Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

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JOURNALOFVIROLOGY, June1986, p.757-764 Vol. 58, No. 3

0022-538X/86/060757-08$02.00/0

CopyrightC) 1986,AmericanSocietyforMicrobiology

Localization and Fine Structure of

a

Vaccinia Virus Gene Encoding

an

Envelope Antigen

PIERRE HIRT,' GERHARD HILLER,'tANDRICCARDO WITTEKL*

InstitutdeBiologieAnimale, Universitede Lausanne, CH-1015 Lausanne, Switzerland,' and Max-Planck Institutefor

Biophysical Chemistry,

D-3400

Gottingen,

Federal

Republic

ofGermany2

Received 3 February1986/Accepted 10 March 1986

Themajor antigen on theenvelopeof extracellular vaccinia virusparticlesisapolypeptidewithanapparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). Thegeneencoding

p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation witha monoclonal antibody. The gene is located closetotheright-hand end of theHindlllFfragment. Thecorrespondingregion

of the DNAwassequenced, andanopenreading frame encodingapolypeptide of 41,748 daltonswasobserved. The 5' end of the mRNA, as defined by nuclease Si analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and whichisconserved in all butonelate genesofaranalyzed. It is therefore likely that thissequence

isanimportantregulatory signal for lategeneexpressionin vaccinia virus.

Vaccinia virus is a member of the poxvirus family. The

virionsarecharacterized bytheir large size and typical brick

shape. The genome of vaccinia virus consists of a linear,

double-stranded DNA molecule ofca. 190 kilobase pairs.

This large amount of genetic information is expressed in a

temporally regulated fashion. Earlygenes aretranscribedby

the virion-associated RNA polymerase before complete

uncoating ofthe viral genome. After DNAreplication, late

genes encoding predominantly structural polypeptides are

expressed (14).

In contrast to most other animalDNAviruses, the

multi-plication cycle of vaccinia virus is confinedtothe cytoplasm

ofthe infected host cell, where replication occurs within

discrete cytoplasmic foci termed "factories" (4). Progeny

virus is assembled within or close to the factory areas in a

complex morphogenesis process(reviewed in reference5).

Uncertainty exists with respect to the mechanism of

release ofprogeny virus, a process which is inefficient in mostcell culture systems used for the propagation of

vac-cinia virus. Most investigators therefore recover progeny

virus after experimentally induced cell lysis, which yields

large amounts of infectious virus particles. However, virus

naturally released from the living cell (extracellular virus)

differs antigenically fromvirusrecovered fromtheinterior of

thecell (1).

Recently, cell cultures and virus strains which produce

sufficient amounts ofextracellular virus to permit detailed

biochemicalanalysis havebeen described(17). The

charac-teristic feature of extracellular virus is the presence of an

envelope (1, 24) containing several virus-specific antigens (16, 17), the majorof which is a polypeptide with a molecular

weight of 37,000 (p37K; 8). The polypeptide has been

classifiedasalateone, since it isonly expressedin cells after

the onsetofviral DNAreplication(9).

*Correspondingauthor.

tPresent address: Boehringer GmbH, D-6800 Mannheim 31, FederalRepublic of Germany.

757

In uIuO

IN UITRO

- - aE

TOT IPP IPP TOT

_

U

U,

i---S

FIG. 1. Immunoprecipitation ofp37Ksynthesizedin vivo and in vitro.Vaccinia virus-infected cell monolayers were labeledfor3 h with[35S]methionine and [35S]cysteineat 4h after infection. Total cell lysates were analyzed either directly (lane TOT) or after immunoprecipitationwith theanti-p37K antibody (lane IPP). Total RNAfrom vaccinia virus-infected cells was translated invitro,and the products were analyzed either directly (lane TOT) or after immunoprecipitation with the anti-p37K antibody (lane IPP). A

fluorograph ofa14% polyacrylamide gel isshown.

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758 HIRT ET AL.

A

H i nd fragments

2 35

C NMK F E 01 G LJ H D

4 5

A

I

111 Ii

[11 1

11 L

10 kb

1 2 3 4 5 m m N N K F

.

