Restricted changes in the adenovirus DNA-binding protein that lead to extended host range or temperature-sensitive phenotypes.

JOURNAL OF VIROLOGY,

JUlY

1985, p.206-212 Vol. 55,No. 1

0022-538X/85/070206-07$02.00/0

CopyrightX 1985,American Societyfor Microbiology

Restricted Changes in the

Adenovirus DNA-Binding Protein That

Lead

to

Extended

Host Range or

Temperature-Sensitive Phenotypes

DOUGLAS E. BROUGH, STEPHEN A. RICE, SUSAN SELL,AND DANIEL F. KLESSIG*

Departmentof Cellular, Viral, and Molecular Biology, UniversityofUtah Schoolof Medicine, Salt Lake City, Utah 84132

Received2January1985/Accepted 22March1985

Humanadenovirusfailstomultiply efficientlyinmonkeycellsowingto ablock to late viral geneexpression. Ad2hr400 through Ad2hr4O3are asetof hostrange(hr)mutantswhichwereselected for their abilitytoreadily

grow in these cells at 37°C. The mutations responsible for this extended host range have previously been

mappedtothe5' portionofthe geneencodingthe72-kilodaltonDNA-bindingprotein (DBP). DNA sequence

analysesindicate thatallfour hrmutantscontain the samealterationatcoding triplet 130, which changes a

histidinecodon to atyrosinecodon. Theseresultsextend thoseofAnderson et al. (J. Virol.48:31-39, 1983), which suggested thatonly this change inthe DBPamino acid sequencecanexpand adenovirushost range to

monkey cells. Thehrphenotypedoesnotappear torequirephosphorylation of this tyrosineresidue, since no

phosphotyrosinewasdetected in DBPisolated fromAd2hr400-infected monkey cells. The hr mutantsAd2hr400 throughAd2hr4O3, however,arecoldsensitive for growthin monkey cells. The mutantAd2ts400, which was

derived from Ad2hr400, represents asecond class of hrmutantswhichcan growefficiently in monkey cells at

32.5°C. Thecold-resistant hr mutation of Ad2ts400 haspreviouslybeen mapped tothe 5' region ofthe DBP

gene(mapunits 63.6 through 66). DNAsequence analysisof this region shows thatthis mutantcontains the

original hr alterationatcoding triplet 130aswell as asecondalterationatcoding triplet 148, which changes an alanine codonto a valine codon. Wesuspect that thealterations atamino acids 130and 148 change the structureof the amino-terminal domain oftheDBP,allowing ittobetterinteractwithmonkey cellcomponents required for late viral geneexpression. Ad2ts400 alsocontains a temperature-sensitive mutation which has

previously been mappedtothe3' portion oftheDBPgene (mapunits61.3 through 63.6).Sequenceanalysis of thisregion indicates that the DBPcoding triplet413 has been altered. Thischangefromaserine codonto a

proline codon is thesamealterationreportedin thepreviouslysequencedDBP mutantsAd5ts125 (W.Kruijer etal., Nucleic Acids Res. 9:4439-4457, 1981) and Ad5tslO7 (W. Kruijeretal., Virology 124:425433, 1983.). Thus itappearsthat onlyaverylimited number of changes in either the 5' orthe3' portion of the DBPgene cangive risetothe hrortemperature-sensitive phenotypes, respectively.

The nonstructural adenovirus 72-kilodalton (kd)

DNA-binding protein (DBP) is a multifunctional phosphoprotein

encoded in early region 2A ofthe adenoviral genome

(co-ordinates 61.6 through 66.5) (10, 13, 30, 41, 55). DBP is

required for viral DNA replication and appears to play a

central rolein regulatingviralgeneexpression. Twoclasses

ofDBPmutants,eachaffecting verydifferentactivities and

mapping in distinctregions oftheearlyregion2Agene,have

been isolated.

