In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein.

0022-538X/89/083345-08$02.00/0

Copyright X31989, AmericanSocietyforMicrobiology

In

Vitro Processing of

Dengue

Virus Structural Proteins:

Cleavage of

the Pre-Membrane

Protein

LEWISMARKOFF

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892

Received 15 February 1989/Accepted 19April 1989

Processing of dengue virusstructuralproteinswasassessed in vitro. RNAtranscripts forcell-freetranslation wereprepared fromclonedDNA (dengue virustype4, strain814669genome) encodingcapsid, pre-membrane

(prM), and the first 23 amino acids of envelope (E). Processing of a 33-kilodalton precursor polypeptide encodedbywild-typeRNAtranscriptsoccurredonlyin thepresenceofaddedmicrosomalmembranes. Under theseconditions, cleavageatthecapsid-prM and prM-E sitesandglycosylation ofprM occurredinassociation with translocation. Amino acid sequence analysis confirmed that translation initiated at the predicted N

terminus ofthecapsid and that capsid-prMcleavage occurred atthe predicted site forthe action of signal

peptidase following a candidate signal sequence (hydrophobic residues 100 to 113) in the dengue virus precursor. Mutations were introduced into the dengue virus DNA template by site-directed mutagenesis, altering nucleotide sequences encoding the capsid and the candidate signal for prM. The phenotypes ofthe mutantswerededuced by analysis oftheproducts of cell-free translationofthe respectiveRNA transcripts. The resultingobservationsconfirmedthatcleavageatthe capsid-prM and prM-Esites iseffected entirely by signal

peptidaseand that the candidate signalis requiredfor translocation.

The four serotypes of dengue virus are members of the

flavivirus family, which now includes more than 70 different viruses. Flavivirus particles are composed of three structural

proteins: agenome-associatedcapsid protein, a

membrane-associated (M) protein that is derived during virus

matura-tion by internal cleavage from a glycosylated precursor protein (prM), and a membrane-anchored hemagglutinating

envelope glycoprotein(E). E is themajor antigenic

determi-nantof freevirus responsible forserotype specificity(18-20,

31,32).

FlavivirusgenomicRNAs are10,500 to 11,000nucleotides

long and include a single open reading frame encoding a

precursorpolyproteinapproximately 3,400aminoacidslong (5-7, 10, 14-16, 18, 22, 26, 33). Structural proteins are

derivedbycleavage from the N terminus of the precursor in thefollowingorder: capsid,prM, and E (2-4, 18,22, 24, 28).

Since some viral proteins are glycosylated and virus

parti-cles are assembled in the endoplasmic reticulum (11, 19),

processing of the flavivirus precursor polyprotein must

in-clude not only cleavage but also translocation ofcleavage

products. An association betweencleavage ofcapsidand E

proteins and cotranslational translocation has been

sug-gested bythe resultsofanin vitrostudy(25). Inaddition,the

dengue virus capsid, prM, E, and NS1 were apparently normally cleaved and glycosylated when expressed in a

vaccinia virus recombinant containing a 4-kilobase (kb) fragmentof cloned dengue virus type 4 DNA encoding the

capsid, prM, E, NS1, and NS2a (34). Therefore,

nonstruc-tural proteins (NS2b through NS5 [14]) not encoded in the vaccinia virus recombinant are not required to process the

structuralproteins and NS1.

As expected, the locations ofpotential signal and

trans-membrane segments in the flavivirus precursor are

con-served. Within N-terminal sequences encoding structural

proteins,the precursor containsan uninterrupted 14-amino-acid hydrophobic segment(residues 100 to 113 in the

pre-cursorsequence).This sequenceprecedestheNterminus of prM determined by amino acid sequencing of labeled pro-teins isolated from virus-infected cells (2, 4) and is the

candidate signal sequence fortranslocation ofprM(18, 19, 33). Thecandidatesignal is preceded by a conserved pair or triplet ofbasic amino acids (Arg-Lys-Arg; residues 97 to 99 in the dengue virus type 4 sequence [33]). It has been

suggested that these conserved residues might constitute a proteolyticsite(18, 33). Inaddition, thepredicted C termini

of both prMand E arehydrophobic segments(residues246

to 279 and 735 to 773, respectively) asymmetrically inter-rupted in each case by a single Arg. These are candidate transmembrane segments for prM and E and signals for translocation of therespectivedownstreamglycoproteins, E and NS1 (14, 18, 22, 33).

To establish the association between cleavage of struc-turalproteinsfrom the precursor andtranslocation,

process-ing of a truncated dengue virus precursor protein was

studied in vitro. RNA transcripts were prepared from a 911-base-pair fragment of denguevirus type4DNA, includ-ingthecomplete capsid and prM nucleotide sequences and sequencesencoding thefirst23 amino acids of E. Cleavage ofthe encoded precursoratthecapsid-prMand prM-E sites

was dependent upon the presence of microsomes during cell-free translation and appeared to mimic the in vivo

events.Todeterminethemechanism of cleavage, mutations

of nucleotide sequencesencodingthe capsidand the candi-dateprMsignalwere introducedintodenguevirus DNAby site-directedmutagenesis.The resultsof cell-free translation of mutant RNA transcripts are also described. An intact

signalat residues 100to 113 wasrequiredfor translocation and cleavage ofboth prM and E. Mutations of the

capsid

sequence had no effect on processing, and cleavage ap-pearedto beentirely mediatedby the signal peptidase.