'

~~~~~200-So"'I"'4

_{_} ₉ _-U

.*Rrn.

II

U

,- 69

,-46

U

up

b

_C

FIG. 2. Gel analysis of immunoprecipitated cell-free translation products. Lanes 1 to 5 and N, M, K, and F in panels B and C show the immunoprecipitated polypeptides made by RNA selected on pools of restriction fragments or on individual restriction fragments,

respectively, asindicatedintheHindlIl map of the vacciniavirus DNA shown in panel A. The arrows indicate the positions of twoSallsites within the unique sequence of theHindlll B fragment. Afluorograph of a 14% polyacrylamide gel is shown. The molecular weights (in thousands) of polypeptides used as size markers (lanes m) are indicated. kb, Kilobases.

Amonospecific antibodyhasrecentlybeen usedtofurther

characterize p37K and also to study the mechanism by

whichvaccinia virusacquiresits envelope (9). After

synthe-sis, p37K, which is unglycosylated but contains palmitic

acid, accumulates in Golgi membranes. These membranes

with viral protein(s) incorporated are then used to enwrap intracellular vaccinia virus with a double-walled coat (9).

These virus-carryingvesicles then appeartobetransported to the cell surface, wheretheiroutermembrane fuses with

the cytoplasmic membrane, thus releasing a virus particle

surroundedby asinglemembrane.

Inthiscommunicationwereportthemapping ofthe gene

encoding p37K on the vaccinia virus genome. We also

presentthe fine structure of the gene and itsflanking regions.

MATERIALS ANDMETHODS

Virus and cells. The WR strain of vaccinia viruswasused inthisstudy. Rabbitkidneycells(RK-13) and African green

monkey kidney cells (CV-1) were grown in Eagle minimal

essentialmediumsupplementedwith5%fetal calfserumand inDulbecco modifiedEaglemediumsupplementedwith10% fetal calf serum, respectively. Cells were infected with 10 PFUof vaccinia virus per cell.

Monoclonalantibodyeevl. The material usedfor immuni-zationconsisted oftheBrij-solubleandtrichloroacetic

acid-precipitable components of crude extracellular vaccinia

vi-rus preparations. Spleencells from successfully immunized

Lourats werefused with theratmyelomaline YB2/0(11)to J. VIROL.

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VOL.58,1986~~~~~~~VACCINIA VIRUS ENVELOPE ANTIGEN 759

A

Hind III F

9~~~~~~~~~~O r9C F

1 2 3 4 5 6 7 6

1 kb

1 2 34'5 67 6 m 5 6 7 8 m

" '200"W

S.

92

Sa

U,',W

l..-iI"

46

350

iMP

[image:3.612.133.488.71.460.2]

B

C

FIG. 3. Immunoprecipitationof invitro-translatedpolypeptides.TotalRNAwasselectedby hybridizationtoelectrophoretically separated DNArestrictionfragments(B)ortosubcloned DNAfragments (C).Lanes 1to8 show theimmunoprecipitated polypeptides obtained from RNAselectedonthe DNAsegmentsindicated inpanelA. Afluorographofa14%polyacrylamide gelis shown. The molecularweights(in thousands)ofsizemarkers (lanes m)areindicated. Symbols: IHindIII; F' Sall; F. EcoRI;~,BamHl.

createhybridomasinaccordance with theinformationgiven in a recent review (7). A selected clone produced eev1 antibodies,whichspecificallyandselectively immunoprecip-itated the previously recognized acylated p37K of extracel-lular virus (manuscriptinpreparation).

For immunoprecipitation experiments purified eev1 anti-bodies were covalently coupled to Sepharose (Pharmacia,

Uppsala, Sweden) by standardprocedures.

Preparationof labeled cell extracts. Monolayer cultures of CV-1 cellsin 3-cmdisheswereinfected with vacciniavirus.

At 4 hafter

infection,

the

growth

mediumwas

replaced' by

1

ml of medium

containing

1/10 of the

regular

methionine

concentration and 20 p~Ci each of

[35S]methionine

and

[35S]cysteine.

After an additional 3 h ofincubation, cells were washed with

phosphate-buffered

saline and thenlysed

by

theaddition of 0.5mlof0.0625 M Trishydrochloride (pH

