Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

JOURNALOFVIROLOGY,Mar. 1992, p. 1535-1542 0022-538X/92/031535-08$02.00/0

CopyrightC1992, American Society for Microbiology

Cleavage of the

Dengue

Virus Polyprotein

at

the

NS3/NS4A

and

NS4B/NS5

Junctions

Is

Mediated

by Viral

Protease

NS2B-NS3,

Whereas

NS4A/NS4B

May Be Processed by

a

Cellular

Protease

ANNIECAHOUR, BARRY FALGOUT, AND CHING-JUH LAI*

Molecular ViralBiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and

Infectious

Diseases,

Bethesda,

Maryland

20892

Received13 September 1991/Accepted3December 1991

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus

polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5,vNS4A-NS4B-NS5, and vNS4B-NS5wereconstructed.

These recombinantswereusedtoinfectcells,and the labeledlysateswereanalyzed by immunoprecipitation.

Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produceduncleaved polyproteins. These findings indicate that NS2B is required for processing ofthe downstream nonstructural proteins, includingtheNS3/NS4Aand NS4B/NS5junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavagesite follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junctionintheabsence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5

expressed onlythepolyprotein, earlier results demonstrated that cleavageatthe NS4A/NS4Bjunction occurred whenananalogousrecombinant, vNS3-NS4A-84%NS4B,wasexpressed. Thus, itappearsthat uncleaved NS3

plus NS5 inhibitNS4A/NS4B cleavage presumably because the putative signalsequence isnotaccessiblefor recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5

polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with

vNS2B-30%NS3orwithvNS2Bplus v30%NS3. These resultsindicatethat cleavageatthe NS4B/NS5junctioncanbe

mediatedby NS2B and NS3intrans.

The four serotypes ofdengue virus are members of the

Flaviviridae,afamily ofsome 70 viruses,mostofwhichare

transmittedby mosquitosorticks(45). Manyflavivirusesare

human pathogens and cause a variety of diseases such as

yellow fever, dengue, Japanese encephalitis, or tick-borne

encephalitis. Among flaviviruses, dengue viruses have the highest incidence of infection and the widest geographic distribution (16). Forthis reason, intensive efforts are

cur-rently beingdirectedatresearchondenguevirus. Like other

members of the flavivirus family, dengue virus contains a

positive-strandRNAgenomeapproximately 10 kb inlength (27). Complete nucleotide sequences ofthree dengue virus serotypes and several other major flaviviruses have been determined (11, 12, 17, 19, 24, 25, 29, 30, 34, 43, 47). The results of amino acid sequence data or alignment with the established protein sequences of other flaviviruses indicate that the dengue virus RNAgenome codes fora long

poly-proteinwith the order ofNH2-anchored C (anchC-pre-M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH (2-5, 8, 11, 28, 33, 39, 40, 43, 46). This body of sequence information indicates that flaviviruses share the same

ge-nome organizationand presumablythe same mechanism of

gene expression and viralreplication (7, 42). The flavivirus

polyproteinisapparentlycleavedco-andposttranslationally

togenerate the individual viral proteins.

Analysisof amino acid sequences nearthecleavage sites of flavivirus polyproteins suggests that there are several

cleavage mechanisms ofprotein processing. The cleavage

*Correspondingauthor.

site at each of the anchC/pre-M, pre-M/E, EINS1, and NS4A/NS4B junctions follows a long hydrophobic region,

andcleavageat these sites is believedto be mediated by a

host cell signal peptidase. Evidence supporting signal-di-rected cleavage thatgenerates the three structural proteins has been obtained from in vitro translation and processing studies (26, 28, 37). Cleavage at the NS1/NS2A junction, presumably mediated by a novel protease, requires an

eight-amino-acid sequence at the NS1 C terminus and the downstreamNS2A(13, 18).Thecleavage sitesatthe NS2A/ NS2B, NS2B/NS3, NS3/NS4A, and NS4BINS5 junctions share a common sequence motif in which apair of basic amino acids(RR, KR, RK),orQRattheNS2B/NS3 junction indengue viruses, precedeseitherG, S,orA(34, 39).This class ofprocessing events is thought to be mediated by a

