Polypeptide structure and encoding location of the adenovirus serotype 2 late, nonstructural 33K protein.

Vol. 45, No. 1 JOURNALOFVIROLOGY,Jan. 1983,p.251-263

0022-538X/83/010251-13$02.00/0

Polypeptide Structure and Encoding Location of the

Adenovirus Serotype

2 Late,

Nonstructural

33K

Protein

ELIZABETH A. OOSTEROM-DRAGONt AND CARL W. ANDERSON* BiologyDepartment,Brookhaven NationalLaboratory, Upton,New York11973

Received 19July1982/Accepted4October 1982

Radiochemical microsequence analysis of selected tryptic peptides of the

adenovirustype233K nonstructuralprotein has revealed the precise region ofthe

genomicnucleotide sequencethat encodes this protein. The initiation codon for

the 33Kprotein lies 606 nucleotidestotheright of theEcoRI restriction site at 70.7

mapunits and 281 nucleotidestotheleft of the postulatedcarboxyterminalcodon

of the adenovirus 100K protein. The coding regions for these twoproteins thus

overlap; however, the 33Kprotein is derived fromthe +1 frame with respect to

the postulated 100K reading frame. Our results contradict an earlierpublished

report suggesting that these two proteins share extensive amino acid sequence

homology (N. Axelrod, Virology 87:366-383, 1978). The published nucleotide

sequence of the Ad2 EcoRI-Ffragment(70.7 to 75.9mapunits)cannot

accomo-dateinasingle reading frame thepeptide sequencesof the 33K protein that we

have determined. Sequence analysis of DNA fragments derived from virus has

confirmedthepublishednucleotide sequence in all criticalregionswithrespect to

thecoding region for the33Kprotein. Consequently,ourdataareonly consistent

with the existence of an mRNA splice within the coding region for 33K.

Consensus donor and acceptor splice sequences have been located that would

predict the removal of 202 nucleotides from the transcripts for the 33Kprotein.

Removal of these nucleotides would explain thestructureofapeptidethat cannot

otherwise be directly encoded by the EcoRI-F fragment. Identification of the

precise splice points by peptide sequencing has permitted a prediction of the

complete amino acidsequence for the 33Kprotein.

Adenoviruses havenonenveloped, icosahedral

virions composed of a linear double-stranded

DNA ofabout 23 x 106 Mr encased in a shell

constructed from 10 to 12 different

virus-en-coded proteins (29). In addition to these

struc-turalproteins,lateviral transcriptsarebelieved

to encode at least three nonstructural proteins

thatare believed tofunction in virion assembly

and maturation. A maturation proteinase

activi-tyhasbeen described (9) thatprocesses atleast

four virion polypeptides andthatis believed to

be encoded within late mRNA region L3 (see

Fig. 2) (1, 17, 21), but the polypeptide

corre-sponding to this activity has not yet been

ob-served. Twononstructural proteins are

synthe-sized in large amounts late afterinfection from

mRNAs produced from late region L4. The

100,000-molecular-weight(100K)polypeptide is

believedtofunction in hexoncapsmer assembly

andtransport(14-16,26, 27).A33Kpolypeptide

tPresentaddress:CODON, Brisbane,CA 94005.

is also encodedby this region, but its function is

notknown, and its possible relationship to the

100Kproteinsequencehasbeensuggested.

Be-fore functions can be definitively ascribed to

either the 100Kor33Kpolypeptides, their

rela-tionship to each other and to their

genome-encoding nucleotide sequences must be

deter-mined.

Althoughabundantatlate times afterinfection

of HeLacells, the 33Kproteinwasoverlooked

byearly studies ofadenovirus-specific proteins

(4, 36),probably because of its low methionine

content. Thefirstdescription ofaca. 33,000Mr

polypeptide specific to extracts of

adenovirus-infected cellswasby Russell and Blair (31), who

described amajorvirus-specific phosphorylated

polypeptide of39,000 Mr found in the nuclei of

adenovirus 5-infected cells late after infection. This componentwassubsequently observedby

Levinsonetal.(22) andby Axelrod (6). Axelrod

compared the tryptic peptides from the three

major adenovirus-specific nonstructural

phos-251

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252 AND ANDERSON

phoproteins, the early 72,000-Mr DNA-binding

protein, the lOOK protein, and the 33K protein

and concluded that the lOOK protein and the

33K protein wereclosely related in amino acid

sequence. Lewiset al. (23) mapped by in vitro translation of hybridization-selected adenovirus

mRNA a 38,000-Mr polypeptide to the EcoRI-F

(70.7 to75.9 mapunits [m.u.]) and -D (75.9 to

83.4m.u.)restriction fragmentsofadenovirus2. But notuntil the much later

hybridization-trans-lation studies of Ricciardietal. (30)andMilleret

al.(25)wasthe33Kpolypeptideshown to be an

independently codedadenovirus product. Even

these studies didnotshow that the in vivo and in

vitro 33K products were necessarily related in sequence.

Recently Gambke and Deppert (14, 15)

pre-pared antiserum to purified adenovirus 2 33K

protein and found that thisserumdid not

cross-react with the lOOK protein. This result led

Gambke and Deppert to reexamine the

conclu-sionof Axelrod that theseproteinswereclosely

related; their own tryptic mapping studies

showed that theseproteinswereprobably

unre-latedinaminoacidsequence. Using their

antise-rum we have shown that the in vitro product

mappedtothe EcoRI-Ffragment of the

adenovi-rus2 genomeis indeed the sameasthe in vivo

33K productdescribed byAxelrod, Gambke and

Deppert, and others. Radiochemical

microse-quenceanalysis of the major tryptic peptides of

33Kprotein isolated from adenovirus 2-infected

HeLa cells has allowed us to predict the

com-plete amino acid sequence of this protein by

comparing partial peptide sequences with the

knownnucleotidesequenceof the EcoRI-F

frag-ment (13). Surprisingly, this analysis showed

that the 33K protein is encoded by an mRNA

that is spliced within the coding region. This

result explains why the 33K protein could not

previously be relatedcorrectlytothenucleotide

sequence.