One mutant class consists of

temperature-sensitive

(ts)

mutants and is typified by the adenovirus type 5 (Ad5)

mutantsAd5ts125(17)andAd5tslO7(50).Studies withthese

mutantssuggest arole fortheDBP in viral DNAreplication,

earlygeneexpression, virus assembly, and cellular

transfor-mation.At the nonpermissivetemperature these ts mutants

areunabletoreplicate viralDNA(17, 29),presumably owing

tothethermolabilenatureofthesingle-stranded DNA

bind-ing activity of the tsDBP (56). Furthermore, in

Ad5tsl25-infected cellsthenormal turnoff of earlygenemRNA

expres-sion does not occur at the nonpermissive temperature (11, 12). The negativeregulation of earlygeneexpression

medi-ated by wild-type (wt) DBP appears to act at the level of

transcription forearly region4 (26, 45) and at the level of

mRNAturnoverforearlyregions 1A and 1B(8). Studies on arevertantofAd5tslO7,

R(ts107)202,

suggest an additional role for DBP in virus assembly (46). R(ts107)202 has

re-* Corresponding author.

gained a wtphenotype in human HeLacells but remains ts

for growth in human 293 cells. This mutant appears to

replicateDNAandexpresslategenesnormallyin293 cellsat

thenonpermissive temperature but failsto assemble virion

particles. Finally, DBPappears toplayarole inmodulating

the efficiency oftransformation ofratcells, since Ad5tsl25

transforms ratcells with three- tofivefold greaterefficiency

thandoes wt virus(24,57).

Thesecondclassofearlyregion2Amutantsarehostrange

(hr) mutants, which have been selected for the ability to

grow efficiently in monkey kidney cells (1, 32, 35). The wt

adenovirusdoesnotgrowwellinmonkey cells,eventhough

virus adsorption (20), earlygeneexpression (3, 20, 23), and

DNA replication(23, 27, 48)all occurnormally. The defect

appears toinvolveablocktolategeneexpression, sincethe levels of late viral proteins arediminished (9, 23, 33). This

reduction can, in most cases, be accounted for by the

decreased levels of late mRNA's(19, 33)which result from reduced transcription (J. Johnston, K. P. Anderson, and D. F. Klessig, manuscript in preparation). An exceptionis

thecapsidfiberprotein, inwhich altered mRNA splicing or

poorutilizationof the message,orboth, also contributesto the block in protein synthesis (4, 5, 34). Mutations in the

early region 2A gene allow adenovirus to overcome the

block to late gene expression and grow

productively

in

monkeycells.

Geneticmappingand DNAsequencedeterminationof the

twomutantclasses and their revertantshave

suggested

that 206

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the DBPcontainsdistinctfunctionaldomains (2, 36-39, 49), sincethetsmutations andtheir second siterevertantsmapin

the 3' segment of the DBP gene, whereas the hrmutations mapin the 5' segment. Furthermore, analysisof the mutant

Ad2ts400, which contains mutations of both types, has shown that the hr DBPcanovercometheblocktolategene expression in monkey cells even when the viral DNA replication activity of the protein is nonfunctional (49). The twofunctional domains of DBPmay correspond tothetwo physical domains revealed by the sensitivity of DBP to

severalproteolyticenzymes. TheC-terminal 44-kd fragment

retains DNA binding activity (31)and is functional inan in

vitro DNA replication system (6). The smaller, heavily

phosphorylated N-terminal 26-kdfragment (31) presumably

carries the activity required for late gene expression in

monkey cells (49)and probably in humnan cellsas well.

Herewe reportthe DNAsequence analyses of Ad2hr400 through Ad2hr4O3, fourindependently derived hrmutantsof Ad2 thatgrowefficiently inrnonkey kidney cells. Our results

extendthose of Andersonetal. (2), who suggested that only

a specific change in the DBP amino acid sequence can

expand adenovirus host range to monkey cells. However, thesequence analysis of Ad2ts400 shows that anadditional

change inthe N-terminalsegmentof DBPcanallow the virus

to grow even more efficiently in monkey cells than do

Ad2hr400through Ad2hr4O3. Characterization of the altera-tion responsible for the tsphenotype of Ad2ts400, in

con-junction with similar analyses of other DBP ts mutants

(37-39),suggeststhatonlyaverylimitednumber ofchanges can impart thetsphenotype.

MATERIALS ANDMETHODS

Cells, virus, and infections. Propagation of cellsand isola-tion of virus and viral DNA were performed as previously

described(49). Viral infectionswereperformed monolayers aspreviously described (49).