virus-coded protease inthe cytoplasm (21, 35). It has been proposed on the basis of limited sequence homology to serine proteases that NS3 is a viral protease (la, 15). The N-terminal third of flavivirus NS3 contains three appropri-ately spacedconserved amino acids(Hisatposition 51, Asp

at94, and Serat 135 indengue type4 virus [DEN4] NS3),

proposedto be the catalytic triad. In addition, amino acid

sequences predicted for the substrate-binding pocket are

also present. Experimental evidence supporting this pro-posal has been obtained by using in vitro translation and

processingofpolyproteinprecursors (10, 31, 44).Our labo-ratorystudied the viral functions thatarerequiredfor these processing events in vivo, using recombinant vaccinia

vi-ruses expressingvariousportionsof the DEN4polyprotein. We observed that DEN4 polyproteins that contain a large

deletionwithin NS2Bwerenotcleaveddespitethepresence

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1536 CAHOUR ET AL.

-4 NS5(900 aa)->

I I I I

SmaI Pst I Hind III

- -

-ATCccc AS4| CG GCCC CTGCGAGA GGCAAAGC TTi CCCTAC|CCCT CGG SACGTIC TCT CCGTTTCSGAT

pATH2/NS5-1 pATH3/NS5-2

2SrpBAIS5-1(aa1-199) TxpZ/NS5-2(aa 199-757)

FIG. 1. Sequences of DEN4 NS5 cDNA in TrpE expression

vectors. Sequences at thejunctions between the bacterial TrpE vector, pATH2 or pATH3, and the NS5 cDNA fragment to be

expressedare shown. The two plasmid vectorsdiffer only in the

readingframein thepolylinker region.NS5sequencesbetween the

SmaI and PstI sites were cloned in the corresponding sites of pATH2tocreatepATH2/NS5-1;NS5 sequencesbetween thePstI

andHindIII siteswere cloned inpATH3to create pATH3/NS5-2. Polylinkersequences and non-dengue virus sequencesare inbold

letters. Thelengths of aminoacids (aa)encodedin the expressed

NS5segmentsareindicated.

of the entire NS3 sequence. These results showed that

NS2B, in addition to

NS3,

is required for cleavage at the

NS2A/NS2B and NS2B/NS3junctions and presumably for

cleavageattheNS3/NS4Ajunction (14).Wefurther showed that NS2B is capable of acting in trans. Only indirect evidenceindicatingthatNS3 canact in transwasobtained. The current study was initiated to examine the cleavage mechanisms utilized for processing of the remaining

non-structural proteins, i.e., NS4A, NS4B, and

NS5,

from the

NS3-NS4A-NS4B-NS5regionof the DEN4polyprotein.We presentdatademonstratingthatcleavageattheNS4A/NS4B junction is apparently mediated by a signal peptidase-like

hostproteaseand thatNS4B/NS5 cleavagecanbemediated

byboth NS2B andNS3 in trans.

MATERIALS ANDMETHODS

Expressionof

TrpE/NS5

fusionproteinsandpreparationof antisera. ThebacterialTrpE expressionvectorspATH2and pATH3 (41)were kindlyprovided byG. Ketner(TheJohns Hopkins University, Baltimore, Md.). Initially, weprepared a full-length NS5 cDNA fragment by polymerase chain reaction(PCR), using appropriate primers thatintroduceda