MATERIALSANDMETHODS

Cells and virus.TheoriginsofourHeLacells and of serotype 2adenovirus stocks and the propagationof these have been described previously (2, 32). The adenovirus stock used fortheseexperimentsis direct-ly related to those usedby the Cold Spring Harbor Laboratory for DNA sequence studies(38). Adenovi-rus DNA was prepared from purified virions as de-scribedpreviously(32).

Purification of radioactively labeled 33K protein. Nearly confluent 90-mm culture dishes ofHeLacells wereinfected with 250PFUof adenovirus per cell.At 24 to26 hafterinfection, the infected cultureswere

placed into medium containing the appropriate 3H-amino acid(1 mCi per dish) or [35S]methionine (250

,uCiperdish). After incubationat37°C for4 to 6h, the radiolabeled cultures were fed with 5 ml of normal Dulbecco modifiedEaglemediumsupplementedwith 2% calfserumperdish,and incubationwascontinued

until approximately 48 h after infection. Harvested cultures were washed with ice-cold phosphate-buff-ered saline and then with0.01 MTris-hydrochloride (pH 7)-0.5 M NaCl-0.002 M MgCl2 (TSM buffer). Aftersuspension in 1.0 ml of ice-cold TSM buffer per dish, thecultureswerelysedby the addition of Noni-det P-40 to0.5% and phenylmethylsulfonyl fluorideto 300,ug/ml.Thenuclearpellet, obtained by centrifuga-tion, was washed once inTSM buffer containing0.5% Nonidet P-40 suspended in sodium dodecyl sulfate (SDS) sample buffer (4), and heated for 5 min in a boiling-water bath. Nuclearextracts werefractionated byelectrophoresis through slab-type SDS-polyacryl-amidegels (4). Proteinswerelocated bystaining with Coomassie brilliant blueorby autoradiographyof the dried gels. A dried gel slice corresponding to the position of the 33K protein was excised and eluted electrophoreticallywithanISCOsampleconcentrator

(3,33).

Digestion with trypsin and peptide fractionation by reverse-phase(C18) high-pressure liquid chromatogra-phy. Samples to be digested with trypsin were first extracted with acetone-acetic acid-triethanolamine (90:5:5)threetimes andacetonetwicetoremoveSDS (18). After the addition of 1 mg of apomyoglobin,

samples wereperformicacid oxidized (4) and

lyophi-lized. For digestion with trypsin, samples were dis-solved in 0.9 ml ofwaterand 0.1 mlof1MNH4HCO3; 10 ,ug oftosylamido 2-phenylethyl chloromethyl ke-tone-treated trypsin (Worthington Diagnostics, Free-hold, N.J.)wasadded.After 2 h of incubation at 37°C, asecond 10 ,ug oftrypsinwasadded, and incubation wascontinued foratotal of 6 h. Samples were then frozen andlyophilized.

Fractionation by reverse-phase chromatography wasperformed byusinga250-by 4.6-mm,10-p.m RP-18 column (E. M. Reagents, Inc.) equilibrated with 95%buffer A(10 mMammonium acetate, pH 5.4) and 5% buffer B (100% acetonitrile). Samples were dis-solved in 0.3 mlof20%formic acid andcentrifugedat 12,000 x g for 15 min before application to the column. The columnwasdevelopedwitha discontinu-ouslineargradientof acetonitrile(seeFig.3)at aflow rateof 2.0ml/min; 1.0-mlfractionswerecollected. The position ofapomyoglobin peptideswas monitoredat 214 nm; these positions were used to facilitate the alignmentofradioactivepeptideprofilesfrom separate experiments. In general, separations varied by less than +1 fraction. A portion of each fraction (0.05to 0.15ml) wasassayed forradioactivity with 3.5 mlof scintillation fluid (Aquasol; New England Nuclear Corp., Boston, Mass.). Appropriate fractions were pooled,1mgofapomyoglobinwasadded, and pooled fractionswerelyophilizedbefore amino acid sequence analysis.

Amino-terminal sequence analysis. Radiochemical microsequenceanalysis wasperformed witha Beck-man 890 C sequencer and a 0.1 M Quadrol buffer programasdescribedpreviously(3, 5, 33).Polybrene (3 mg) was included with all peptide samples. Se-quencer performance was routinely monitored by analysisof carrierapomyoglobin.

DNAsequenceanalysis. DNA sequenceanalysiswas performed bythe method of Maxamand Gilbert(12, 24). Appropriate restriction enzyme-generated frag-ments werelabeled with[32P]ATPandpolynucleotide kinase.

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ADENOVIRUS 2 33K PROTEIN 253 Immunoprecipitation. Immune complexes were

ad-sorbedtoFormalin-fixedStaphylococcusaureuscells

(The Enzyme Center, Boston, Mass.) and collected by centrifugation (19). Complexeswerereleasedby

boil-ing in 2% SDS-0.1 Mdithiothreitol sample buffer as

previously described(26).

RESULTS

Identification of the adenovirus 33K protein. The adenovirus 33K protein isamajor nonstruc-tural virus polypeptide produced at late times afterinfection; itcanreadily beidentified in the

pattern of Coomassie brilliant blue-stained bandsafterSDS-polyacrylamidegel electropho-resisof infected cell nuclearextracts(Fig. 1A). To confirm the identity of the 33K protein,

radioactively labeled nuclearextracts were

im-munoprecipitated with antiserum specific for the adenovirus 33K protein (the kind gift of W. Deppert), and the precipitateswereanalyzed by

SDS-polyacrylamidegelelectrophoresis. Figure

1B shows that theonly product

immunoprecipi-tated from [35S]methionine-labeled or3 P04-la-beled nuclear extracts of infected cells is a

product of ca. 33,000 Mr that corresponds in

mobilitytothemajor nuclearcomponent identi-fiedasthe33Kprotein in Fig. 1A. Figure 1B also shows that theelectrophoreticallypurified prod-uct used for the peptide and sequence analysis described here is radiochemically pureand can

be immunoprecipitated by 33K protein-specific

antiserum.