6.8)-3% sodium dodecyl sulfate-15% glycerol-1%

2-mercaptoethanol-0.015% bromophenol

bluefor subsequent analysisof

polypeptides

by

SDS-polyacrylamide

gel

electro-phoresis. For

immunoprecipitation

cells were lysed by the

addition of1 ml of10 mM Tris hydrochloride (pH 7.4)-l%

Triton X-100-0.5% sodium

deoxycholate-0.1%

SDS-150

mMNaCl-1 mM EDTA. After3 hat

40C,

insoluble material

was removed

by

centrifugation

at 100,000 X gfor 90 minat

40C.

Imimunoprecipitation. Polypeptides

were

immunoprecipi-tated from tota cell extracts or from in vitro translation

mixtures asdescribed previously(28).

RNA extraction. Earlyand late RNAs were purifiedfrom vaccinia virus-infected cellsasdescribed in detail elsewhere

(28).

Hybrid selection ofRNA. RNAspecific todefinedregions

of the vaccinia virusgenomewas

purified by hybridization

to

cloned DNA restriction fragments as described previously

(28).

In vitro translation. Total and

hybrid-selected

RNAswere translated ina

message-dependent

rabbitreticulocyte lysate assay as described

previously

(28).

Nuclease Si analysis. The procedure for

mapping

RNA transcripts by nuclease Si analysis (2) with 5'-end-labeled DNA fragments as hybridization probes (25) has been

de-scribedpreviously (28).

DNAsequencing.DNAfragmentswerecloned into

single-stranded

phage

vectors (13) and sequenced

by

the chain

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760 HIRT ET AL.

A mRNA p37 k

L E L

F~ F¶ - f

91g--250 bp

3. a

41.

B

1

2 3

3

4

-m p

t

e I p I p

t

Ie

m p I p p

I

Es~~~~~~~~~~~~~~~~~__

622

d

622 45

527

4S

527 *

403 am

_ __ 403

309

3093

242 238 f

217 to 201t

242

FIG. 4. Nuclease Si mappingof RNAtranscripts. (A)ADNAsegmentfrom theright-handHindIIIsite of theHindIIIFfragmenttothe

extremeleft-handSallsitewasused fortranscriptional mapping.Variousfragmentsderivedfrom thisregionwerelabeled at the 5' end(open circles) and usedashybridization probes. Eachprobe(1to5)wasanalyzedeitherdirectly (BandC,lanesp)orafterhybridizationtotRNA

(B, lanes t) or to early (B, lanes e) or late (B and C, lanes 1) RNA from infected cells. Si-resistant material was analyzed on a 6%

polyacrylamide sequencing gel.The sizes(in bases)oflengthmarkers(lanes m)areindicated.Symbols:

I,

HindIII; y,Sall; ?,BglII;-A,

HincII; I, ClaI; y,EcoRI. (NotethatonlytheBgII, HincII, andClaIsitesofparticularinterestareindicated;this regionof the DNA

contains additional restriction sites for theseenzymes.) Themapposition and direction oftranscriptionof thep37K mRNA,aswellasof

additionalearly (E) and late (L) RNAs,is shownbythearrows.The broken lineatthetipof eacharrowindicatesthatthe3' end of theRNA hasnotbeenmapped;vertical broken lines indicate the relative positionsofparticularrestriction sites in theappropriate Si probe. termination method (21). In some cases, the chemical

deg-radationmethodwas used(12).

RESULTS

Immunoprecipitation of in vitro- and in vivo-synthesized p37K. To identify p37K, a characteristiccomponent of the

envelopemembrane of extracellular vacciniavirus,wemade

use ofa newly establishedrat monoclonal antibody (eevl) whichselectivelyreactswith thispolypeptidein

immunopre-cipitationaswellasin Westernblotexperiments (G. Hiller, manuscriptinpreparation).

When synthesized in vivoin the presence ofradioactive palmitic acid, p37Kwaslabeled(9). Acylationofproteinsis

aposttranslationaleventwhich does notoccurincelllysates

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VACCINIA VIRUS ENVELOPE ANTIGEN 761

used for in vitro protein synthesis. We therefore examined

the ability of monoclonal antibody

eev1

to react with in vitro

translation products directed by total RNA from vaccinia

virus-infected

cells.

As a control, the in vivo-synthesized

polypeptide