Cloning of adenovirusDNA.TheEcoRIB restriction frag-ments of Ad2hr400 through Ad2hr4O3 and Ad2ts400 were

cloned in the EcoRI restriction site of pBR322. Plasmid DNAwasprepared by the method of Tanaka and Weisblum

(53). The KpnI-SstI fragment (map units [mu] 61.3 through 69.9)wasisolated from theplasmid containing the Ad2ts400

EcoRIB fragment. A1-,ugportion of this DNAwasdigested

with0.05 U of Bal 31 nuclease (Bethesda Research Labora-tories) for 1minat16°C in 100 ,ul(total volume) of the buffer suggested by Maniatisetal.(42). This DNAwasthentreated

with the Klenow fragment of DNA polymerase (42) and cloned into the XbaI site ofM13mpl9 (42) in both orienta-tions by using XbaI linkers (P-L Biochemicals, Inc.). De-letionsof the parent M13 clones were generated with

exo-nuclease III (New England BioLabs) by the method of Henikoff (28), JM105 was transformed with the various

M13mpl9plasmids by the method described in the 1982 P-L Biochemicals handbook (Manual for the M13

Cloning/Sequencing Kit, p. 9). ,B-Galactosidase-negative

cloneswere screened onLB plates supplemented with 0.03

mM

isopropyl-p-D-thiogalactopyranoside

(Sigma Chemical Co.) and 0.03% 5-bromo-4-chloro-3-indolyl-D-galactoside (Boehringer Miannheim Biochemicals) as described

previ-ously (44). M13 clones were grown in LB medium. The protocol of Messing (44)wasusedtoisolate M13 replicative

form DNA. M13mpl9waskindlyprovided by J. Messing.

DNA sequenceanalysis. The DNAsequence for the DBP geneofAd2hr400wasdeterminedby themethod ofMaxam

andGilbert (43). Restriction enzyme digests (New England

BioLabs) of plasmid DNAwereperformed under the

condi-tions recommended by Maniatis et al. (42). The resultant DNA fragments were separated on agarose gels and

re-covered byelectroelution (42). The isolated fragments were

dephosphorylated with bacterial alkaline phosphatase and

then 5' end labeled with polynucleotide kinase and

[y-32P]ATP (Amersham Corp.) (43). After cleavage with

ap-propriate restriction enzymnes to yield asymmetrically la-beled fragments, the fragments were fractionated by

acrylamide gel electrophoresis and eluted (43). The isolated fragments were subjected to the chemical degradation

re-actions of Maxam and Gilbert, except that 4% formic acid

was used in place of 4% pyridine formate for the

adenine-plus-guanine reaction. Each treated samplewasanalyzed by

electrophoresis through denaturing 0.4 mM gels (40cmby 20

cm) of three acrylamide concentrations, 20%, 10or8%, and

6%. After electrophoresis, each gel was subjected to auto-radiography at -70°C with Kodak XAR film. In general, about 150 nucleotides of contiguous sequence could be determined foreach labeled fragment. Thegeneral strategy is outlined in Fig. 1A. The BgIII-EcoRI fragment (mu 63.6 through 70.7) from plasmid DNA was first digested with

HpaIIorXhoI,and after5'endlabeling, the second

restric-tion enzyme digestion was done with BstEII. The BglII

fragment (mu 60.2 through 63.6)wasfirstdigested with SstII

or BstEII and then with BstEII and KpnI or BglI,

respec-tively. The KpnI-XhoI fragment (mu 61.3 through 66) was

firstdigested with BstEII and then with HpaII.

The sequence around the coding triplet 130 was

deter-mined in the mutants Ad2hr401 through Ad2hr4O3. The BglII-EcoRI fragment (mu 63.6 through 70.7) from each plasmidwas first digested with HpaII, 5' end labeled, and

A.

SstI[ BstEfl BgIZ BstElE

A. I

Hpafl Xhol

I I

61.6mop units 63.6 66 66.5

EN.