Invitro 33Kproduct labeled with

[35S]methio-ninewasprepared by translation of RNA

select-edby hybridizationtothe EcoRI-F(70.7to75.9 m.u.) orHindIII-H (73.6to79.9m.u.) fragment ofadenovirus 2 DNA(28, 30). Among the prod-uctsfrom thesetranslationswas amajor

compo-nent withamobilityby SDS-polyacrylamide gel

electrophoresis essentially identical to that of 33K protein from infected cells (data not shown). This component was excised from a

preparative SDS-polyacrylamide gel, digested with trypsin, and compared with similarly

pre-pared 33K protein from infected cells by

re-verse-phase liquid chromatography (data not shown). Both preparations had single major

me-thionine-containingpeptides that eluted from the

column atidentical positions. Subsequent stud-ieshave shown that this elution position

corre-spondstothat ofthesingle methionine-contain-ing peptide predicted to be present in the 33K protein (see below and Table 1). Thus, the in vivo 33K product must be encoded within the region from 70 to 80 m.u. on the adenovirus 2 genome(Fig. 2).

Inspection of the nucleotide sequence

corre-sponding to late mRNA within the EcoRl-F fragment of adenovirus 2 (13) reveals four mod-erately long open reading frames that could

potentially encode the 33K protein. The first

open reading frame begins at the left-hand

boundary of the EcoRI-Ffragment (70.7m.u.),

is 296 codons long, contains one methionine

codon, and is believed to encode the

carboxy-terminal portion of the adenovirus 100K late,

nonstructural protein (13). The second open

frame begins at nucleotide 307 (N307; to the

right of70.7), is 295 codons long, overlaps the

first openframe, and contains four methionine

codons. The thirdopenframe begins atN1006,

partially overlaps the second open frame, and

containsnomethionine codons. The fourthopen

frame begins at N1519 and has been found to

encode the precursor to virioncomponent VIII

(Oosterom-Dragon and Anderson, unpublished data).

33K protein has ablocked amino terminus.The

33Kprotein is knownto map tothe leftof virion

component VIII (25). The five methionine

co-dons located in the firsttwoopenreading frames

are each followed within 20 codons by one or

more leucine oralaninecodons. Consequently,

if the amino terminus of the 33K protein were

notblocked, direct amino-terminal

radiochemi-cal sequence analysis of [3H]leucine- or

[3H]al-anine-labeled 33K protein wouldyield

radioac-tivity inacharacteristicpattemof residues that

wouldidentify theencodinglocation of the

ami-noterminus of the protein. Several such

experi-ments were performed with [35S]methionine-,

[3H]leucine-, or[3H]alanine-labeled33Kprotein

isolated as described above, and no significant

releaseofradioactivitywasobtainedfromanyof

the first 20 Edman cycles. Analysis of carrier

apomyoglogin included with the radioactive

samples assuredusthatthesequencer was

func-tioningproperly. Since wehaverarely failedto

obtain sequenceinformationbythis methodfor

proteins without blocked amino termini (3, 5,

33),weconcludethatthe 33Kprotein,likemost

of the latevirionproteins, hasa blockedamino

terminus that precludes direct sequence

deter-mination.

Sequence analysis of 33K protein tryptic

pep-tides. Adenovirus 2 33K

?rotein,

sepa-rately with different H-amino acids or

[35S]methionine,

electropho-resis, performic acid oxidized,anddigested with

trypsin as described above. The 20o formic

acid-soluble peptides wereappliedto a

reverse-phasecolumn forfractionationbyhigh-pressure

liquid chromatography. Examples of the

pat-terns ofradioactivetryptic peptides obtainedare

givenin Fig. 3. Eachdifferent amino acid label

producedauniquepattern of well-resolved

pep-tides; some peptides appeared to contain only

one of the radioactive amino acids used, and

othersweredetectedby several different amino

acid labels. Appropriate fractions from

individ-VOL.45, 1983

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FIG. 1. Identificationof the adenovirus33Kprotein.(A)Coomassiebrilliant blue R-250-stained 17.5% SDS-polyacrylamide geluponwhichwereelectrophoresedthefollowing: 1,nuclearextractof mock-infectedHeLa

cells; 2, nuclearextractofadenovirus 2-infectedHeLacellsharvested32h after infection; 3, the corresponding cytoplasmicextractofadenovirus 2-infectedHeLacells. Thepositionoftheadenovirus33Kproteinaswellas

thepositions ofseveral other viral proteinsareindicatedatthe left of the figure. A cellular protein withamobility

similarto theadenovirus33Kprotein is found in nuclear extractsof uninfected HeLa cells, butthis cellular protein isnotrecognized by antibodytotheviral33Kprotein.Cellular protein synthesis wouldhave been largely

inhibited atthetimeinfected cellswerelabeledfor the preparationof radioactive33Kprotein.(B)

Autoradio-gramofanSDS-polyacrylamide geluponwhichwerefractionated immunoprecipitates obtained with antibody

specific for the 33K protein. The samples were as follows: 1, [j5S]methionine-labeled nuclear extract of adenovirus2-infected HeLa cellslabeledca.26to32hafter infection;2,immunoprecipitate of the [I5S]methio-nine-labelednuclearextractobtained with normal rabbitserum;3,asin lane2,butobtained withserumspecific

fortheadenovirus33Kprotein; 4, nuclearextractof adenovirus 2-infectedHeLacetlslabeled with32Pi26to32h afterinfection;5,immunoprecipitateof the

32Pi-labeled

5,butwith antiserumspecific for the adenovirus33Kprotein (Fig. 3); 8, immunoprecipitate of [3H]alanine33K

protein withnormal rabbit serum; asin lane 8, but withantiserum specific for the adenovirus 33Kprotein. Immune complexes were collected with Formalin-fixed S. aureus cells as previously described (26). The

polyacrylamide gel wastreated with En3Hance(NewEngland NuclearCorp.) before fluorographyonKodak

AXR-5film.

ualexperimentswerepooled andlyophilizedfor

subsequentsequence analysis.