MATERIALSANDMETHODS

Preparation of recombinant pTZ/Sal. A 4,040-base-pair

(4-kb)fragment of cloned dengue virus type4 strain 814669 DNA bounded by BglII restriction endonuclease

cleavage

sitesatpositions88and 4128wassubcloned into theplasmid vector pTZ18U (Pharmacia, Inc.,

Piscataway,

N.J.) at the

BamHI site within the pTZ18U polylinker in plus-sense

3345

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Bglllfragment, dengueDNA

r1'

NS1

Sal I

IN

SalI .

Salfragment, dengue DNA

"I.

pTZ18U NI- -C jprM \ E

f Hind III linearpTZ/SalDNA

polylinker

FIG. 1. Derivation of Sal DNA in the pTZ/Salconstruct. A4-kbfragmentof cloneddenguevirus(type 4,strain814669)DNApreviously expressed in a vaccinia virus recombinant (34) was cloned into the plasmid vector PTZ18U at the BamHI site. pTZ18U-dengue virus

recombinant DNAwasdigested withSall, excising 3'-terminalsequencesfrom thedenguevirus DNA. The resultantpTZ/Salrecombinant

DNAlinearized by HindIII digestion, usedasthetemplate for synthesis of RNA transcripts,isdepicted. X, Noncodingsequencesindengue virus DNA; T7 RNA polymerase promoterinpTZ18U.

relationshiptothe T7RNApolymerase promoter sequence

contained in thevector.3'-Terminal nucleotide sequencesin

the dengue virus DNA were excised by digestion of the

recombinant vectorDNA with the restrictionendonuclease

Sall,whichcleaves thedenguevirus DNAatnucleotide 999 andpTZ18UDNAatauniqueSallsite in thepolylinker3'to dengue virus DNAsequences. The Sall-digestedDNA was religated to construct pTZ18U-dengue virus recombinant DNAwhichincluded only the5' 911nucleotides (88to999)

of the4-kbdenguevirus DNAfragment. These 911

nucleo-tidesdefine the Sal fragment of dengue virusDNA.pTZ18U recombinant DNA containing the Sal fragment of dengue virus DNA isdesignated pTZ/Sal (Fig. 1).

Preparation of pTZ/SalssDNA and site-directed mutagene-sis of dengue virus DNA sequences. The vector pTZ18U

contains the fl origin of replication in opposite

transcrip-tional orientationto the T7 RNApolymerase promoterand totheampicillinresistancegene.Toproduce pTZ/Sal single-stranded DNA (ssDNA), we followed a standard protocol. Toproducemutations in dengue virusssDNA,weemployed the site-directed method of Zollerand Smith (35). Klenow

fragment was obtained from Promega Biotec, Madison, Wis., andT4 DNAligasewas obtained from New England BioLabs, Inc., Beverly,Mass.Oligonucleotide primerswere preparedon anApplied Biosystems380A DNASynthesizer.

The presence of a desired mutation was confirmed by primer-directed DNA sequencingofthepertinent region in

dengue virus DNA (21).

Synthesis of RNA transcripts from recombinant vector DNA. pTZ/Sal DNA and clonedmutantrecombinant DNAs were linearizedby digestion with the restriction endonucle-ase HindlIl (Fig. 1). Synthesis of RNA on 2 to 5 ,ug of linearized DNA was carried out by a standard procedure

using20to 40U of T7RNApolymerase (Promega Biotec).

Cell-freetranslation of RNA transcripts and polyacrylamide

gel analysis ofproducts. Cell-free translationwasconducted in a 25- or 50-,ul final volume using a supplemented rabbit

reticulocyte lysate pretreated with micrococcal nuclease

(Promega Biotec). A typical 25-pAl reaction mixture

con-tained17.5,ul of rabbit reticulocyte lysate, 0.5 ,ul ofa1 mM amino acid mixture minus methionine, 0.5 p.l of RNase

inhibitor(RNasin; Promega Biotec), 2.5 ,ul of

[35S]methio-nine(15 mCi/ml, >1,000Ci/mmol;Amersham Corp.,

Arling-tonHeights, Ill.), and 0.2to0.5 ,ug of RNA inwater.Protein synthesiswasconducted in thepresence orabsence of added caninepancreaticmicrosomes (1.0to1.5

RI

replacingwater

in a 25-pu reaction mixture; Promega Biotec). To prepare

proteins for sequencing, 1 mM amino acids minus

methio-nine and leucinewas addedtothe reactionmixture inplace of amino acids minus methionine, and the reaction was

carried out in the presence of 25 ,uCi of [3H]leucine (1

mCi/ml, 150 Ci/mmol; Amersham) and [35S]methionine. Syn-thesized proteinswere analyzed bysodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels

con-tained 0.2% SDS and 15 to 17% acrylamide. The 17% gels wereelectrophoresed at140constantvoltsfor 16 h atroom

temperature, dried, and exposed to XAR-2 film (Eastman Kodak Co., Rochester, N.Y.).