SmaI site preceding the ATG codon for initiation ofNS5 synthesisandaKpnIsiteatthe 3' terminus ofdengue virus cDNA. Full-length dengue virus cDNA clone 2A was used

asthetemplate (23).Twosubcloneswereseparatelyinserted

into the appropriate vector forexpression ofNS5 (Fig. 1). Plasmid

pATH2/NS5-1

contained the SmaI-PstI fragment (nucleotides[nt]7560to8152),andpATH3/NS5-2contained the PstI-HindIII fragment (nt 8152 to 9829). Plasmid

con-structswereusedtotransformEscherichiacoli C600. Trans-formantsweregrowninM9CAmediumcontaining ampicil-lin and induced with ,-indoleacrylic acid (10 ,ug/ml) (20). Bacteriawere pelleted and resuspended inTEN buffer (50 mM Tris HC1 [pH 7.5], 0.5 mM EDTA, 0.3 M NaCl) containing 2 mg of lysozyme per ml, disrupted by freeze-thawing, and treated with 3 mg of DNase I per ml. The

insoluble fraction was semipurified by three cycles of

cen-trifugation and washing with TEN buffer containing 0.1% NonidetP-40. Thepelletwasdissolvedby heatingat37°C for 30 minin Laemmli buffer (0.01 MTris HC1 [pH 6.8], 20% glycerol,5mMEDTA,4%sodiumdodecylsulfate[SDS], 50 mM ,-mercaptoethanol,0.002%bromophenol blue)

contain-ing 20 ,ug of phenylmethylsulfonyl fluoride per ml

(22).

Samples were separated on SDS-polyacrylamide

gels

and stained with Coomassie blue. Fusion

protein

bands were

excised, washed with distilled water,

homogenized

in

phos-phate-bufferedsaline(PBS)containing0.1%

SDS,

and emul-sified with an equalvolume of

complete

Freund's

adjuvant.

This mixture was used to inoculate New Zealand White rabbitsintradermallyalongthebackat100to300

pug

of each fusion protein per dose. Booster inoculations

using

the fusionprotein-polyacrylamide gel homogenate,emulsified in incomplete Freund's adjuvant, were performed at 4 and 8

weeks following theprimary immunization. Serum

samples

were collected beginning2 weeks afterthe lastinoculation.

Construction of recombinant vaccinia viruses. DEN4 cDNAfragmentscodingfor various

polyproteins

within the

NS3-NS4A-NS4B-NS5 domain were inserted into the

vac-cinia virus intermediate transfer vector pSCll(BglII)

(13).

TheN-terminal amino acidpositions of

NS2B,

NS3,

NS4A,

NS4B, and NS5 have been determined by alignment of the deduced DEN4 polyprotein sequencewith thatofflavivirus

Kunjin (14). To construct pSCll/NS4A-NS4B-NS5 and pSC11/NS4B-NS5, initiallyaDEN4 cDNA

fragment

coding

for NS4A-NS4B was prepared by polymerase chain

reac-tion, using full-length DEN4 2A cDNA as the

template,

oligodeoxyribonucleotide (oligo) 2850

(5'-GCCGGATCCA

CCATGAGTATAACTCTCGAC-3') tointroducethe initia-tion codonprecedingthe firstaminoacid of NS4A, and

oligo

2851 (5'-TCCTGGATCCTACCTCCTAGGGGTTTGTGC-3')toprovidea stopcodon. Similarly,DEN4cDNA

coding

for NS4Bwaspreparedby usingthe 2AcDNAtemplate and oligo 2445 (5'-AAGATCTATGTTGATCTACGTCATATTG AC-3'), which containsaninitiation codon preceding thelast

17amino acidsof the putative 19-amino-acid signal ofNS4B, andoligo 2851as primers. ThePCRproducts wereinserted separately into pSCll(BglII). The DEN4 insert in these pSC11 derivatives wasextended to include the entire NS5 coding sequence by fragmentexchange with

pSC11/NS2B-NS3-NS4A-NS4B-NS5, usingthe BsmI site at nt 7544 and the XhoI site in pSC11 DNA. DEN4 cDNA inserts in the

constructsofpSC11/NS4B-NS5 expressingshortenedforms of NS5 such as 50%NS5 (terminating at nt 8912) and 22%NS5(terminatingat nt8141) were obtainedby

polymer-ase chain reaction, using plasmid pSC11/NS4B-NS5 as the

template

and appropriate primers. Plasmid pSC11/NS2B-NS3-NS4A-NS4B-NS5 or pSC11/NS3-NS4A-NS4B-NS5

wasobtained by fragmentexchange between

pSC11/NS2A'-NS2B-NS3-NS4A-NS4B-NS5 and pSC11/NS2B-NS3 or

pSC11/NS3-84%NS4B

(14) at the unique BstBl site at nu-cleotide 5069 and the XhoI site in pSC11. PlasmidpSC11/