Individual peptides were applied tothe spin-ning cup sequencer for automated sequential

Edmandegradation. This analysis gives the

po-sition(s) of the radioactive amino acid with

re-specttotheamino-terminaltrypsin cleavage site

that produced the peptide. Examples of the analysis of several 33Kpeptidesaregiven in Fig.

4and 5. Thepatternsof[3H]leucine and [3H]pro-line released from peptides designated L12 and P10 are given in Fig. 4A and B, respectively.

Thesetwopeptides elutedatthe sameposition

during chromatography. Leucine was found in A

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VOL.45, 1983 ADENOVIRUS 2 33K PROTEIN 255

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FIG. 2. Mapof the adenovirus 2genome.The adenovirus 2genomeisrepresented bytwothinparallellinesin

themiddle ofthepicture. Mapunits aremarked below these lines;the positionsof EcoRIcleavagesitesare

indicated by solid trianglesabove theselines;the letterdesignationofthe EcoRIfragmentsisgivenbetweenthe lines. Lines above and below the genome representation indicate RNA transcripts or mRNAs; promoter

positionsareindicatedbyabracket. Individual late mRNAbodysequencesfrom the5 lateregionsLi throughL5

areindicated bythick lines(10).Theapproximatelocationencodingseveralproteinsis indicatednearthetopof

thefigure.The lowerpartof thefigure showsamapofjustthe EcoRI-Ffragment (70.7to75.9m.u.).Theregions encoding the 100K protein (13),the 33Kprotein (this manuscript), andtheprecursortovirioncomponentVIII

(Oosterom-DragonandAnderson,unpublished data)areindicated.The exactlocationof the amino terminusof

P-VIII hasnotbeen determined. Solid diamonds indicate thepositionsofHpaIIrestrictionsites;theopencircle designatesthepositionof theHindlllrestriction site at72.8m.u. Numbersalongthe bottom oftheEcoRl-F fragment representation indicate distance in nucleotides fromthe EcoRI site at 70.7m.u.

amino acid positions 1 and 3 of this peptide, whereas prolinewasfound inpositions 4, 5, 6, 7, and 8. Inspection of the EcoRI-F nucleotide

sequence revealedonly one location that could encode a peptide with this pattern of leucines

and prolines. The location begins 624 nucleo-tides fromthe EcoRI-Fcleavagesiteat70.7m.u.

in thesecond openreading frame. Immediately preceding the leucine codon at N624 are three

lysine codons,acondition consistentwith

cleav-age by trypsin at this location in the protein. Similar analysis ofthe peptide A7-P7 (Fig. 4C andD) shows itto beuniquelyencoded by the

sequence beginning at N813, also within the secondopenreadingframe.These resultsprove

thataportion of the 33Kproteinisencoded by the open reading frame between N307 and N1190. Since the lOOKprotein is believedtobe encodedbythe firstopenreading frame (13, 25),

this result strongly suggests that the 33K and lOOK proteins cannot be directly related by amino acid sequence. To confirm this

expecta-tion, we prepared [3H]leucine-labeled lOOK

trypticpeptides and chromatographed them

un-derconditions identicaltothose used for

separa-tion of the 33Ktryptic peptides. No significant coincidence of radioactiveprofileswasobserved (datanotshown).

Partial sequenceanalysis of the four peptides

for which dataarepresented in Fig. 5 revealed surprisingly that these peptidesare allencoded in the thirdopenreading frame. PeptideP4-V3is encoded beginning atN1258, peptide A3 is

en-coded beginning at N1219, peptide F8 is

en-codedbeginningatN1303,andpeptideL5-A5 is encodedbeginningatN1435(Table 1).The data presented in Table 1, therefore, prove thatthe 33Kprotein is also partiallyencodedbythethird

open readingframe.

Peptide A6 was found to have alanines at positions1,2, 5,7, 8, 9, 10, 11, 12, 13, 15,and16

(Fig. 4). This pattern of 11 alanines cannot be encoded by a contiguous sequence within the EcoRI-Ffragment;however,the last 10 alanines

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ADENOVIRUS 2 33K PROTEIN 257

TRYPTICPEPTIDES OF ADENOVIRUS 2 33K PROTEIN

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FIG. 3. High-pressure reverse-phase, liquid chro-matographyof thetryptic peptidesof theadenovirus 2 33K protein. SDS-polyacrylamide gel-purified 33K protein,labeled in vivo with[3H]alanine(A),

[3H]pro-line (B), [3H]valine (C), or several other amino acid isotopes ([3H]isoleucine, [3H]leucine, [3H]lysine,

[35S]methionine, [3H]phenylalanine, or [3H]tyrosine;

datanotshown)wasdigestedwithtrypsinasdescribed in the text, and thetrypticdigestswerefractionatedby reverse-phase high-pressure liquid chromatography. The programmed gradientproffle used for all experi-ments is shown in (A); the flowrate was2.0ml/min,

and 1.0-ml (30-s) fractions were collected. For the

threeexperiments shown, 100 Fl of each fractionwas countedwith 3.5 ml ofAquasolpersampleinaSearle Mark IIIscintillation counter. The countsper minute from these100-1Llsamples have been plotted directly without background subtraction. The effluent was also monitored at214nm to detecttryptic peptides derived from the apomyoglobin present as carrier in each sample (profiles not shown). Each radioactive peak has beenassigned a name derived from the isotope used (single-letter amino acid code) and the relative elution order for that peptide. Appropriatefractions were pooled, mixed with apomyoglobin, and lyophi-lizedinpreparation for radiochemical sequence analy-sis.Approximate positionsoftryptic peptides predict-edfrom the nucleotide sequence(Table 1)that have

exactly match thesequencepredicted beginning

at N1120 in the third reading frame.