Protease and endo F digestion of proteins. To digest

pro-teins with thermolysin (Boehringer Mannheim Biochemi-cals, Indianapolis,Ind.),5[lofacell-free synthetic reaction

mixture was added to 5 1ld of TC buffer (10 mm Tris hydrochloride [pH 7.5], 10 mMCaCl2), 1 ,ulofthermolysin (1-mglml solution in TC buffer) wasadded, and the reaction mixture was incubated for 60 min at 4°C. Thermolysin activitywasterminatedby the additionofa10-foldexcessof EDTA.

Endoglycosidase F (endo-,B-N-acetylglucosaminidase F [endo F])wasobtainedfromBoehringerMannheim. Twoto five microliters ofa cell-free synthetic reaction mixture or >5,000cpmofaneluted[35S]methionine-labeled proteinwas

incubatedin 25 ofendoFbuffer(Boehringer Mannheim) for 1to3 hat37°C in thepresenceof0.1to0.2 U ofenzyme.

Immuneprecipitation ofproteins. Antibody to peptide 30 (a gift of R.Houghten),a15-amino-acidpeptiderepresenting thepredicted N terminus of the dengue virus type 4, strain

814669 Eprotein(MRCVGVGNRDFVEGV; residues 280to 294 [33]), was prepared in rabbits after conjugation to

keyholelimpet hemocyanin. This antiserum hadan antipep-tide titerof1:1,280 by enzyme-linkedimmunosorbentassay. Proteinssynthesizedinvitrowere incubated for 16 hat 4°C

in thepresenceofantipeptide antibodyatdilutions of 1:10to BglII

Ix

BglII N N1

r.

NS2a

Bgl

II

Hind III'-L-igI

11111 polylinker

---Tlll.

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1:50. The antigen-antibody complexes were then collected on staphylococcal protein A-Sepharose CL4B beads

(Phar-macia). Thebeads were washedin RIPA buffer (0.1 M Tris [pH 7.5], 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100,0.1% SDS) and boiled in Laemmli sample buffer (0.1

MTris [pH 6.8], 10%glycerol, 1% SDS, 0.2 M 2-mercapto-ethanol,0.001% bromphenol blue) prior to SDS-PAGE.

Amino-terminal amino acid sequencing of proteins. Pro-teins doubly labeled ([3H]leucine and [35S]methionine) as

describedabove were separated by SDS-PAGE on a gel that had been preelectrophoresed in the presence of running bufferplus 1 mMmercaptoacetic acid. A2%agarose

stack-ing gel was used instead of acrylamide, and 0.1 mM mercap-toacetic acid was added to the running buffer. Proteinswere eluted from polyacrylamide and sequenced in a Beckman

Model 890MProtein Sequencer.

RESULTS

Cell-free translation of wild-type RNA transcripts. Sal DNA in the pTZ/Sal construct includes the 13 3'-terminal nucleotides of the dengue virus 5' noncoding region and sequences encoding the capsid (amino acids 1 to 113), prM (amino acids 114 to 279), and the first 23 amino acids of E

(amino acids 280 to 302) in the dengue virus serotype 4 precursorpolyprotein (33). RNA transcripts prepared from

HindlIl-linearized pTZ/Sal DNA were expected to include 21 nucleotides of the pTZ polylinker 5' to dengue

virus-specificsequences as wellas 5'-terminal GGG appended to

all transcripts by T7 RNA polymerase (8). In addition, transcripts included polylinker sequences 3' to the dengue

virus insert which encode a common C terminus

(Gln-Ala-Cys-Lys) in cell-free translation products (Fig. 1). The products of cell-free translation of wild-type (wt) RNA transcripts are shown in Fig. 2A.

[35S]methionine-labeled proteins were directly applied to the gel for SDS-PAGE under reducing conditions. In the absence of added microsomal membranes (Fig. 2A, lane 0), a single major

productwasevident, with a molecular size of approximately 33kilodaltons(kDa). This is in agreement with the predicted

size ofthe uncleavedtranslationproductof full-lengthpTZ/ Sal RNA (306 amino acids; estimated molecular mass,

33,660 daltons). Minor lower- and higher-molecular-mass species were also detected. Higher-molecular-mass species are likely to be the product of RNA transcribed from

contaminating uncleaved circular pTZ/Sal DNA.

