) and (E) and

(G)mock-infectedKBcells, 40.5°C. (A), (B), and (C)

were from one gel analysis and can be compared directly; (D), (E), (F), and(G) were from a separate experiment.

J. VIROL.

IC

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.501.56.245.63.244.2] [image:8.501.264.452.282.499.2]

(9)

[image:9.501.52.450.82.211.2]

ts MUTANT OF ADENOVIRUS TYPE 7 1167

TABLE 5. Interference by mutant Ad7 on replication of heterologous adenoviruses at elevatedtemperatures

Yieldof heterologousvirus inpresenceof:

Ad7 (Hu) Ad7(19)

Heterol- Ad7

ogous vi- pres- 33°C 40.5aC 33°C 40.5aC

rus ent

PFU/ml _duction'Fold re- PFU/l Fold re- PFU/ml Fold re- PFU/ml

reduc-a m duction duction P

tion

Ad2M 0 1.0 x 108 - 3.9 x 107 - 3.6 x 106 - 1.0x 107

-+ 8.0 x 107 1.2 2.2 x 107 1.7 2.0 x 106 1.8 5.5 x 105 18

Ad2 0 6.0 x 107 - 7.2 x 105 - 3.9 x 107 - 3.9 x 105

-+ 3.6 x 107 1.6 4.1 x 105 1.7 5.0 x 106 7.8 2.0 x 104 19

a Fold reduction=yieldofheterologous virus in absence of Ad7/yield of heterologous virus in presence of

Ad7.

TABLE 6. Effect of heterologous adenoviruses on replication of human Ad7 Yieldof:

Heterolo- Ad7 (Hu) Ad7 (19)

gousvirus 33°C 40.5°C 33°C 40.5°C

present_________________________________________________

PFU/ml _ductionFold re- PFU/ml Fold re-_duction PFU/ml Fold re- PFU/ml Fold

re-duction duction

None 4.7 x 107 - 2.7 x 107 - 4.2 x 107 - 1.5 x 105

-Ad2M 7.3 x 106 6.4 2.4 x 105 112 5.0 x 105 84 2.4 x 105 0

Ad2 4.3 x 105 109 1.7 x 106 15.8 5.9 x 106 7.1 7.0 x 104 2

a Fold reduction =yield of Ad7inabsence of heterologous virus/yield of Ad7 in presence of heterologous virus.

considerably less effect on replication of Ad7

(Hu) at 330C. Co-infection with Ad2 caused a

decrease in Ad7 (Hu) yields at both 33 and

40.5°C (109-fold and 15.8-fold, respectively).

Ad2 did not inhibit Ad7 (19) replication to as

great an extent (7.1-fold at 33°C). Asexpected,

since Ad7 (19) was ts for replication, the

co-infecting heterologous adenoviruses had little effect on yields at 40.5°C. The basis for the

inhibitory effects on Ad7 replication by Ad2M

and Ad2 iscurrentlyunknown.

DISCUSSION

This report characterizes a naturally

occur-ring ts mutant of

weakly

oncogenic human

Ad7. Thismutant, Ad7 (19), appearstobe de-fective in a late gene function that interferes

with the assembly ofvirus particles. Particles

made at the permissive temperature exhibit

greater thermolability than do WT virions,

which suggests that the defect may involve a

virion structural polypeptide. Analysis by

PAGErevealedthat the major structural poly-peptides (hexon, penton, fiber, and core) are

synthesizedat40.5°C.

Precise quantitation ofvirus-specific protein

synthesis was difficult, because the inhibition

ofhost protein synthesis by the mutant, was

notefficienteven atlate stagesafter infection.

This apparent lack of inhibition of cellular

mac-romolecular synthesis has not been described

forassemblymutantsof otheradenovirus

sero-types.Thepossible regulatory role of the hexon

polypeptide is intriguing. Previous studies (2)

suggestedarelationship between the synthesis

of capsid proteins and the inhibition of host

proteinsynthesis. Although the suggestion was

made thatthe fiber antigenmight perform this

function(2, 18), ithas been shown that detecta-ble levels of fiber are not present at the time

hostprotein synthesisbegins to decline (1, 29,

38). Hexonpolypeptides, however, are

detecta-ble at that time (1, 29), and our results are

consistent with the possibility that the hexon

may be involved in inhibition ofhost protein

synthesis.