Further-more, prolines found in peptide P5 and the

valine found in peptide V4, two peptides with

elution positions identicaltotheposition of A6,

also exactly matched this sequence (Table 1).

This result strongly suggested that the coding

region for the carboxy-terminal portion of the

33Kprotein beginsatleastasfar leftin the third

openframeasN1120, and thatan event

connect-ing the second and third open reading frames

mightoccurimmediately tothe leftof this point.

Analysis of peptides fromopenreading frame 2

indicatedthat the lastconfirmed residue in this

frame is the proline (residue 20) in peptide P7

(Fig. 4D) encoded by N870toN872. Therefore,

the connectingevent must occur to theright of

this site and within thenext 249nucleotides.

Two separate explanations for the above

re-sults are possible. An error might have been

made in the sequence between N872 and

N1120, orthemRNAforthe 33Kprotein might

bespliced within the coding region.

Partialnucleotide sequence analysis of the

ade-novirus 2 EcoRI-F fragment. To address the

possibility that a sequencing ambiguity may

have causedan apparentshift in the33Kprotein

reading frame, weindependently determined the

sequence of selected portions of the EcoRI-F

fragment. The sequence of 249 nucleotides

around the HindIIIsite at72.8 m.u. was

deter-mined by labeling HindIII-cleaved genomic

DNAwith[32P]ATPandpolynucleotide kinase,

isolating the relevant EcoRl subcut fragments

(70.7 to 72.8 m.u., 72.8 to 75.9 m.u.), and

treating these fragments by the method of

Maxamand Gilbert (12, 24). The 153 nucleotides

totheleft of N736 (to N583) and the 1%

nucleo-tidestotheright of N736 (to N932)werefoundto

be identicaltothepublishedsequence(13). Ina

similar way, except starting with the isolated

genomic EcoRI-F fragment, the 188 nucleotides

totheleft of the HpaII siteatN1554(to N1365)

and the 160nucleotides tothe leftof theHpaII

site at N1134 (to N975) were also found to be

identical to the published sequence. The

697-nucleotide sequence independently confirmed

by usinclude the sequence immediately before

the alanine codon at N1120. Thus the two

co-donsimmediately preceding this alaninemustbe

Ser-Thr- andnot(Lys/Arg)-Ala- asissuggested

by our direct peptide sequence analysis. Our

sequenceanalysishasconfirmedall but 43of the

nucleotides between N872 (the Pro in P7) and

N1120(thesecond Ala in A6).

beenidentifiedby sequence analysisare indicatedat

the top of(A). Predictedtrypticpeptidesarenumbered from the aminoterminus(Table1).

VOL.45,1983

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TRYPTIC PEPTIDES OF THE ADENOVIRUS 233K PROTEIN LQLPPPPPTDEEEYWDSQAEE LATVPSIATTSAPQAPPALPVR

peptide L12 C peptide A7

A 3H-Leu 1 4 3H-Ala

AAPTAPAAAAAAATAAVTQK

E peptide A6

140r

;_ IV*_J v ;J IV v v v, Iv ;

RESIDUE RESIDUE RESIDUE

FIG. 4. Radiochemical microsequence analysisof adenovirus 2 33Kprotein tryptic peptides. Tryptic peptides ofradioactivelylabeled 33K proteinwerefractionated by reverse-phasechromatography as describedinthe

legendtoFig.3.Appropriate pooledfractionswereappliedtoaBeckman 890Csequencertogetherwith 2mgof apomyoglobinand 3mgofpolybrene.Panelpairs depictthetotalyieldofradioactivityin theamino acid fraction after eachEdmandegradation cycleforcorresponding peptides. Peptidesarenamedasdescribed inthelegendto

Fig. 3,and theisotopewith which eachpeptidewaslabeled is indicatedinthepanel. Peptideelutionpositions

canbe ascertained fromFig. 3orTable 1. The amino acidsequencepredictedfrom thenucleotide sequence

coffespondingtoeachpeptide pairisgivennearthe top of theupperpanelinthesingle-letteramino acid code. PeptidesA6 and P5(panelsE andF) correspondtothetryptic peptidethatspanstheintemalsplice junctionin

the33K mRNA(see text).Theamountofradioactivity appliedtothesequencerwasasfollows:(A) 3,900cpmof

[3H]leucineinL12; (B) 5,300cpmof[3H]prolineinP1o;(C) 1;520cpmof[3H]alanineinA7; (D) 13,500cpmof

[3H]prolineinP7; (E) 36,300cpmof[3H]alanineinA6;and(F) 6,650cpmof[3H]prolineinP5.

33K mRNA isinternally spliced.The structure ofpeptide A6 cannotreadilybeexplained by a

frameshifteventinthevicinity of N1120. There-fore, we examined the possibility that the 33K mRNA isspliced in amannerthatconnects the tworeadingframes andexplainsthe structureof peptide A6. The nucleotide sequence between

N1112 and N1120 (TCTCTACAG) conforms welltothe consensus splice acceptor sequence

YNYYYNCAG (34) (Fig. 6).Examination of the

sequencetothe left of N1120revealeda poten-tialsplice donor siteatN918. Asplicefrom this position tothe C at N1121 would result in the removal of 202 nucleotides from the transcript for the 33Kprotein, wouldpreservethe alanine

codon that appearedtobeencodedby N1120to N1122, and would cause this codon to be

pre-ceded in the mRNAbyanArg-Ala codon pair.