Lower-molecular-mass proteins could have resulted from internal initiation of translation or from internal cleavage of the

33-kDa polypeptide. Evidence favored internal initiation;

separate experiments failed to demonstrate a

precursor-product relationship between the 33-kDa and lower-molec-ular-mass proteins, suggesting that cleavage of the

full-length polypeptide did not occur in the absence of

microsomes (datanot shown).

When RNAtranscriptswere translatedin the presenceof canine pancreatic microsomal membranes, seven proteins were detected. Four high-molecular-mass products (A, B, and CinFig.2A, lanes 0.5to2., and C' inFig. 3A, left lane) ranged insize from 32 to 38 kDa. Protein C appeared to be

identical in size to the unglycosylated 33-kDa full-length product. Three low-molecular-mass proteins varied in size

fromabout 24to28kDa (a,b,andcinFig. 2A, lanes 0.5, 1., and 2.). Increasing portions of membranes added to the

reaction mixture were associated with both enhanced pro-duction ofproteinsa, b,andc relativetothe precursor and

diminished total synthesis.

A. B.

piMembranes

M

o75i1.

2

30

-30

Kd-+ :Mm M - + - +:Pr

A B

i...'h-.C

6

1.s" NW a

c c

21Kd-12

Kd-.-FIG. 2. (A) Cell-free translation ofSalRNA inthe absence(lane 0)orpresenceofincreasing portions (in microliters) of microsomal membranes (lanes 0.5 to 2.). One-half microgram of RNA tran-scribed in vitrowasaddedto17.5 ,ulof micrococcal nuclease-treated rabbit reticulocyte lysate plus 0.5 p.l ofRNasin, 0.5 p.l of 1 mM amino acids minus methionine, 2.5 ,u1 of [35S]methionine, and microsomes asindicated, to afinal volumeof25 ,ul. The cell-free translation reaction mixture was incubated for 60 min at 30°C. Three-microliter samples of each reaction mixture were addedto Laemmli sample buffer, heated to 90°C for 3 min, and directly applied to a 17% gel for SDS-PAGE. 14C-labeled low-molecular-mass marker proteins were co-electrophoresed (lane M). The gel wasdriedandexposedtoKodakXAR-2film.Kd, Kilodaltons. (B) Proteasesensitivity ofinvitro translationproducts.Cell-free trans-lation was carried out as described above in the absence (-) or presence (+) of microsomes (Mm). After incubation, aliquots of each reactionmixture weresubjectedtodigestionwiththermolysin for60min at4°CinTCbuffer(Pr+).Protease-digested aliquotswere coelectrophoresed withaliquots of undigested product (Pr-). Elec-trophoresis was done as described in the legend to panel A. M, 14C-labeledmarkerproteins.

Translocation of cell-free translation products. Resistance

toprotease digestion ofcell-freetranslation productsunder

conditions that maintained the structural integrity of the

endoplasmic reticulum demonstrated that they had been translocated (Fig. 2B). As acontrol, the 33-kDafull-length polypeptide synthesized in the absence of membranes was completely protease sensitive. Under the same conditions, membrane-dependent products B, C, a, b, and c were protected from protease digestion. Protein C' was not

de-tected in this experiment. Protein A appeared partially

protease sensitive. Protease digestion removed protein A

and introduced a new high-molecular-mass product that migrated slightlyslower thanproteinC. This resultcould be

explained by a transmembrane orientation of protein A which rendersanextralumenalpiecesusceptibletoprotease. Digestion of glycoproteinswith endo F. Todetect N-linked

glycosylation of translocated proteins, the mixtureofA,B, C, and C' with a, b, and c was digested with the enzyme endo F (Fig. 3A). The products of

digestion

were four

polypeptides designated 1, 2, 3,and4indescendingorder of

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B.

wt

eF:- +

A

b

c 9443

wt a b wt

- -+ - + +:eF

_

C' 4w1

a

.-

50Kd

a

f

b

C.

4 403

-21.5KtY _ 44

4

3OKd

14Kd

1 2 3 4 5 6

FIG. 3. (A)Digestion ofwtcell-freetranslati C, and C' plus a, b, and c) with endo F tc

carbohydrate.Invitro translation ofwtRNAtrar outin thepresenceof1.5,ulof added microsome,

legendtoFig. 2A.Total productswere subjecte 0.2 U of endo F for 2 hat37°C. Aliquotsofun

endo F-digested (eF+) proteins were coelectro labeled marker proteins as in Fig. 2. (B)Identi translation products a and b in relation to en(

cell-free translationproducts preparedasdescril Fig. 2Awere separated on a preparative gel. I productsaandbwerelocatedbydirectexposur film, excised, and electroeluted. Products a

separately digested by endo F. The electroph proteinsaand b(lanes2and4)and their endo and 5) were compared with total proteins (lai digestedtotalproteins (lane6)duringSDS-PAG] ofcell-free translation product c in relation tc

[35S]methionine-labeled protein c was prepare

other cell-free translation products, digested by paredwith total wt and endoF-digested wt pr above. Lane 1,wtproducts (cisindicated);lan