Levine and Ginsberg (19) showed that both

fiber and hexon antigenscan bind to KB cell

and adenovirus type5 (Ad5) virus DNAin

vi-troandinhibitDNA-dependentRNA

polymer-aseactivity. However,the functioningofthese

proteinsinvivoremains unproven. Thelack of

bindingto cell DNAby the aberrant Ad7 (19)

hexonwould substantiate these theories.

Analysisonnon-dissociating gelsof

cytoplas-mic extracts from infected cells revealed an

alteredmobilityfor the hexon capsomeres,

sug-gestingthat thets lesionresidesin the hexon

VOL. 21, 1977

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

gene. The altered

mobility

could be due to a

change in the amino acid composition of the hexonpolypeptide such that its chargewas al-tered.Slight variationsinthe

migration

in

gels

of hexons from three different adenovirus

sero-types (types 2, 3, and 5) have been reported;

concomitant amino acid analyses revealed a

lower glutamic acid

composition

in the least

electrophoretically

mobile adenovirus type 3 (Ad3) hexons (25).

The possibility that the Ad7 (19) mutation that affects assembly might involve a lack of

viral polypeptidetransportisunlikely, but the

accumulation in the cytoplasm of large

amounts of

amorphous

material detected

by

electron microscopy remains

unexplained.

The

transport of the hexon and penton

antigens

does not appear to be

altered;

these antigens

weredetectedinthe nucleus

by

indirect

immu-nofluoresence. PAGE analysis of nuclear

frac-tions from infected cells also showed the

pres-ence of the hexon

polypeptide

in the nucleus.

However, it ispossible that the altered hexon

mayhavemodifiedthetransport ofother

poly-peptides

involvedinthe maturationprocess. It should be noted that the hexonantiserumused

in this studywould not detect hexon

polypep-tidesthat might accumulate in the

cytoplasm

(32). Since intact hexons are found

predomi-nantly in the nucleus, it is possible that the

hexons being analyzed are actually nuclear

hexonsthat leak intothe cytoplasmduringthe

extractionprocedure. Asimilarprocedure does

extract the intranuclear SV40 tumor antigen

(37).

The characteristics of the Ad7 (19) mutant

arereminiscent of the adenovirustype 5classI

mutants which fallinto four complementation

groups (42). ClassI mutantsareassembly

mu-tants;they shownomajordefectsinthe

produc-tionofcapsid antigens (28) andno marked

re-ductioninthe synthesis ofcapsidpolypeptides

at the restrictive temperature (30).

Thermola-bility of the type 5 mutants and synthesis of

capsomereshavenotbeenreported. Of the class

Itype 5 mutants,some could undergo intertypic

complementation with WTAdl2 inHela cells;

the resultantprogeny of crosses between several mutants with defects in hexon transport or pro-duction exhibited phenotypic mixing. In that

system,theAdl2did not inhibit theAd5

multi-plicationin Hela orhamster embryo cells, but the

effect

of the Ad5 mutants on thereplication

of WTAdl2was nottested (42, 43). These

obser-vationsare of interest, since the Ad7 (19)

mu-tantappears to affect the multiplication of

het-erologous adenoviruses in mixed infections in

KBcells, and phenotypic mixing between these

serotypes also appears to occur (M. K. Estes

andJ. S. Butel,unpublisheddata).

Although theAd7mutantdescribed here was

a natural isolate, its defect appears different

thanthe natural block inassemblyobservedat

42°C with adenovirus 2 (17, 24). In that system,

there was a failure of the hexon polypeptides (monomers) to assemble into capsomeres

(tri-mers) at 42°C (17). The number of capsomeres

detected on non-dissociating gels at 42°C was

much decreased. The trimers and monomers,

however, exhibited identical tryptic peptides,

suggestingthat something other thanjust the primary structure is involved in the assembly

of Ad2at42°C. Our datashow that theAd7(19)

capsomeres are made at 40.5°C, but their

al-tered migration on the non-dissociating gels

suggests that the Ad7 (19) block in assembly

may reflect achange in the primary structure

of the hexon polypeptide, which affects the

hexoncapsomeres. There are obviously

multi-plesteps in assembly; further studies are

neces-sary to determine if the observed biological

changes with Ad7 (19) are due to a direct or indirect effect of the altered hexon polypeptide.

ACKNOWLEDGMENTS

Wewould like to thank Betty Altenburg for performing theelectron microscopic work. We are also grateful to Phil Hopperand Peggy Sansone for excellent technical assist-ance.