Thispredicted splice is precisely consistent with the observed structureofpeptide A6. No better potential splice donor sitewasfound by

exami-nation of the nucleotide sequencebetween N872

and N1120.

If the33K mRNA is indeedsplicedas suggest-edabove, the 202-nucleotide splice should have beenseenbyheteroduplexorSi nuclease

analy-sis ofregion L4 mRNAs, and certain peptides predicted tobe encodedby either the secondor

thirdopenreadingframe shouldnotbe identifi-able. In fact, a spliced L4 mRNA has been

observed both by Si analysis (8; S. Berget, unpublished data quoted in reference 25) and by heteroduplex analysis (10). Furthermore, we

havenotbeen abletofind severalpeptidesthat

we would have expected to observe during

reverse-phase chromatography of 33K tryptic peptides. For example, the proline-containing peptide Gln-Pro-Pro-Leu-Ala-Gln-Glu-Gln-Gln-Gln-Arg, predicted by thesecond openreading frame before the start of the third openframe,

has not been found among the proline-or ala-nine-labeledtryptic peptides.

N

x

0

Fn

w

E

06

0

x

3 0

w

E

U.

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ADENOVIRUS 2 33K PROTEIN 259

TRYPTIC PEPTIDES OF THE ADENOVIRUS 33K PROTEIN

N

0

x 0

Fn w

E

CL

0

0

n

w

a

w E

UL

10

RESIDUE RESIDUE RESIDUE

FIG. 5. Amino-terminal sequenceanalysis of adenovirus 2 33Kproteintryptic peptides. Sequenceanalysis wasasdescribedin the legend toFig.4.The amount ofradioactivity applied to the sequencer was as follows: (A) 8,300 cpm of[3H]prolinein P4; (B)4,550cpmof[3H]valineinV3; (e) 9,500 cpmof[3H]alanineinA3; (D) 1,200

cpm of [3H]phenylalanine in F8; (E) 5,450 cpm of [3H]leucine in L5; (F) 5,150 cpm of[3H]alanine in A5.

Additionalsequencingexperiments with other labels and otherpeptidesarenotshown;asummaryof all amino acidpositions directlyconfirmedbysequencingisgivenin Table 1.

To exclude the unlikely possibility that all

predicted peptides from the intervening

se-quence were lost during reverse-phase

chroma-tography, we prepared tryptic peptides of

[3H]isoleucine-labeled 33K protein and

subject-edthe complete mixture toamino-terminal

se-quenceanalysis (Fig. 7). The secondopen

read-ing frame can potentially code for five

isoleucines,threeof which wouldcomefrom the

predicted intervening sequence. The third open

frame could potentially encode six isoleucines,

threeof which also wouldcomefrom the

inter-vening sequence. The isoleucine codons in the

predicted intervening sequence are situated so

thatradioactivitywould beexpected in the 2nd,

3rd, and 14th residues from second reading

frame codons, and the 16th, 27th, and 31st

residues from third reading frame codons.

Ra-dioactivity would be expected in residues 1

(twice), 7, 8, and possibly3 from the remaining

isoleucine codons. Figure7 shows the presence

of isoleucine in residues 1,2, 7, andpossibly 8,

butnotsignificant radioactivityinresidues3,14,

and 16. Thepresenceofisoleucine in residue 2 is

explained by an unusual trypsin cleavage that

occursafter thetyrosineinpeptidet18(Table 1).

Trypsin is known to occasionally cleave after

tyrosine,and the existenceof the

carboxy-termi-nal fragment of this peptide has been

indepen-dently confirmed by reverse-phase

chromatog-raphy and partial sequence analysis. The

carboxy-terminal fragment elutes at

approxi-mately thesamepositionast27(fraction 62,data

not shown). Thus, at least two isoleucine

pep-tides from the expected intervening sequence

peptideswerenotfound,andallof theexpected

isoleucines from the peptides predicted to be

encoded beginning at N570, except for the

iso-leucineatposition3(seebelow),wereobserved.

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PREDICTED ADENOVIRUS 2 33K PROTEIN INTERNAL mRNA SPLICE POINTS

... Thr ThrGly Thr Arg Ala gly lys ser lys gln pro... Frame 3

... .ACCACT GGA ACC AGG GCC GGT AAG TCT MG CAG CCG... 1 strand (3' side)

901 911 921 931 nucleotide number

A GGU RAG 5' Splice consensus sequence

lice

... ile thr thr val ile ser thr Ala Pro Thr Ala Pro Ala Ala... Frame 1

... ATT ACT ACCGTC ATC TCT ACA GCC CCT ACT GCA CCG GCG GCA... 1 strand (3' side)

1101 1111 1121 1131 nucleotide number

Y- mYY -CA G 3' Splice consensus sequence

FIG. 6. Predicted internalsplice sites in the 33KproteinmRNA.Portions of the nucleotide sequence within the EcoRI-Ffragment are shown. The corresponding openreadingframe amino acid sequence is also given. Nucleotidesarenumbered from theEcoRIcleavage siteat70.7m.u.(13). Theconsensussplicesite sequences arefrom Sharp (34).

carboxy terminus of the 33K protein.The

amino-terminalandcarboxy-terminal sequences of the

33Kprotein have notbeen determined directly,

but several indirectarguments canbeappliedto

deduce their encoding locations. None of the

expected peptides predicted by the sequence

between N411 and N606 has been observed,

including the isoleucine-containing tryptic

pep-tide predictedby N570 to N587. Only one major

methionine-containing 33K tryptic peptide was

observed by reverse-phase chromatography,

andthis peptide elutedattheposition of peptide

t4(Fig. 3). This 33-amino-acid peptide is

expect-ed to contain two methionine residues at

posi-tions 29 and 30from the amino terminus (Table

1). Sequence analysis of the mixture of

[35S]methionine-labeled33Ktryptic peptidesdid

not reveal amethionine in the first20residues.