3,endoF-digestedc;lane4,totalwtproteins di~ Theidentityof endo Fpolypeptide 4 with endo

c(c eF)is indicated. 14C-labeled markerprote phoresed (left lane). kd, Kilodaltons.

molecular size(33, 31, 22, and19kDa,res were consistent with the expected sizes c

polypeptides representingthefull-lengthpr

prM, prM + E, and prM. A polypeptide ( with that of the cleaved capsid (12 to ]

detected. Translation products were next fromapreparative gelanddigestedwithen B, C, and C' collectively yielded polypo

Proteins AandBwerereduced insizebyi

showing that they were glycosylated. Pr appearedidenticaltopolypeptides 1 and 2

Glycosylation of proteinsa,b, and c was a (Fig. 3B and C). Glycoprotein a yielde Glycoproteinsb andcbothyielded

polypep

teins a and b were each approximately 6 polypeptides 3 and 4, respectively. Glyc4

kDalargerthan its endoFdigestion produ(

ItappearedthatendoFremovedtwocarb( (GlcNAc2-Man9-Glc3, where GlcNAc

cosamine, Manis mannose, andGlc isglu

eachfromproteinsaand b andonefromc

precursor,potential glycosylation sites liee

prMsequence (discussed elsewhere).

c. Sequencing and immune precipitationof endo F digestion

wt _ wt products. The N and C terminiof endo F digestion products

+ +:eF 1 through 4 were subsequently identified. Initially, the N-terminal 30 residues ofpolypeptides that had been labeled with both [35S]methionineand [3H]leucinewere sequenced. By alignment of the labeled residues, the N-terminal se-quences ofpolypeptides 1 and 2 were demonstrated to be

identical to thatpredictedfor thedengueviruscapsid protein

*

(data

notshown).

Sequence

data for

polypeptide

3 is shown

in Fig. 4A. Within the first 30 residues, methionine was

detected inposition12 and leucinewasdetected inpositions 3, 11, 23, and 24. The results of sequencing the first 30

cb~ residues ofpolypeptide 4 were identical. The locations of ceF- * 54 methionine and leucine residues within the N termini of

polypeptides3 and 4wereinagreementwith thosepredicted for the N-terminalsequenceofthedenguevirusprM (2, 4, 5, 18, 33), showingthatspecific cleavageat thecapsid-prMsite 1 2 3 4 occurred when microsomes were present during cell-free ionproducts(A, B, translation.

remove N-linked To detect

cleavage

ofthe first23amino acidsof Efromthe

nscriptswas carried C terminus of the truncated precursor, we prepared rabbit

nas

describedin the antibody to a peptide representing the 15 N-terminal

resi-dtodigestionwith dues ofEand demonstrated thatantipeptide antibody

pre-digested (eF-)and cipitatedE fromalysateofdenguevirus-infectedcells. The

phoresed with 14C- same antibody preparation specifically immune precipitated

ification of cell-free polypeptides 1 and 3 (Fig. 4B, lane3), indicating thateach

do F digestion. wt retained the C-terminal E sequences. Precipitation by

pre-bedinthelegendto immune serum is shown as a negative control (lane 4). In

retoKodakXAR-2 contrast, polypeptides 2 and 4 were not immuneprecipitated

and

b were then

by

the

antipeptide

antibody andappeartorepresent species

aretic mobilities of cleaved at the prM-E site. The endo F polypeptides were

Fproducts (lanes 3 therefore identifiableby apparentmolecularsize, N-terminal

ne 1) and endo F- sequence, and C-terminus-specific immune precipitation

E.(C)Identification (Table 1):polypeptide 1= capsid+ prM+ E(the full-length

endo F products. precursor);polypeptide 2 = capsid+ prM;polypeptide 3 =

-d, separated from prM + E; andpolypeptide4 =prM. Consequently,proteins

tendo Fs

and corn- a, b, and c were verified as specific glycosylated cleavage

e2,isolatedc; lane products of the 33-kDa precursor. Protein a was identified as gestedwith endo F. prM + E bearing two carbohydrate residues. Proteins b and F-digested product c were identified as alternate glycosylated forms of prM. inswerecoelectro- Cleavagewas membranedependent at both thecapsid-prM andprM-E sites, and cleavage products weretranslocated.