61.6map units 63.6 66 66.5

-

--FIG. 1. Strategy usedtosequencetheDBPgene.Theregion of theadenovirusgenome (mu 61.6through 66.5)that encodestheDBP is depicted as a large open bar. Arrows indicate the direction of sequencing. (A) DNA sequence ofthe Ad2hr400 DBP was deter-minedbythechemical degradation method ofMaxamand Gilbert (43). Purified restriction enzymefragments ofDNA from pBR322 clones containing the EcoRIB fragment (mu 58.5 through70.7) of

Ad2hr400weredigested with various restrictionenzymes,labeledat both ends with polynucleotide kinase, cut with other restriction enzymes to generate asymmetrically labeled fragments, purified, and sequenced. Relevant restriction sites are shown. (B) DNA sequencing of Sangeretal.(51)wasusedtodeterminetheAd2ts400 mutant DBP DNA sequence (mu 61.6through 66). Thick arrows

represent regions sequenced by using a 15-nucleotide universal primerandtemplates from theparentM13mp19clones carrying the KpnI-SstI(mu61.3through 69.9)fragmentordeletions of it gener-ated by exonuclease III treatment (28). Thin arrows represent regions sequenced by the parent template and synthetic 20-nucleotideprimers.

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208 BROUGH ET AL.

then digested with BstEII to yield asymmetrically labeled

fragments forsequence determination.

The sequence of Ad2ts400 DBP was determined by the

dideoxynucleotide-primer extension method ofSangeretal.

(51), withmodifications described intheOctober 1983New

England Nuclear handbook (Step-by-Step Protocolfor

35S-Sanger Sequencing) for using [a-35S]dATP as radioactive

label. The M13 single-stranded DNA usedastemplate was

isolated by the protocol described in the 1980 Bethesda

ResearchLaboratories handbook(M13Cloning/Dideoxy Se-quencing Instruction Manual, p. 29). A 15-nucleotide uni-versal primer (New England BioLabs) was used for

sequenc-ing the full-length insert and the M13 deletion variants

obtained from exonuclease III treatment. Gaps left in the

DBPsequencebecauseof lack ofappropriateM13 deletions

were filled by using 20-nucleotide primers synthesized and

purified by preparative gel electrophoresis (Applied

Biosystems, Inc.). Regions of DNA sequence containing

putative mutationsor sequence ambiguities were subjected

tosequenceanalysis oftheopposite strand with appropriate

20-nucleotide primers. The general sequencing strategy is

illustrated inFig. 1B.

Phosphoamino acid analysis. Ad2- or Ad2hr400-infected

CV-1 cells were labeledfrom 22 to 24 h postinfection with

100 ,Ci ofH332PO4 in 0.2 ml of Dulbecco modified Eagle

medium minus phosphate supplemented with 2% dialyzed

calf serum. Proteins were extracted at 0°C by a high-salt

Nonidet P-40 extraction (15) in the presence of30 ,ug of

phenylmethylsulfonyl fluoride and 100 ,uM ammonium

metavanadate toinhibit phosphataseactivity(22). DBP was

immunoprecipitated

withapolyclonal rabbit antiserum made

against native DBP andelectrophoresed on a7.5% sodium

dodecyl sulfate-polyacrylamide gel. The proteins were

electroeluted from the gel bands, passed over small

Sephadex G-25 columns to desalt, and then subjected to

partial acid hydrolysis (14). The hydrolyzed samples were

lyophilized,

solubilized in water, and dried a secondtime.

Thesampleswerethensolubilizedin 10 ,ulofwater

contain-ing phosphoserine, phosphothreonine, and phosphotyrosine

as markers and were spotted onto chromatography paper

(Whatman 3MM). Two-dimensional, high-voltage paper

electrophoresis was performedasdescribed by Clinton and

Huang (14), except thatelectrophoresis was atpH 1.9 for2

hin thefirst dimensionand atpH3.5for40 minin the second

dimension. As acontrol, epidermal growth factor-activated

protein kinase fromhumanepidermoid carcinoma A-431cell

membraneswas used to label anendogenous

membrane-as-sociatedproteinattyrosine

(54).

This membranepreparation

waskindly provided by S. Wiley.