Sequence analysis of cyanogen

bromide-cleaved, [3H]alanine-labeled 33K mixed

pep-tides revealed no significant radioactivity in the

first 20 residues (datanotshown). Thus cleavage

after methionine residues produced no

un-blocked amino termini. These results allsuggest

that the 33K protein contains only the two

methionines expected in peptide t4. Cleavage

after these methionines by cyanogen bromide would leave an amino-terminal glutamic acid

residue which would be expected to cyclize

underthe strongacid conditions used. The

me-thionine codon atN606 to N608 isfollowed by

analaninecodon. Where this sequence occurs at

known eucaryotic protein synthesis initiation

sites (5, 37), the initiation methionine has been

found to be removed, and the amino-terminal

alanine becomes acetylated. Thus, our findings

arecompatible with initiation of33Ktranslation

at the N606-608 methionine codon. The se-quencesurroundingthis AUGalso matches the

mostfrequentlyfound eucaryotic translation

ini-tiation sequence, CANNAUGG (20). If

initia-tion were to occur at the only other available

methionine codon in the second open frame

(N411 to N413), we would notexpect it to be

removed post-translationally, and we would

have expected to find evidence for the

methio-ninepostulatedtobe encodedbyN606 to N608.

Furthermore, the sequences surrounding this

AUG codonsuggest that itmight not be a strong

initiation site for translation even if present in

the mRNA(20).

The last codonconfirmedby peptidesequence

analysis in the thirdopen frame is the Phe codon

ADENOVIRUS 33K PROTEIN

3H-Ile TRYPTIC PEPTIDES

'4

0

0

U,

0 5 10 15 20

RESIDUE

FIG. 7. Amino-terminal sequence analysis of the mixed [3H]isoleucine-labeled 33K tryptic peptides. Adenovirus 2 33Kproteinlabeled with[3H]isoleucine wasdigestedwithtrypsin. Thelyophilized digest con-taining 20,000 cpmwasdissolved in88% formic acid andapplied to the sequencer together with 2 mg of apomyoglobin and3mg ofpolybrene.Sequencingwas asdescribed inthetextandinthelegendtoFig.4.

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ADENOVIRUS 2 33K PROTEIN 261

TABLE 2. Predicted amino acid composition of adenovirus 2 33K proteina

Predicted amino acidcomposition of proteins

Nonpolarb Noncharged, polarb Charged,polar"

Amino No. of % Amino No. of Amino No.of

acid residues acid residues acid residues

Gly 5 2.2 Ser 18 7.9 Asp 12 5.3

Ala 34 15.0 Thr 19 8.4 Glu 22 9.7

Val 7 3.1 Asn 4 1.8 Lys 14 6.2

Ile 4 1.8 Gln 11 4.8 Arg 17 7.5

Leu 20 8.8 Cys 2 0.9 His 1 0.4

Met 2 0.9 Tyr 4 1.8

Phe 4 1.8

Trp 3 1.3

Pro 24 10.6

aPredicted number of amino acids, 227; calculated polypeptide chain molecular weight, 24,892.

bNonpolar residues, 45.4%; noncharged, polar residues, 25.6%; charged, polar residues, 29.1%.

from t27atN1459toN1461. Elevencodons after this Phe codon, the sequencepredicts an UAG termination codon. The existence of this termi-nation codon was confirmed by our nucleotide sequence analysis. Because no obvious splice donor site islocated between N1461 and N1493,

we conclude that the 33K protein is likely to terminate with aspartic acidaspredicted by the

nucleotide sequence.

Composition of the 33K protein. The predicted

sequence of the 33K protein is given in Table 1

in the form of the expected tryptic peptide

sequences. Thepredicted amino acid

composi-tion is given in Table 2. The protein has ahigh

content of alanine (15%), proline (10.6%), and glutamic acid (9.7%). The 33K protein has previ-ously been showntocontainphosphoserine (6), and a preliminary analysis of the 32P04-labeled

tryptic peptides by reverse-phase chromatogra-phy indicates thatapeptide with the mobility of

peptide t4 is phosphorylated. This 33-amino-acid peptide contains sixserine residues;wehavenot yetdetermined whichone(s) is phosphorylated. Additional 32Pradioactivitywasobserved in the

column flow-through. We have not yet deter-mined whether this materialrepresentsasecond

phosphorylation site orphosphate released

dur-ing trypsin digestion. A potential cyclic AMP-dependent kinase phosphorylation site (37)

ex-ists in peptide tll (Table 1). No other peptide that is retarded during reverse-phase chromato-graphy was found tobe labeled by 32P04 (data notshown). At leastonepotential glycosylation

site(37) exists in the carboxyterminal portion of the 33K protein (Asn-Arg-Ser-); however, we

have no experimental evidence to suggest that this site is actually glycosylated.

DISCUSSION

We have shown that the adenovirus 2 33K protein is a unique, late, nonstructural virus

protein that is encoded bytwoseparateportions

of the EcoRI-F restrictionfragment (70.7to75.9

m.u.). Sequence analyses of radioactively

la-beledpeptides andcomparison withpredictions

made from thepublished nucleotidesequenceof

this region (13) have allowed us to deduce the

precise sites for an internal mRNA splice, the initiation site for translation, and the termination

sitefor 33Kprotein synthesis. Thisinformation

wasnot(13-15)and couldnothavebeenreadily

deduced from the nucleotide sequence alone

givenour presentunderstanding of the

molecu-larsignals required forgene expression.