These results confirmand extend data whichsuggestedthat

pectively). These thesignal peptidaseisresponsiblefor cotranslational

cleav-)f unglycosylated ageof the flavivirusstructuralproteins (25); however, they

ecursor,capsid+ do not clarify the significance of the conserved potential consistent in size proteolytic site in the capsid sequence or localize signal 14 kDa) was not function in the truncatedprecursor. Experimentsthat follow

separately eluted address thesequestions.

do F. ProteinsA, Cell-freetranslation oftranscripts bearingmutations in the

eptides 1 and 2. capsid sequence. Two in-frame deletion mutations were

endoFdigestion, introduced into the capsid nucleotide sequence in pTZ/Sal -oteins C and C' DNAbysite-directedmutagenesistotest thepossibilitythat

(datanotshown). cleavageofthecapsidatthe conservedpotential proteolytic lsodemonstrated site(Arg-Lys-Arg; residues 97 to99) isrequiredin associa-d polypeptide 3. tion with signal recognition. The mutant dIC polypeptide )tide 4.Glycopro- lacked an internal 74 amino acids ofthe capsid sequence kDa larger than (residues 13 to86), which should haveabrogated any

auto-oprotein c was 3 proteolytic activity of the capsid or any conformational ct,polypeptide4. requirement for normalprocessing. Mutant dlC-pM lacked ohydrateresidues thetrypsinlike site itself(residues 97 to 99; Fig. 5). In the

is N-acetylglu- absence of microsomes, the predominant product of dlC

icose; Mr- 3 kDa) transcripts was a protease-sensitive 25-kDa polypeptide, :. Inthe truncated consistent in size with that expected for the full-length -ntirelywithin the unglycosylated product of the nucleotide sequence (Fig. 6A).Also notedasaminorspecieswasa22-kDaprotein,the A.

MYt

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B.

anti-ERIP./

+4

-Ii

eF: - + a 4

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1.2 11 1 i i I II I7

1.1

II

O3H-DPM

0 35S-DPM

A-

B- C-

C,-

a-

b-

c-a.

I.

-30kd

-21.5kd

-12kd 0.1 SIL S T1 I I DG111lII I Ii,,I RGI II I I Ii TI G

F SLST RDG E PLM VA KHE RG R PL L F KTTEG*

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*Predlc*ted N-terminal secuence of orM _{1 2 3 4}

FIG. 4. (A) Sequencing of polypeptide 3. Cell-free translation was carried out in the presence of membranes and [3H]leucine and [35S]methionine labels. The reaction products were digested with endo F and separated on a modified 17% SDS-polyacrylamide gel.

Polypeptide 3waslocated byexposureof the geltoKodak XAR-2 film andexcised. The proteinwaselectroeluted, andasample containing 2.7x 104dpm of 3H and 4.9x 104dpm of 35Swasfractionatedon aBeckmanModel 890M Protein Sequencer. Thirty residuesweresequenced

from the Nterminus(radiolabel in fractions 1to30is shown, lefttoright). The resultsarecompared with the predictedsequenceof the N terminus ofthe dengue virustype4strain 814669 prM (33). The first fraction is aligned with the N-terminal F(single-letter code), and labeled Mand Lresiduesareindicated in boldface. (B)Immuneprecipitation of endo F-digested cell-free translation products by antibody specific

forC-terminal Esequences. [35S]methionine-labeled cell-free products (lane 1)weredigested with endo F (lane 2). A samplewasincubated

for 16hat4°C with rabbit antibodytoa15-amino-acid peptide representing the predicted N terminus of E (lane 3)orwith preimmuneserum

(lane 4). The immune precipitates werecollected by using staphylococcal protein A-Sepharose CL4B beads, eluted from the beads, and coelectrophoresed with undigested and digested total proteins. eF-, Not digested with endo F; eF+, digested with endo F; anti-E+, radioimmunoprecipitation (RIP) with rabbitantipeptide antibody; anti-E-,RIPwith preimmunerabbitserum.kd, Kilodaltons.

expected sizeof a productinitiating internally at Met-112 of

the wt capsid sequence. The Met-112 codon lies within a

consensus nucleotide sequence favoring initiation (12). In the presence of microsomes, translation of mutant diC transcripts produced three translocated proteins which

[image:5.612.54.298.633.714.2]

comigratedwith theproducts ofwttranscripts (proteinsa,b, and c)already identified as glycosylated forms ofprM + E and prM. Similarly, the three low-molecular-mass cell-free translation products of mutant dIC-pM transcripts in the presence orabsenceof microsomes were not distinguishable from those of the wt by size or proteasesensitivity (Fig. 6B;

TABLE 1. endoF-digested productsof cell-free translation ofwtRNAtranscripts

endo F Molecular RP Translation eo size(kDa) N-terminal RIP"

product(s)

peptYde

bySDS- sequence antE Identity

peptidePAGE anti-B

A, B,C,C' 1 33-34 Capsid + Cb+ prM + E

2 31-32 Capsid - C + prM

a 3 22 prM + prM+ E

b,c 4 19 prM - prM

aRIP,Radioimmunoprecipitation. bC,Capsid.

compare to Fig. 1B). To confirm theiridentity, processed mutant dlCand mutantdIC-pM products were subjected to endo F digestion and subsequent immune precipitation by

rabbit antibody to E sequences at the C terminus of the

precursor(datanotshown).Thisanalysisdemonstrated that dlC anddlC-pM products comigrating withwtglycoproteins

a,b,andc wereidenticaltothem. Inconclusion, cleavageof thecapsid andglycosylation andtranslocation ofcapsidless proteins were not detectably altered by the capsiddeletion mutations.