NS2A'-NS2B-NS3-NS4A-NS4B-NS5

was constructed ear-lierby insertion of theDEN4 sequencebetween theStuI site

at nt 3616 and the 3' flanking PstI site (following the C/G sequence), blunted by T4 polymerase, at the SmaI site of

pSC11

(46a). The yellow fever virusNS2B-32%NS3 cDNA fragment (nt 4181 to 5180 of the yellow fever virus se-quence), encoding a 333-amino-acid polyprotein, was pre-pared by polymerase chainreaction, using appropriate

prim-ersandplasmid pES/6,aderivative of clone pGX4EI (36),as

the template. All constructs described were verified by restrictionenzyme mapping and by sequencing of 50 to150

nt across the insertion sites. Both CV-1 and TK-141 cells

were propagated in Eagle's minimum essential medium

supplemented

with 10% fetalcalf serum. Recombinant vac-cinia viruses were constructed by using the wild-type vac-ciniavirusstrainWRandthe pSC11/DEN4DNA derivatives as described earlier (6, 13). Recombinant vaccinia virus

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PROCESSING OF DENGUE VIRUS NONSTRUCTURAL PROTEINS 1537

vSC8, containingthe lacZgene but no dengueviruscDNA, wasusedas anegative control (6). Recombinant virus stocks were preparedfrom infected CV-1 cells, and the virus titer was determined by plaque assay on monolayers of CV-1

cells. The nomenclature for the recombinant virus con-structsandtheirencodedpolyproteins followed the previous

practice (14).

Radiolabeling and immunoprecipitation. CV-1 cells were

infected

with recombinant vaccinia viruses or the control

vaccinia virus vSC8 at a multiplicity of infection of 5 PFU per cell diluted in Eagle's minimum essential medium

con-taining 2% fetal calf serum (MEM2). At 16 to 24 h postin-fection, the medium was replaced with methionine-free MEM2 for 1 h and then with methionine-free medium

containing

L-[5S]methionine

(100 ,uCi/ml; specific activity,

-800Ci/mmol) for2h. Mediumwas removedafter

labeling,

and thecells were rinsed twice with cold PBS and lysed in RIPA buffer (1% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 0.1 MTris-HCl [pH7.5], 0.15 M NaCl)containing 20 ,ug of phenylmethylsulfonyl fluoride per ml. The labeled

lysate of DEN4-infected cells was kindly provided by M.

Bray ofourlaboratory.

Immunoprecipitation

was performed by using NS5-spe-cific antisera raised against

TrpEINS5

fusion proteins or

DEN4hyperimmune mouseascitic fluid(HMAF). Briefly, a

50- to

100-pd

aliquot ofalabeled lysatewas mixedwith the

NS5-specific antiserumor HMAF (3 to 5

IlI)

and incubated onice for 2 h. Thenanexcessof Pansorbinwasadded to the

mixture, and after incubation on ice for another 1 h, the

immune precipitates were collected by centrifugation and

washedtwice withRIPAbuffer containing2% SDS.

SDS-polyacrylamide gel electrophoresis. Immune

precipi-tates wereresuspended in Laemmli buffer and boiled for5

min before loading. Sampleswere analyzed by electropho-resison anSDS-8% polyacrylamide gel

(acrylamide/bisacry-lamide ratio of60:1.6). Gels were treated for fluorography and usedtoexpose X-rayfilm.