RESULTS

Nucleotidesequence analysis ofAd2hr400toAd2hr4O3.The

mutants Ad2hr400 through Ad2hr4O3 were isolated after

nitrous acid mutagenesis ofAd2 and subsequent selection

for variants which could grow productively in the

estab-lishedmonkey kidneycelllineCV-1 (35).Previously, genetic

mapping ofthe mutants by markerrescue placedthe

muta-tions inthe 5' portion ofthe DBP gene located between mu

63.6 and 66 (36). To determine the precise location and nature of the mutation in each isolate, DNA sequence

analysisof the DBPgenewasperformed.The strategy used

for

determining

thesequenceoftheentire mutantDBPgene

(mu 61.6 through 66.5) from Ad2hr400 is

given

in Fig. 1A.

Only one nucleotide difference was observed in the mutant

DBP gene compared with the Ad2 DBP DNA sequence

publishedby Kruijer et al. (39). A thymine residue replaces

G

A+G

C+T

G-

_

C

A>C

A-A_

...4..;.

C-G-

I-.*

...

c-

S

W.ikiiii

A-

-I-G-U

A-FIG. 2. Maxam and Gilbert (43) sequence analysis of the Ad2hr400 DBP gene.Aportion of the sequencing gel autoradiogram thatcontainsthesingle nucleotide change (*),ascompared withwt Ad2, at position 1 ofcoding triplet 130is shown. Thechangeofa

cytosineto athymineincoding triplet130changesthecodonfrom histidinetotyrosine. Thesamechangewasalso observed in the hr mutantsAd2hr401throughAd2hr4O3.

acytosineresidue at thefirstposition of coding triplet130 in

the DBP gene (Fig. 2), changing a histidine codon to a tyrosine codon.

Thenucleotide sequence in theregion foundtobe altered

in Ad2hr400wasdetermined forthethree otherhr mutants,

Ad2hr4O1through Ad2hr4O3. All three mutants werefound

tohave

precisely

thesame nucleotide change asAd2hr400. No other change was observed in the

region

examined,

whichencompassed 100 to 150 nucleotides surrounding the

mutation. Thusallfourmutantsexaminedhereaswellasthe

phenotypically

similar mutants Ad5hr4O4 (38) and

Ad2+NDhr600

through

Ad2+NDhr603

(2) have

precisely

the

same mutation.

Therefore,

all nine

independently

isolated

adenovirus mutants selected for the

ability

to grow in

monkey cells haveacquiredthesame amino acid changein

the DBP, a substitution of

tyrosine

for histidine at amino

acid 130.

Phosphoamino acid analysis ofAd2hr400 DBP. To

deter-minewhether the hrmutantDBP

might

become

phosphory-lated on tyrosine in monkeycells, we examined the

phos-phorylation patterns of the DBP isolated from Ad2- and

Ad2hr400-infected CV-1 cells. Infected cells were labeled

from22to 24h postinfectionwith [32P]orthophosphate, and

the DBP was isolated by

immunoprecipitation

ofthe cell

extracts with polyclonal antiserum directed

against

DBP. The

immunoprecipitated proteins

wereeluted fromasodium

dodecyl

sulfate-polyacrylamide gel,

acid

hydrolyzed,

and

subjected

to

high-voltage

paper

electrophoresis (Fig.

3A and

B).Phosphoserineand

phosphothreonine

werefoundinboth

wtand hr DBPs in agreement withprevious analyses ofwt

DBP synthesized in infected HeLa cells (7). However,

phosphotyrosine

was notdetected in either themutantorwt

proteins. Phosphotyrosine

wasdetected in the

positive

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MUTATIONS IN THE ADENOVIRUS DNA-BINDING PROTEIN

Origein

I

.P-Tyr

-

'P-Thr

~P-Se

r

FIG. 3. Two-dimensional separation ofphosphoamino acids. The positions ofthe origin, phosphotyrosine (P-Tyr), phosphothreonine (P-Thr), andphosphoserine(P-Ser) markers, and [32P]orthophosphate(Pi)areshown.H332P04-labeledDBPfrom Ad2-infected CV-1 cells(A) orAd2hr400-infected CV-1 cells (B) waspurified and subjected to acidhydrolysis. As apositive control, A-431 cell membrane fractions labeled in vitro (C)wereacidhydrolyzed inparallel.Theresultingproducts were separatedbytwo-dimensional high-voltage electrophoresis (seetextfor details).

trol consisting ofin vitrophosphate-labeled A-431cell

mem-braneswhich containepidermal growth factor receptors and

one or moreproteinswhicharephosphorylatedat atyrosine residue (Fig. 3C) (54).