Cell-free translation oftranscripts bearingmutationsinthe

prM signal. Toidentifythe signalsequencein the truncated

precursor,two mutantDNAswerepreparedwithalterations inthecandidatesignal (residues 100to 113) (Fig. 5). Mutant dlss DNA had an in-frame internal deletion of sequences

encoding 10 amino acids of the hydrophobic segment

(resi-dues 102to 111). Mutantsubss bears a substitution ofthe codon for Ile at position 109 by a codon for Lys, which interruptsthehydrophobicsequence.The results of cell-free translation of dlsstranscriptscomparedwithwtareshownin Fig. 7A, and the results of protease digestion of subss products compared with wt are shown in Fig. 7B. The phenotypes of these two mutants were indistinguishable. The signal-minus precursor synthesized in the presence of microsomeswasnotprotectedfrom proteasedegradation,as

A.

-I

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DNA

wt X

Map

r-Capsid

Bgl II

Modification

. Env

r-' preM

Sal I

Deletecapsidsequences fromaa13throughaa86 Aaa1a-"-a86

RKRK

R _

AaalO2-8a111

K

Delete -Arg-Lys-Arg preceding preM signal

sequence

Delete intemal 10aas(102-p 111) from preM signal

sequence

Substitutelle1i8within preM signal sequence

by Lys

FIG. 5. Diagrams of Sal DNA andmutationsproducedinSalDNAbysite-directedmutagenesisofsingle-strandedpTZ/SalvectorDNA.

aa,Amino acid. _, Consecutive hydrophobic residues; 3, noncoding region.

shown for subss. Failure ofglycosylation and cleavage is

shown for both dlss and subss. The 33-kDa truncated pre-cursor wasunaltered, despitethepresenceof microsomes in thetranslation reaction mixture. Interruptionordeletion of

A.

dIC

:Mm

+ -+: Pr

w s1p-iw,

+ ;

MP,

the hydrophobic segment at once prevented translocation, glycosylation,andcleavage of theprecursorateither poten-tial site. These results confirmed a signal function for the hydrophobic segment atthe capsid-prM juncture.

DISCUSSION

In vitro translation of RNA transcripts prepared from cloneddenguevirus DNAencodingthecapsid, prM,and the

A.

wt Mm: - +

qW...-'

diss

l

Bwt subss

Mm: - + - +

Pr:- + - + + +

a,

.. _.

.,. it

2 1Ka ..:.. .:::.:::..

*.,... 30

Kd-21Kd

[image:6.612.123.499.72.315.2]

21 Kd

FIG. 6. Cell-free translation of dlC (A)and dlC-pM (B) mutant RNAtranscripts and proteasesensitivity of the products.

Transla-tion wascarried out as described in the legend to Fig. 2A in the

presence (+) or absence (-) of 1.5 1.l of microsomes (Mm).

Thermolysin digestionwascarriedoutasdescribed in the legendto

[image:6.612.91.286.419.655.2]

Fig. 2B. Pr-, Undigested products; Pr+, thermolysin-digested products. Productswere coelectrophoresed with "C-labeled stan-dards(lane M), showninpanelB. Kd, Kilodaltons.

FIG. 7. Cell-free translation of dlss (A) and isubss (B) mutant RNA transcripts and protease sensitivity of the subss product.

Conditions were as described in the legend to Fig. 6. Mm-,

Microsomes absent; Mm+, 1.5 of microsomes present; Pr-, productsnotdigestedwiththermolysin; Pr+, products digestedwith

thermolysin. Kd,Kilodaltons.

dlC

dl C pM

diss

subss

r

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[image:6.612.326.564.454.675.2]

N-terminal23amino acids of E resulted in the

synthesis

ofa

precursor polypeptide which was cleaved. Cleavage

prod-ucts

representing

the

capsid

+prM, prM+ E,andprMwere

identified,

and glycosylated forms of prM + E and prM

bearing

two

carbohydrate

residues weredetected(proteinsa

and

b).

The33-kDain vitroprecursorcontainedone

consen-sus site

(Asn-X-Ser/Thr)

fortheaddition ofN-linked

carbo-hydrate,

at Asn-182 in prM. However, prM contains an

additional

site, Asn-X-Cys (in

the conserved sequence

Asn-Lys-Cys-Thr;

residues145to

148),

whichhasbeen shownto

have

carbohydrate

acceptorfunctioninvitro (1)and in vivo

(23).

We presume that both Asn residues 145 and 182 are

glycosylated

in

proteins

a and b. A third glycosylated

cleavage product,

prM

bearing

a

single carbohydrate

resi-due,

wasalso detected

(protein

c).