was

immunoprecipitated from infected cell

ex-tracts in parallel. In both cases, a single polypeptide of the expected size was detected (Fig. 1). The in vitro translation product showed a slightly increased electrophoretic mobility as compared with the in vivo-synthesized polypeptide; this increase can best be explained by the lack of posttransla-tional modification of the in vitro-synthesized product.

Mapping of the p37K gene within

HinduI

restriction

frag-ments. RNA from vaccinia virus-infected

cells

was selected

by hybridization to cloned

HindIII

restriction

fragments

of

the vaccinia virus genome. Five nitrocellulose membranes containing the indicated fragments (Fig. 2A) were used in a first experiment. After hybridization, the filters were

washed, and

bound

RNA was then eluted and translated.

After immunoprecipitation, the products were analyzed by

polyacrylamide

gel

electrophoresis

(Fig. 2B). The mRNA

encoding p37Kwasclearly selected byfilter1containing the

HindIII

fragments N, M, K, and F. These fragments were therefore used separately to select RNA, and the polypep-tide was obtained from RNA selected on the HindIII F

fragment (Fig.

2C).

Localization of the p37K gene within the

HindIll

F

frag-ment. To further map the gene, we prepared a detailed restriction map of the HindIII F fragment (ca. 13.5 kilobase pairs) (Fig. 3A). The recombinant plasmid was cleaved with

EcoRI,

BamHI, HindIII, and

SalI,

and the resulting

frag-ments were then separated by gel electrophoresis and trans-ferred to nitrocellulose membranes. The membranes were cut such that each piece contained a given restriction frag-ment. The DNA was used for hybrid selection of RNA. Selected RNA was translated in vitro, and p37K was

iden-tified by immunoprecipitation (Fig. 3B). A

SalI

fragment of

ca. 840 base pairs (bp) (Fig. 3B, lane 7) efficiently selected the mRNA. Smaller amounts of the mRNA were also

selected on a

SalI

fragment of 230 bp (Fig. 3B, lane 6), and

trace amounts were selected on all other fragments. Since about 1,000 nucleotides are sufficient to encode a

polypep-tide with a

molecular

weight 37,000, it is somewhat

surpris-ing that the mRNA was selected on such a long stretch of

DNA. This result may be

explained

at least in part by the

fact that

late-infection

vaccinia virus transcripts are very

heterogeneous in length, do not contain discrete 3' ends (27), and may exceed in length the minimal coding requirement severalfold. However, some cross-contamination of restric-tion fragments or inaccuracy in cutting the membranes or both cannot be excluded. A small polypeptide was also immunoprecipitated from the in vitro translation products of RNA selected on fragment 2. This polypeptide presumably

has an antigenic

site

in common with p37K and is apparently

detectable only when its mRNA is sufficiently enriched. The experiment nevertheless clearly indicated that the

coding sequence of

p37K

is located at the right-hand end of

the

HindIII

F fragment. Four DNA fragments from this region were therefore subcloned, and equimolar amounts of

each recombinant

plasmid

were

immobilized

on

nitrocellu-lose filters and used to select RNA. After in vitro translation,

p37K was

immunoprecipitated

(Fig.

3C).

Again, a large

amount of p37K was obtained from RNA selected on the

SalI

fragment of ca. 840 bp (Fig. 3C, lane 7), and

succes-sively decreasing

amounts

were obtained from RNA

se-lected on the adjacent left-hand fragments. Thus, hybrid selection of RNA clearly showed that a large portion of the

HIND III FRAGMENTS

F

L...j 10 kb

4-Ikb

2 840 Sal

Sal

I It ~ ~~~~~~IXba

Alu

I Dra

Bgl

---I A Sau

IOO bp

A 40 4-3 u WA do

B. 4 °0