This work was supported by Public Health Service re-search grant CA 10,893 from the National Cancer Institute. M.K.E. is therecipient of Public Health Service postdoc-toralfellowship 1-F22-CA 03209 from the National Cancer Institute, and J.S.B. is the recipient of Faculty Research Award PRA-95 from the American Cancer Society.

LITERATURE CITED

1. Anderson, C.W., P. R. Baum, and R. F. Gesteland. 1973.Processingofadenovirus 2-induced proteins. J. Virol. 12:241-252.

2. Bello, L.J., and H.S. Ginsberg.1967.Inhibitionof host proteinsynthesisintype5adenovirus-infectedcells. J.Virol. 1:843-850.

3. Benyesh-Melnick,M., and H. S. Rosenberg. 1964. The isolation ofadenovirus type 7 from a fatal caseof pneumonia and disseminated disease. J. Pediatr. 64:83-87.

4. Boeye,A., J.L.Melnick, and F. Rapp.1965. Adenovi-rus-SV40"hybrids": plaquepurification intolinesin which the determinant for the SV40tumorantigen is lostorretained. Virology 26:511-512.

5. Boeye, A., J. L. Melnick, and F. Rapp. 1966. SV40-adenovirus"hybrids":presence oftwogenotypesand the requirement of theircomplementation for viral replication. Virology28:56-70.

6. Borum, T. W., M. D. Scharff, andE.Robbins. 1967. Preparation of mammalianpolyribosomes with deter-gent NonidetP-40. Biochim.Biophys. Acta 149:302-304.

7. Butel, J. S., and F. Rapp. 1966. Replication in simian cellsof defectiveviruses in anSV40-adenovirus "hy-brid"population.J.Bacteriol. 91:278-284.

8. Butel, J. S., and F. Rapp. 1967. Complementation be-tween a defective monkey cell-adapting component and humanadenoviruses in simian cells. Virology 31:573-584.

9. Ensinger, M. J., and H. S.Ginsberg. 1972. Selection

on November 10, 2019 by guest

http://jvi.asm.org/

(11)

ts MUTANT OF ADENOVIRUS TYPE 7 1169 and preliminarycharacterization of

temperature-sen-sitivemutantsoftype5adenovirus.J.Virol. 10:328-339.

10. Fenner,F., B. R.McAuslan, C. A. Mims, J. Sambrook, and D.0.White.1974.Thebiology of animal viruses.

AcademicPressInc., New York.

11. Hama, S., and G. Kimura.1972.Temperature-sensitive

mutantsofhuman adenovirustype12.Jpn. J. Micro-biol.16:337-338.

12. Henry, P., P. H. Black, M. N. Oxman, and S. M. Weissman. 1966. Stimulation of DNA synthesis in

mousecell line 3T3 by simian virus40. Proc. Natl. Acad. Sci. U.S.A.56:1170-1176.

13. Hirt, B. 1967. Selective extraction ofpolyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369.

14. Ishibashi, M.1971.Temperature-sensitive conditional-lethal mutants ofan avianadenovirus (CELO). I. Isolation andcharacterization. Virology 45:42-52. 15. Laemmli, U. K. 1970. Cleavageofstructuralproteins

during the assembly of the head of bacteriophageT4. Nature (London) 227:680-685.

16. Ledinko, N. 1974. Temperature-sensitive mutants of adenovirustype12defectiveinviral DNA synthesis. J. Virol.14:457-468.

17. Leibowitz, J., and M. S. Horowitz. 1974. Synthesis and assembly of adenovirustype 2 polypeptides. II. Re-versible inhibitionof hexonassemblyat42°. Virology 61:129-139.

18. Levine, A. J., and H. S. Ginsberg.1967. Mechanism by which fiber antigen inhibits multiplication oftype5 adenovirus. J. Virol. 1:747-757.

19. Levine, A. J., and H.S. Ginsberg.1968.Roleof

adenovi-russtructural proteins in the cessationof host-cell biosynthetic functions. J. Virol. 2:430-439. 20. Lundholm, U., and W. Doerfler. 1971.

Temperature-sensitive mutants ofhumanadenovirustype12. Vi-rology45:827-829.

21. Maizel, J. V. 1967. Acrylamide gel electrophoresis of proteins andnucleic acids,pp.334-362.InK.Habel

and N. P.Salzman(ed.), Fundamental techniquesin virology. Academic Press Inc., New York.

22. Maizel, J. V., Jr., D.0.White, and M.D.Scharff.1968.

Thepolypeptidesofadenovirus.I.Evidence for multi-ple proteincomponentsinthevirionand comparison of types2,7Aand12.Virology 36:115-125. 23. Martinez-Palomo, A., J. LeBuis, and W. Bernhard.