Cleavage

ofthe precursor

waswholly dependentonthe presenceofmicrosomesduring

translation. Evidence that in vitro cleavage events are a

model of in vivo processing ofthe dengue virus precursor

polyprotein

was

provided

by sequence analysis of the N

terminusofprMproduced invitro; its sequencewas

identi-caltothe

predicted

Nterminus ofprM deducedby

compar-ing

thedengue virus sequencetothose ofotherflaviviruses for which the N-terminal sequenceofprMhas been

directly

determined (2, 4, 33). The

approximate

accuracy of the

cleavage

event atthe prM-Esitewasestablishedbyimmune

precipitation

ofendo

F-digested

products with an antibody

directed

against

the

predicted

first 15 amino acids ofE(33).

The

capsid-prM

andprM-E cleavage sites inthe contextof

hydrophobic

sequences thatprecedeeach (5, 18, 19, 33)are

highly

favored sites for the action of signal peptidase (27),

the

presumed cleavage

enzyme.

To test the

hypothesis

that

signal peptidase

is

uniquely

responsible

for

cleavage

of the

capsid

and prM and to

identify

the

signal,

the phenotype ofmutant dengue virus

DNA

containing

eitheran altered capsidsequence orsignal

sequence

(residues

100 to 113) was determined in cell-free

translation

experiments.

The

capsid

mutationswere

gener-ated to rule out the

possibility

that the capsid is initially

cleaved at or upstream from a

pair

of basic amino acids

(residues

98 and 99) which precede the signal for prM, in

associationwith

recognition

ofthat

signal.

TheSindbisvirus

core

protein,

which lies N terminal to

pE2

and El in the

Sindbis virusstructural

polyprotein,

initiates

processing by

a similar

cleavage

event which is

autocatalyzed

(9). Mutants

with a

large

internal deletion of capsid sequences (mutant

dIC)

or a deletion of the

putative cleavage

site (mutant

dlC-pM)

were

analyzed.

Themutationin the dlC

polypeptide

shouldhave abolished any

enzymatic activity

ofthe

capsid

and removed alternate

trypsinlike cleavage

sites upstream

from residues 98 and 99. However, neithermutation of the

dengue

virus

capsid

hadadetectableeffecton

processing

of

the precursor

polypeptide, suggesting

that

cleavage

of the

capsid by

an

activity

other than thatof

signal peptidase

isnot

a

prerequisite

for

processing.

Asaconsequence,it wouldbe

predicted

that the

signal

segment is the C terminus of the

mature

capsid protein. Signallike

function ofthe candidate

segment

(residues

100to

113)

wasestablished

by analysis

of mutant

polypeptides

witha

large

internal deletion of

hydro-phobic

residues(mutant

dlss)

orwith an

interruption

in the

hydrophobic

sequence

by

aninternalsubstitutionofa

single

hydrophilic

amino acid fora

hydrophobic

amino acid

(Ile

-*

Lys;

mutant

subss).

Both mutations of the

putative signal

prevented

translocationofthe

polypeptide.

Failureof

trans-locationwas associated with failure of

processing,

suggest-ing

that access of intralumenal

signal peptidase

to the precursorwas

required

for

cleavage.

Both mutant

polypep-tides contained the hydrophobic segmentatthe C terminus ofprM (residues 246 to 279) thought to be the signal for E (14, 18, 22, 33). This segment did notmediate translocation of the truncated precursorpolypeptide encoded bypTZ/Sal transcripts, perhaps because the sequences downstream (residues 279 to 302) are insufficient in length to drive translocation (17) or because, in fact, translocation of E is dependent on the signal for prM. These findings together

suggestthatprocessing of theflavivirus precursor is initiated by cotranslational recognition of the prM signal segment. Translocation affords access of intralumenal signal peptidase to hydrophobic segments preceding the N termini of both prMand E. Signal peptidase effects cleavage at thepredicted sites.

The cleaved capsid protein was not detected in these experiments. The capsid should have been labeled at a reduced specific activity with respect to prM, since sequence datapredict that it contains only 5 methionines among 113 residues, whereas prM contains 10 methionines among 166 residues. This disparity alone would probably not account for our failure to locate the capsid among labeled translation

products. Once cleaved, the capsid may be unstable due to

proteolysisor maybe poorly resolved under the electropho-retic conditions employed. Chimeric glycoproteins contain-ing the capsid were identified, and these appeared to be translocated (proteins A and B). The finding of uncleaved productscontainingthe capsid and prM may be an artifact of

inefficiency ofcleavage duringtranslocation in vitro. How-ever,achimera of the capsid and prM, the 30-kDa glycopro-tein NVX, has been detected among flavivirus proglycopro-teins

synthesized during infection (13, 29, 30), suggesting that

cleavage ofthecapsid may alsobe inefficient in vivo.

ACKNOWLEDGMENTS

I thank Bangti Zhao and Ching-Juh Lai forthe generousgift of cloneddengue virusDNA,Suzanne Emerson andPeter L. Collins for manyhelpful discussions, John Coligan and MichaelRaumfor protein sequencing,MyronHill for preparation ofoligonucleotides, and ChristinaFonseca andLindaJordan for editorial assistance.

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