FIG. 5. Sequencing strategy. A detailed restriction map of the DNA that was sequenced is shown. Various restriction fragments

were either cloned into single-stranded phage vectors and se-quenced by the chain termination method (A) or labeled at the 5' end (circles at end of arrows) and sequenced by the chemical degrada-tion method (B). The direcdegrada-tion and extent of sequencedetermination

are indicated by arrows. kb, Kilobases.

coding sequence of p37K maps within aSalI fragment of840

bp located near the right-hand end of theHindIlI F fragment.

Mapping of the p37K mRNA. The p37K mRNA was

mapped by nucleaseS1 analysis. Five 5'-end-labeled

frag-ments derived from the region to which the p37Kgene had

been mapped by hybrid selection of RNA were used as

hybridization probes (Fig. 4A). Each probe was analyzed

directly (Fig. 4B and C, lanes p) or hybridized totRNA as a

control (Fig. 4B, lanes t) or to early (Fig. 4B, lanese) or late

(Fig. 4B and C, lanes 1) RNA isolated from vaccinia

virus-infected cells. After hybridization, single-strandedDNA was

digested with nucleaseS1, and resistant material was

ana-lyzed by gel electrophoresis.

With probe 1, a SalI-ClaI fragment, the entire probe was

protected with both early and late RNAs. In addition, late

RNA also protected a fragment of 360 nucleotideswhichwas

also obtained with the longer probe 2 butwhich was labeled

at the same end. The map position of this late RNAisshown

in Fig. 4A. It is unlikely that this transcript represents the

p37K mRNA, since a cloned fragment from this region was

negative in the hybridization selection procedure (Fig. 3C,

fragment 8). The map positions of additional major earlyand

late RNAs detected with probe 2 are also indicated in Fig.

4A. With probe 3, the fragment which very efficiently

selected the p37K mRNA, late RNA protected the entire

probe and, in addition, a smaller fragment of ca. 750

nucle-otides. Since probe 3 was labeled at both ends, it was not

possible to determine the direction of transcription of this

late RNA, which most likely represents the p37K mRNA.

Probe 3 was therefore cleaved asymetrically with either

HincII (probe 4) orBglII(probe 5; note thatonly one BglII

site had been cleaved). The digested fragments (without

isolating the individual fragments) werethenanalyzed either

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762 HIRT ET AL.

I 10 20 30 40 50 60 70 80 5'

OTCOACTTTORT0RRRRTTTTROCORTRTROCCORTOATRTTCTRORTCOTTORTRGRRCRAORTOTATRROTTTTTRTOTTRRCTRA

89 99 109 119 129 139 149 159 169 179

ATO TOO CCR TTT OCR TCO GTR CCT 0C0 GAP OCR RAR TOT AGO CTO OTA GAR RCA CTA CCA GAR ART ATO GAT TTT AGA TCC GAT CRT TTA [let Trp Pro Phe Ala Ser

UVl

Pro Ala Glg Rio Lys Cys Arg Leu Val Olu Thr Leu Pro Olu Asn

[let

Asp Phe Rrg Ser Rsp His Leu

180 189 199 209 219 229 239 249 259 269

RCA RCA TTT GAR TOT TTT ARC GAR ATT RTC ACT CTR OCT ARO PAR TAT ATA TRC ATA OCR TCT TTT TOT TOT RAT CCT CTO ROT ACO ACT Thr Thr Phe Glu Cys Phe Asn Olu lie lie Thr Leu Rio Lys Lys Tyr lie Tyr lie Ala Ser Phe Cys Cys Rsn Pro Leu Ser Thr Thr

270 279 289 299 309 319 329 339 349 359

AGO GOR OCO CTT RTT TTT OAT RAR CTA RAR GAO OCR TCT GAR RAR 000 ATT RAR RTR ATA OTT TTO CTR OAT GAR COR 000 RAR ABA RAT

Rrg Oly Rla Leu lie Phe Asp Lys Leu Lys Olu Rla Ser Olu Lys Gig lie Lys lie lie Val Leu Leu Asp Olu Arg Gly Lys Rrg Rsn

360 369 379 389 399 409 419 429 439 449

CTO GOR GAO CTA CAR ROT CRC TOC CCO ORT RTR RAT TTT ATA RCC OTT ART ATA OAT RR RAR RAT RAT OTO GOR CTA CTR CTC GOT TOT Leu GIy Olu Leu Gin Ser HIs Cys Pro Asp lIe Rsn Phe lie Thr Val Asn lIe Asp Lys Lys Rsn Asn Val Olg Leu Leu Leu Glg Cys

450 459 469 479 489 499 509 519 529 539

TTT TOO OTO TCA OAT OAT OAR ROR TOT TAT OTA OGR ARC OCO TCR TTT ACT 0OG GOA TCT PTR CRT ACO RTT AAR ACO TTR GOT OTR TAT Phe Trp Val Ser Asp Asp Olu Arg Cys Tyr Val Gly Asn Ala Ser Phe Thr Gly Gly Ser Ile Hls Thr Ile Lys Thr Leu Gly Val Tgr