The proposedsequenceof the 33Kproteinis

227 amino acids long; the calculated molecular

weight of this amino acid chain is 24,892, a

numberconsiderably smallerthanthe size

esti-matedfrom SDS-polyacrylamide gel

electropho-resis. Similarly anomalous behavior has been

noted for the adenovirus 2 ElA proteins (35).

We believe that the high proline and glutamic

acidcontentof theseproteins maycontributeto

theirlower-than-expectedmigrationratesduring

gel electrophoresis. In fact, we estimate the

molecular weight of the 33K protein by gel

electrophoresistobe closerto39,000Mrthanto

33,000 Mr.Toavoidconfusion,wehaveretained

the name "33K protein" until an appropriate

functionaldesignation canbegiventothis

prod-uct.

The amino acid sequence ofthe 33K protein

has some unusual features. The regionbetween

amino acid residues 103 and 118 (within t9,

Table 1) contains 12 alanines, 7 of which are consecutive. This sequence is encoded

immedi-ately after the internal mRNA splice and is

followedatresidue139 to 146by anotherrunof

five alanines and three glycines. Regions very

rich inproline (residues 9 to 13), glutamic acid

(residues16 to53) orbasic amino acids(residues

3 to5, 90 to 95, and 122 to 136)are alsofound.

Analysis oftheproteinsequencebythemethods

ofChou and Fasman(11)suggests the presence VOL.45, 1983

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OOSTEROM-DRAGON AND ANDERSON

of several short regions of probable a-helix.Two

of thesearein theamino-terminal portion

(resi-dues 22to38, and residues41 to 52)before the

mRNA splice point. The corresponding region

of the nucleotide sequenceprobably alsocodes

for the carboxy-terminal portion of the 1OOK

protein; 103 codons are predicted to

simulta-neouslycode for bothproteins. This is the only

region of the adenovirus 2 genome thus far

identifiedthatsimultaneously encodestwo

prod-ucts unrelated in amino acid sequence. The

carboxy terminus of the100Kproteinis

predict-edto occurjust before the 33K mRNA splice point.

Why is the 33K mRNA spliced? Asfar asis

known, neither strand of the sequencebetween

N918 and N1120 contains signals for other

eventsthatmight be incompatable witha

func-tional coding sequence for the 33K protein. A

promoter sequence for region E2 transcripts does occur onthe opposite strand (7), butat a

positioncorrespondingto coding sequencesfor

thecarboxyterminus of the 33Kprotein (Fig.2).

No termination codons in eitherreading frame

usedby the 33Kproteinoccurwithin theregion

removed by splicing. Thus we have found no

obvious explanation for the internalsplicein the

33K mRNA except to connect the two reading

frames andtoalter the amino acidsequencethat

would otherwise result.Perhaps the 33Kprotein

was once two separatefunctions,orperhaps the

regionbetween N918 and N1120performssome

function which has not yet been recognized.

WhilemanycellularproteinmRNAsarespliced

within coding sequences, theonly other

adeno-virus products with mRNAs knowntobespliced

withincodingsequences aretheearlyregion1A

products (35). It has been suggested, however,

that the mRNAsforcomponentIVa2andfor the

region E2Bproducts maybespliced justwithin

the amino-terminal coding region (Jeff Engler

and BruceStillman, personalcommunication).

The 33K mRNA presumably corresponds to

the L4 mRNA in which a splice has been

ob-servedby heteroduplexanalysis (10).The

struc-tureof this mRNA isnotknown precisely, but

presumably another splice also connects

se-quences to theleft ofN606(the initiationcodon)

tothetripartite leadersequence. The nucleotide

triplet CAG found in the spliceacceptor

concen-sus sequence occurs six times between N606 and N411; the codon for the first AUG in the

amino-terminal openreading frame is atN411.

Fourof these CAG sequencesare preceded by

pyrimidine-rich sequences whichare very

simi-lar to the concensus splice acceptor sequence. Incontrast, thefourCAGsequences thatoccur

betweenN606and thedouble-methionine codon

atN708 are preceded by purine-rich sequences

and donotappeartoresemble

splice

The function ofthe 33Kprotein is unknown.

To ourknowledge,no mutantshavebeen

identi-fiedwhichmapwithinthe predicted33Kcoding

sequences. Severaltemperature-sensitive

muta-tions are known that map within sequences

encoding the lOOK protein. Two of these,

H5tsll5 andH5tsll6, havevery similar

pheno-types, belong to a single complementation

group,butmapabout 1 m.u. apart (27; E.

Oos-terom-Dragon, Ph.D. thesis, Yeshiva

Universi-ty, Bronx, N.Y., 1980) H5ts116 maps to

theleft of the EcoRI restriction siteat 70.7 m.u.,

whereasH5tsll5mapswithintheEcoRI-F

frag-ment(70.7to 75.9m.u.).The similar phenotypes

of these two mutants suggest that they are

defective in only the same function. Thus we

believe that the H5tsll5 mutationis likely to be

found before N606 and the start ofsequences

which alsoencode the 33Kprotein. Although we

havenotbeen abletodeduce the function of the

33K protein from knowledge ofthe nucleotide

sequenceswhich encode it, this knowledgemay

allow the construction of mutants which will

helptoelucidate its function.

ACKNOWLEDGMENTS

Wethank Medora Hardy and Sydell Lamb for excellent

technical assistance. We are grateful to John Dunn and William Crockett for assistance with the sequencing of DNA fragments, and to Marshall Elzinga, Nicholas Alonzo, and JeanneWysocki for assistance with peptide separation tech-niques and peptide sequence analysis. We thank Keith

Thompsonfor computer analysis of nucleotide and protein sequences.

Thiswork wassupported by Public Health Training grant

T32CA09121 fromthe NationalCancer Institute and by the

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