1967. Electron microscopy of adenovirus12 replica-tion. I. Fine structural changes in the nucleus of

infectedKBcells. J. Virol. 1:817-829.

24. Okubo, C. K., and H.J. Raskas. 1971. Thermosensi-tive events inthereplicationofadenovirustype2 at 42°. Virology 46:175-182.

25. Pettersson, U.1971.Strcturalproteinsof adenoviruses. VI.On theantigenic determinants of thehexon. Vi-rology 43:123-136.

26. Rapp,F. 1973.ThePARA-adenoviruses, p.104-137. In

L. P. Merkow and M. Slifkin (ed.), Progressin

ex-perimental tumorresearch, vol. 18. Karger, Basel, Switzerland.

27. Rapp, F.,S.Pauluzzi,and J. S. Butel. 1969. Variation

inpropertiesofplaqueprogenyof PARA (defective

simian papovavirus40)-adenovirus7.J.Virol. 4:626-631.

28. Russell, W. C., C. Newman, and J F. Williams. 1972. Characterization oftemperature-sensitivemutantsof adenovirus type 5-serology. J. Gen. Virol. 17:265-279.

29. Russell, W. C., and J. J. Skehel.1972.Thepolypeptides of adenovirus-infectedcells.J. Gen.Virol. 15:45-57. 30. Russell, W. C., and J. J.Skehel.1974.Characterization of temperature-sensitivemutantsof adenovirustype 5:synthesis ofpolypeptides in infected cells. J. Gen. Virol.24:247-259.

31. Shiroki, K., J. Irisawa, and H. Shimojo. 1972. Isolation and apreliminary characterization of temperature-sensitive mutantsof adenovirus12.Virology49:1-11. 32. Stinski, M. F., and H. S.Ginsberg. 1974. Antibodyto type5adenovirus hexonpolypeptide: detection of

nas-cent polypeptides in the cytoplasm of infected KB cells.Intervirology4:226-236.

33. Suzuki, E.,and H.Shimojo.1971. A temperature-sen-sitive mutant of adenovirus 31, defective in viral deoxyribonucleic acid replication. Virology 43:488-494.

34. Suzuki, E.,H.Shimojo, and Y.Moritsugu. 1972. Iso-lation and preliminary characterization of tempera-ture-sensitive mutants of adenovirus 31. Virology 49:426-436.

35. Takahashi, M.1972.Isolationofconditional-lethal

mu-tants(temperature-sensitive andhost-dependent

mu-tants)of adenovirustype5.Virology49:815-817. 36. Takemori, N., J. L. Riggs, and C. H. Aldrich. 1968.

Genetic studies with tumorigenic adenoviruses. I.

Isolationofcytocidal (cyt)mutantsof adenovirustype 12.Virology 36:575-586.

37. Tegtmeyer, P., M. Schwartz, J. K. Collins, and K. Rundell. 1975.Regulationof tumorantigen synthesis bysimian virus 40geneA.J. Virol.16:168-178. 38. Walter, G.,and J. V.Maizel,Jr. 1974. Thepolypeptides

ofadenovirus. IV.Detectionofearlyand late virus-inducedpolypeptidesand their distribution in subcel-lular fractions.Virology57:402-408.

39. Weber, J., M. Begin, and G. Khittoo. 1975. Genetic analysisof adenovirus type 2.II. Preliminary pheno-typic characterization oftemperature-sensitive

mu-tants.J. Virol. 15:1049-1056.

40. Williams, J. F., M. Gharpure, S. Ustacelebi, and S. McDonald. 1971. Isolation oftemperature-sensitive

mutantsof adenovirus type5. J. Gen. Virol.

11:95-101.

41. Williams, J. F., and S. Ustacelebi.1971. Complementa-tion and recombination withtemperature-sensitive mutantsofadenovirustype5. J. Gen. Virol. 13:345-348.

42. Williams,J.F.,C. S. H.Young,and P. E. Austin. 1974. Geneticanalysisof human adenovirus type 5 in

per-missiveandnon-permissivecells. ColdSpringHarbor Symp. Quant.Biol. 39:427-437.

43. Williams, J., H.Young,and P. Austin. 1975. Comple-mentation of human adenovirus type 5tsmutantsby humanadenovirus type 12. J. Virol. 15:675-678. VOL. 21, 1977