540 549 559 569 579 589 599 609 619 629

TCT OAT TAT CCC CCO CTO OCC RCA OAT CTT COT ROR ROR TTT OAT ACT TTT ARA 0CC TTT RAT ROC OCR ARA RPT TCR TOO TTG RAT TTA

Ser Asp Tyr Pro Pro Leu Rla Thr Asp Leu Arg Arg Arg Phe Asp Thr Phe Lys Rla Phe Rsn Ser Ala Lys Rsn Ser Trp LeuAsn Leu

630 639 649 659 669 679 689 699 709 719

TOC TCT OCO OCT TOT TOT TTO CCA OTT ROC RCT OCO TAT CRT ATT PRAG RT CCT ATA GOT GOR OTO TTC TTT ACT OAT TCT CCG OAR CRC

Cys Ser Ala Ala Cys Cys Leu Pro Ual Ser Thr Rio Tyr His Ile Lys Rsn Pro Ile Oly Oly Val Phe Phe Thr Asp Ser Pro Glu Hils

720 729 739 749 759 769 779 789 799 809

CTA TTO GOR TAT TCT ROR ORT CTA OAT ACC OAT OTA OTT RTT OAT RAR CTC RAG TCO OCT RAO ACT ROT RTA OAT ATT ORA CRT TTO 0CC

Leu Leu Glg Tyr Ser Arg Rsp Leu Asp Thr Asp Vol Val Ile Asp Lgs Leu Lgs Ser Rio Lys Thr Ser Ile Asp lIe Glu His Leu Rio

810 819 829 839 849 859 869 879 889 899

ATA OTT CCC ACT RCA COT OTC GRC GOT RAT ROC TAC TRT TOO CCC GRC ATT TRC ARC TCC ATT ATA ORR OCR OCC RTT RAT ROR GOR OTT

lIe Val Pro Thr Thr Rrg Val Asp Glg Rsn Ser Tyr Tyr Trp Pro Asp lIe Tyr Asn Ser lie lie Olu Rla Rla Ile Rsn Arg Glg Val

900 909 919 929 939 949 959 969 979 989

RAG RTC ROR CTT CTA OTT GOT RAT TOO ORT RAG ARC GRC OTA TAT TCT ATO OCR ACC OCC RAR ROT CTR GRC OCO TTO TOT OTT CAR RART Lys lIe Rrg Leu Leu Val Oly Rsn Trp Asp Lys Rsn Asp Val Tyr Ser let Rla Thr Rla Arg Ser Leu Asp Rla Leu Cys Vol GIn Rsn

990 999 1009 1019 1029 1039 1049 1059 1069 1079

GAT CTA TCT OTO RAG OTT TTC ACT ATT CR0 RAT RAT RCA ARA TTO TTG ATA OTC GRC GRC ORA TAT OTT CRT RTC ACT TCO OCR RAT TTC Asp Leu Ser Vol Lgs Vol Phe Thr Ile Gin Rsn Rsn Thr Lys Leu Leu lie Vol Asp Asp Olu Tyr UVl His lie Thr Ser Rio Rsn Phe

1080 1089 1099 1109 ll9 1129 1139 1149 1159 1169

ORC GOR RCC CRT TRC CAR RAT CRC GOR TTC OTC ROT TTT ART ROT RTA OAT RAR CR0 CTT OTA ROC ORG OCT RAR ARA ATA TTT ORG ROR Asp GIg Thr His Tyr OIn Asn His Gly Phe Val Ser Phe Rsn Ser lIe Rsp Lys Gin Leu Val Ser Olu Rio Lys Lys lie Phe Olu Rrg

1170 1179 1189 1i99 1208 1220 1230

OAT TOO GTA TCT ROC CRC ROT PAR TCO TTR PAR ATT TAR RRRRRGARRRTAORGACOTRTRGR Asp Trp Val Ser Ser His Ser Lys Ser Leu Lys Ile ***

FIG. 6. Nucleotide sequence of the p37K gene and flanking regions. The sequence starts at the extreme right-handSalIsite of the HindIII Ffragment. Thederived amino acid sequence of the p37K polypeptide is also shown. The mappositionsofthe 5' ends of the mRNA are indicated by the bar.

directly (Fig. 4C, lanes p) or after hybridization to late RNA 230 bp as well as some adjacent left-hand DNA were

(Fig. 4C, lanes 1). The uncleaved fragment (probe 3) was also sequencedbythe strategyoutlinedinFig. 5.The nucleotide includedin the analysis. With probe 3, a majorband ofca. sequence and the derivedamino acidsequenceofthep37K

750 nucleotides was again observed. An intense band gene as wellasthe 5'-and 3'-flanking regionsare shownin

comigratingwiththelargerfragment ofprobe4 wasobtained Fig. 6.

after hybridizationtolate RNA, indicatingthattranscription Location ofthe5'endofthe mRNA. To mapthe 5' endof

oftheputative p37K mRNAis fromrighttoleft. This result thep37KmRNAprecisely,welabeledanAluI-SalIfragment wasconfirmed with probe 5, withwhich the entirefragment (Fig. 5) spanning theputative initiation site oftranscription from the labeled left-hand end to the second BglII site but at the Alul site and used it for nuclease Si analysis. After

nottheright-hand BgIII-SalI fragmentwas protected. hybridization to late RNA, Si-resistant material was

ana-Sequence of the p37K gene. TheSailfragments of 840 and lyzed

alongside

a "sequence ladder"

prepared

from the