Nucleotide sequence analysis ofAd2ts400. Although thehr

mutantsAd2hr400 throughAd2hr403 grow very efficiently in

monkey cellsat 37°C,theyform relatively small plaques on

CV-1 cell monolayers at 33°C, and the kinetics of plaque

formation is dramatically delayed compared with that in

similar infections of CV-1 cells bythe Ad2-simian virus 40

(SV40) hybrid virus, Ad2+ND1. This cold-sensitive

pheno-type is seenonly in monkey cells, since wt and hr viruses

formplaques with similar kineticsonhumancellmonolayers

at 330C. Ad2ts400, a nitrous acid-induced mutant of

Ad2hr400,wasselectedfor itsabilitytogrowreadilyinCV-1

cellsat33°C(49).Thecold-resistant hr mutationresponsible

for thisphenotypewasmapped bymarkerrescueanalysisto

the 5' portion of the DBP gene (mu 63.6 through 66). However, Ad2ts400 also contains a ts mutation in the 3' segmentofthe DBP gene (mu 61.6through 63.6) (49).Its ts

phenotype is similarto thatofAd5tsl25 in that viral DNA

replication andthe turndownofearly geneexpressionatlate

times areblocked atthe nonpermissive temperature.

The DNA sequence ofAd2ts400 was analyzed between

mu 61.6 and 66 by the strategy outlined in Fig. 1B. This

analysis indicated that Ad2ts400 carries three mutations in

the DBP gene as compared withthe wt DBP sequence. All

three changesreplaced cytosine with thymine.Ad2ts400still

maintains the hr alteration at coding triplet 130 which is

presentinAd2hr400. Anadditionalchange wasfound in the

5'section ofthe DBP gene,which by markerrescuemapping

is known to convey the cold-resistant hr mutation. The

alteration wasat position 2 in coding triplet 148 (Fig. 4A),

changing an alanine codon to a valine codon. The single

alterationin the 3'region,towhichthe ts mutation has been

mapped, isatthefirstposition of codingtriplet 413 (Fig. 4B),

changingaserine codontoaprolinecodon. Thismutation is

the same alteration seen in two other ts DBP mutants, Ad5ts125 (38) and Ad5ts107(37).

DISCUSSION

The nitrous acid-induced hr mutants Ad2hr400 through

Ad2hr403, all contain the same histidine-to-tyrosine change

at position 130 in DBP, as do the spontaneously generated

mutants Ad5hr4O4 (38) and Ad2+ND3hr600 through

Ad2+ND3hr603 (2). Thus all nine independently isolated

humanadenovirus mutants, selected forthe abilityto

over-comethe blockto late geneexpression and virusgrowthin monkey cells,haveacquiredthe same mutation. Byasingle

base substitution, the histidine codon can be changed to

codons forseven different amino acids. The substitution of

only atyrosineat

position

130inthe hr DBPs suggests that

this amino acid plays an importantrole in the hrfunction.

The recent discovery ofthe tyrosine kinase activity of

severaloncogeneproducts and of various membranegrowth

factor receptors or their associated components suggests

thattyrosine

phosphorylation

maybe involvedin mediating

avariety of cellularresponses (18, 54). Sincethe hr

pheno-typedepends on thesubstitution oftyrosine for histidine at

amino acid 130 in DBP, the protein was characterized to

determine whetherphosphorylation of this tyrosine residue

G A J C G A T C

C- ._

>~~~

T- _

C- t

C-j

eA-> ^.__

A. B.

FIG. 4. Dideoxynucleotide sequence analysis of the mutant Ad2ts400DBPgene.Thefigureshowsportionsof thesequencing gel autoradiograms ofthe viral DNA noncoding strand which contain thetwomutational sitesinAd2ts400notfound inparentalAd2hr400.

(A) Alteration in coding triplet 148 which is responsible for the cold-resistant hr phenotype. The altered nucleotide (*, G-to-A; C-to-T on the coding strand) changes codon 148 from analanine codon to a valine codon. (B)Mutation in codingtriplet 413 which impartsthe tsphenotype. The altered nucleotide(*,G-to-A;C-to-T

onthecoding strand)changes codon 413 fromaprolinecodontoa serinecodon.

A.

rigi

.P-Tyr

fP-Th

r

P-Ser

Pi

209 VOL.55, 1985

SW

I,Pi

11-1

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210 BROUGH ET AL.

was importantfor the mutant phenotype. Althoughboth hr and wt DBP contained similar amounts of both

phos-phoserine and phosphothreonine, phosphotyrosine was not

detected in either protein. This suggests that tyrosine phos-phorylation is not involved in the hr phenotype. We cannot, however, exclude the possibility that if onlyasubsetof DBP

molecules are phosphorylated on tyrosine, the level ofthis

phosphorylationmight be belowthesensitivity ofourassay,

which could readily detect phosphotyrosine in a control experiment.

The hrmutants Ad2hr400through Ad2hr4O3, which were

selected to grow well inmonkey cellsat37°C, arerelatively

inefficient at late gene expression and growth in

monkey

cells but not in human cells at 33°C. The mutant virus

Ad2ts400 was selected on the basis of its cold-resistant hr

phenotype. Themutationresponsible forthisphenotypehas

beenmapped to the 5'regionofthe DBP gene. Weknowthat

thetsmutationlocatedin the3' segmentofthe DBP geneis

not involved in the cold-resistant hr phenotype, since we

have obtained a virus, Ad2hr4O5, which contains a wt 3'

portion of the DBP gene but retains the cold-resistant hr

phenotype. Interestingly, Ad2hr4O5 forms larger plaques

than do Ad2hr400 through Ad2hr4O3 at37and at39.5°C(S.

Rice and D. Klessig, unpublished results). DNA sequence

analysis oftheAd2ts4005' segmentindicatedthatin addition

tothealteration ofthe histidinecodontothe tyrosinecodon

at position 130, which is found in all of the hr mutants,

Ad2ts400 contained an alteration at the coding triplet 148.

Since an hr mutant containing only achange at amino acid

148 has neverbeen isolated, it seemslikely thatthischange alone is insufficienttoimpartthe hrphenotype. Morelikely,

the alterationatcoding triplet

148

works inconcertwiththe

change atposition130 tofine tune the structure ofthe DBP

so that it can interact more efficiently with monkey cell

factors required forexpression of viral late genes.

The other alteration inthe DBP geneof Ad2ts400 is in the

3'portion ofthe geneandisresponsible forthetsphenotype.

It was previously reported that Ad2ts400 has a phenotype

similar to the mutants Ad5tsl25 and Ad5tslO7 at the

nonpermissive temperature (49). In fact, Ad2ts400 has the

samenucleotide changeas these mutants. The alteration in

coding triplet 413 of Ad2ts400, Ad5tsl25, and Ad5tslO7

changesaproline codonto aserinecodon. Itseemsurprising

that three of the four ts DBP mutants which have been

sequenced containthe samenucleotide alteration. Since we

have used themutants Ad5tsl25 andAdStslO7inour labora-tory, we considered the unlikely possibility that Ad2ts400

arose by a recombination event

involving

Ad5tsl25 or

Ad5ts1O7. We can exclude this

possibility,

since Ad2ts400

containsAd2

serotype-specific

DNA sequences

throughout

the gene,includingtwoAd2-specific differences

(relative

to thewt AdS sequence [38]) locatedwithin 25 nucleotides

of

the ts mutation.

The restriction in the number of mutational sites seen in the N-terminalportion of the DBP is perhaps in retrospect reasonable, given the possible constraints in

protein

struc-ture imposed by the strong selection forgrowth in monkey

cells. In contrast, the conserved changesin the C-terminal

portionareverysurprising. Threeoutof fivetsmutations in

this domain have the same alteration at

position

413. The

exceptions areAd2+NDlts23, which contains an alteration

at coding triplet 282 (39) and Ad2tslll,which is

thought

to

be located between coding triplets 200 and 300

(16,

52).