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VACCINIA VIRUS ENVELOPE ANTIGEN 763

same DNA fragment (Fig. 7). Five major Si-protected

fragments were observed. Since the entire region of the

DNA had already been sequenced, the positions of the protectedfragmentscould be located despitesome problems

in the sequencing reactions shown in Fig. 7. The results

indicated that the 5' ends ofthe mRNA map within about

two to six nucleotides upstream of the ATG translation

initiation codon.

Amino acid sequence of p37K. Following the first ATG

downstreamof the 5' end of theRNA, an openreading frame

which ended with a TAA termination codon at nucleotide

1208 wasobserved.The molecular weight of thepolypeptide

calculated fromtheamino acidcomposition was41,748 and

wasthussomewhat larger than thesizeof p37Kestimated by

polyacrylamide gel electrophoresis.

Hydrophilicity-hydrophobicity analysis of p37K. Since

p37K is tightly associated with Golgi membranes, we

searched for a putative signal peptide and possible

mem-brane anchors by determining the hydropathic index (10)

Sl G

6G+A

C+T C

40

> 20 1

10

0--20

.Z

1 51 101 151 201 251 301 351

FIG. 8. Average hydropathicity of amino acids of the p37K polypeptide.ThemethodofHopp andWoods(10) withawindowof seven amino acids was used to generate the graph. The x axis indicates the amino acid number, and the y axis indicates the hydropathic index.

alongthepolypeptide chain (Fig. 8).No stronghydrophobic

AR

region

characteristic ofa

signal peptide

wasfound atthe N A terminus of

p37K.

Two

hydrophobic regions (amino

acids C A T 130 to 157 and 175 to 192) close to the center of the A A T polypeptide could represent putative membrane anchors. -Rl

T G

qg.p

FIG. 7. Mappingof the 5' end of the mRNAencoi

Alul-Sallfragment(seealsoFig. 5)waslabeledatth,

hybridized tolate RNA from infected cells. Nuclea:

material(lane Si)and theproductsof the sequenceri

G,G+A,C+T,andC)of thesamefragmentwere res

sequencing gel. The map positions of the 5' endE

(indicated by the horizontal bars) are shown at t

sequenceof thenoncodingstrandin the 5' to3' dire

tobottom.

T A u DISCUSSION

R T Incontrast toother viruses whichacquire theirenvelope

R C A

by

a

budding

process

through

the cell

membrane,

vaccinia

cC virus

particles

are

enwrapped

in the interior ofthe cell

by

T

Golgi-derived

membranes.

T In this

study

we

mapped

and

sequenced

thegene

encoding

C A themajorviralantigenpresent on theenvelopeof

extracel-G lularvacciniavirusparticles. This antigenrepresents 5 to7% T of the totalproteinmassof thevirus(8).Hybrid selection of

c RNA,in vitrotranslation, andimmunoprecipitation allowed

C A us to mapthe gene close to the right-hand end ofthe HindlIl A T F fragment. This location corresponds to about 38kilobases A G G from the left-hand end of the 190-kilobase vaccinia virus

C genome. More precise map information was obtained by

C nuclease

S1

analysis,

which also showedthatthe mRNAis

C transcribed fromrighttoleftonthe vaccinia virusDNA.

c T The nucleotide sequence of the

p37K

genereveals several T G

interesting

features. First, the 5' ends of the mRNA as T definedby S1 analysismap withinno more than six nucleo-T tides from the translation initiation codon. Such extremely T short untranslated leaders have been observed in all three C ^ late genes thathave so farbeen sequenced (3, 20, 26) and thus appear to be a

general

feature of vaccinia virus late C genes.A further

highly

conserved feature of lategenesis the G C TAAsequence whichimmediatelypreceeds the ATG

trans-R lation initiation codon. The sequence TAAATG has been

C observedin mostlategenes sequencedelsewhere(3, 19, 26)

A and in several additional late genes sequenced in our

labo-c T ratory

(E.

Van

Meir,

unpublished