Perhaps onlyverylimitedchangesintheC-terminal

domain

ofDBPwill allow this

protein

tofunctionat

32.5°C

butnot at

39.5°C. However, this may not be the entire

explanation,

MUTANTS:hr(IO)

Ad2hr400-403 (a.a. 130) Ad2+ND3hr6OO-6O3(a.a. 130)

Ad5hr4O4 (a.a.130) Ad2ts400 (a.a.130,a. o.148) FUNCTIONS: Lotegeneexpression

ts(4)

Ad2ts400

Ad5ts 125 Ad5ts107 Ad2+NDlts23

(a.a.413) (a.a. 413) (a.a. 413) (a.a.282)

Earlygeneexpression

DNAreplication DNA binding

Virus assembly

Morphologicoltransformation FIG. 5. Schematicrepresentationofthe separatefunctional do-mainsof the adenovirusDBP.TheN-terminal 26-kdfragmentof the

proteincontains the hr mutation which relieves the blockto viral lategeneexpressioninmonkeykidneycells. All of10 independently

derived hrmutantscontain thesamealterationatcodon130,which results inthe replacement ofhistidine by tyrosine. Ad2ts400

con-tainsanadditional hrmutation, locatedatamino acid148,changing

alaninetovaline. This mutationfurther enhances thegrowthofthe

virus inmonkeycells,particularlyatlow

temnperatures.

Thefourts mutants that affect earlygeneexpression, DNAreplication, DNA binding, virus assembly, and morphological transformation are

located inthe44-kdC-terminal domain. Three of these fourmutants have an identical alteration at codon 413, which results in the substitution of serine forproline. Thefourth ts mutationchanges

amino acid282from leucinetophenylalanineinAd2+NDlts23.The two domains may be separated by a hydrophilic hinge region

represented by theconnectingline.

sinceAd2ts400wasisolated afterselection for cold-resistant

hr mutants.

Surprisingly,

one out ofthe two mutants

ob-tained, namely Ad2ts400,

simultaneously

had

acquired

a ts

alteration at amino acid 413 in addition to the alteration at

amino acid 148 which

imparts

the cold-resistant hr

pheno-type. Thus the

repeated

isolation of mutations at

coding

triplet

413

might

suggestthe

high

susceptibility

of this

region

of the gene to

mutagenesis.

The sequence

analysis

ofAd2hr400

through Ad2hr4O3

and Ad2ts400 further supports the model that DBP contains at least two functional domains. The hr mutations at amino

acids130and148 and thetsmutationsat aminoacids413 and

282 are

separated

by

an

extremely

hydrophilic

region

(cen-tered around amino acid

160)

that could act as a

possible

hinge

between the twodomains

(Fig.

5).

The

larger

domain

contains the sites of the ts

alterations,

which affect

early

gene

expression,

DNA

replication,

viral

assembly,

and the

efficiency

of

morphological

transformation. The smaller

do-maincontainsthe hr

alterations,

whichallownormal

expres-sion oflate genes in

monkey

cells. Coinfection of

monkey

cells with wt adenovirus

plus

SV40also allows normal late gene

expression (47),

as does infection with

Ad2-SV40

hybrid

viruses thatcontain

only

theSV40sequences

encod-ingtheC-terminal

segment

ofT

antigen (21, 25,

40).

Thusthe

C-terminal segment ofSV40 T

antigen

and the N-terminal

domainof adenovirus DBP appeartosharesimilar functions.

ACKNOWLEDGMENTS

Thisworkwas supported by PublicHealth Servicegrantno. Al

17315fromthe National InstitutesofHealth andaSearle

Scholar-ship to D.F.K. from the Chicago

Community

Trust. D.F.K. is supportedbyaFacultyResearchAward(no.270)fromthe

Ameri-can Cancer Society, and S.A.R. was

supported

by

a

predoctoral

traininggrant(no.T32GM07464-08)fromtheNationalInstitutes of Health.

J. VIROL.

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[image:5.612.318.549.60.209.2]

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