data).

So

far,

the

only

exception is alategene analyzed byRosel andMoss(20)in

ding p37K.An which thesequence preceding the ATG translation initiation eAluIsite and codon isTAAATA. Further conserved elements of putative

se Si-resistant late promoters alsoinclude stretches of fivetoeight

thymi-oeadcins

(lane8% dine (3; p37K gene, this paper) or adenine (20, 26) residues sof the RNA starting at 16 to 20 bp upstream of the ATG codon. he left on the The open reading frame reveals a polypeptide composed

ctionfromtop of 372 amino acids and having a total molecular weight of

41,748. The difference between the calculated molecular

VOL.58, 1986

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http://jvi.asm.org/

[image:7.576.303.539.63.228.2] [image:7.576.41.281.266.636.2]

(8)

764 HIRT ET AL.

weight and thatestimated fromtheelectrophoretic mobility (8)could beexplained bya signalpeptidewhich isremoved

posttranslationally to produce the mature form of p37K.

However, examination ofthe hydropathic index shown in

Fig. 7 does not supportthis idea. Furthermore,the in vitro

translation product migrates slightly ahead of the in vivo

product(Fig. 1),aresultwhich would alsoargueagainstthe

removal of a signal peptide in vivo. It should be borne in

mind, however, that p37K is modifiedin vivobytheaddition

ofpalmitic acid, which presumably also influences its

elec-trophoretic mobility. The fact thatp37Kacquiresitsaffinity

for membranesby thismodification,similartowhathasbeen

proposed, for instance, for the transforming proteins of

retroviruses(22), also has to be considered.

Examination of the hydropathicity graph reveals that

p37K is relatively hydrophobic, consistent with previous

phase partion experiments and the recognized membrane

association (9). Two hydrophobic regions, each comprising

27 amino acids, are located between amino acids 130to 157

and 175 to 192. These regions could possibly serve as

membrane anchors.

An interesting question is whether p37K is essential for

viral replication or not. The fact that nonenveloped virus

particles harvested from experimentally lysed cells are

in-fectioussuggeststhatitmightnotbe. Furthermore, left-hand

DNA sequences are frequently deleted in orthopoxvirus genomes (reviewed in reference 27), and in one rabbit

poxvirus mutant described by Moyer and Rothe (15) the

deletion extends into the HindlIl F fragment. Although

viable inmostcelllinestested,suchdeletionmutantsmay be

hostrangerestricted(6). Clearly, large portionsof left-hand

DNA sequences are not required for viralreplicationunder

mostexperimental conditionsbut mayencode functions that

modify the biological behavior of the virus. Interestingly,

although both intracellular and extracellular viruses are

infectious, they differmarkedlyin their mode ofadsorption

and penetration (18) in cell cultures. This observation sug-gests that at least the functions which influence certain

biological propertiesarecarried ontheenvelopeof the virus

particle.Nowthat thep37Kgene hasbeenmapped,itshould

be possible to genetically engineervaccinia virusby

insert-ing foreign DNA into thep37K locustoinactivate the gene.

Such experiments have become feasible through the

possi-bility of constructing vacciniavirusrecombinantsby

homol-ogous in vivo recombination (reviewed in reference 23).

Provided that suchrecombinantsareviable, theywould offer

anidealtool forstudyingvirusmultiplicationinthe absence

ofp37Kexpression.

ACKNOWLEDGMENTS

We thank Anne Seiler-Tuyns and Walter Wahli for critically

reading the manuscript, Beatrice ten Heggeler for artwork, and HannelorePagelfor editorialhelp.

This workwassupported bytheSwissNational Science Founda-tion.

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