RESULTS

TrpE/NS5

fusion proteins and NS5-specific sera. In our

earlier

studies,

DEN4 HMAFwas used for

precipitation

of labeled lysates of infected cells, and we observed that

precipitation

of

dengue

virusNS5was

barely

detectable.To

facilitate detection of NS5 orits

polyprotein

precursors, we

prepared

NS5-specific

sera. Since NS5

(103

kDa)

isa

rela-tively

large

protein,

two

subfragments

ofNS5 cDNA were

separately

expressed

in the form of

TrpE

fusion

proteins by

usingabacterial TrpEvector. Asdetailed in

Materials

and Methods and shown

schematically

in

Fig. 1,

fusion

protein

TrpE/NS5-1

contained amino acids 1 to 199 and fusion

protein

TrpE/NS5-2

contained amino acids 199 to 757 of

DEN4 NS5.

Analysis

of the bacterial

lysate

showed that

both

TrpE/NS5-1

and

TrpE/NS5-2

of the

predicted

sizes

(52

and 97

kDa,

respectively)

were detected in the insoluble fraction

(Fig.

2A). These fusion

proteins

were notdetected in the soluble fraction

(data

not

shown).

Rabbit antisera raised

against

each of these fusion

proteins

weretestedfor their

ability

to bind radiolabeled NS5 in

dengue

virus-infected cell

lysate.

Figure

2B shows that both antisera

specifically

precipitated

a 103-kDa labeled

band,

as

pre-dicted for the NS5

protein.

No other

protein

bands were seen in the

precipitates.

These results indicated that both antisera were

specific

for

dengue

virus NS5. Since no

differencein

NS5-binding

efficiency

wasevident betweenthe

A M 1 2 3 4

97

-B (5-I) (5-2)

H Pl PI M _ -trpE/5 -2

68- _ -

-I _ -trpE/5-1

43-

-29-@

N S5

-NS3-_

E-5. NS

I1-_~ 4 -9 2

-69

16-46

;14k-.mmw..

,.Imo- _m

40M

qw

-3O

I8

preM- * - 18

FIG. 2. Characterization of TrpE/NS5 fusion proteins and NS5-specific sera. (A) TrpE/NS5 fusion proteins. The insoluble fraction of theE.coli C600 lysate was analyzed by SDS-polyacrylamide gel electrophoresis, and theprotein bands were stained by Coomassie blue. Lanes: M,protein size markers, shown in kilodaltons on the left; 1, control bacteria; 2, bacteria transformed by pATH2; 3, bacteria transformed bypATH2/NS5-1; 4, bacteria transformed by pATH3/NS5-2; (B) NS5-specific sera. Immunoprecipitation of an

L-[35S]methionine-labeled

lysateofDEN4-infected cells wascarried out totestthe NS5bindingspecificity of the rabbit antiserum raised against the pATH2/NS5-1 or the pATH3/NS5-2 fusion protein. Lanes: H, dengue virus HMAF; PI, preimmune serum; I, postim-munization serum; M,

'4C-labeled

protein size markers, shown in kilodaltons on the right. DEN4 proteins are indicated on the left.

twoantisera, only the antiserum raised against

TrpE/NS5-1

was used in thisstudy.

NS2B is required for proteolytic processing of dengue NS3-NS4A-NS4B-NS5. Because ofourinterest in

identifying

therequirements for dengue

polyprotein

processing

in

vivo,

we constructed a series of recombinant vaccinia viruses

expressing

variouslengths ofthe DEN4 NS2B-NS3-NS4A-NS4B-NS5sequence. DEN4

polyproteins

encodedbythese

recombinant virusesarediagrammed in

Fig.

3A. The

N-ter-minal sequence of

polyprotein

NS2B-NS3-NS4A-NS4B-NS5 or NS3-NS4A-NS4B-NS5 was the same as that of NS2BorNS3constructed earlier(14);Met-Gly

preceded

the first amino acid

(Ser)

ofNS2B, and Met

preceded

the N

terminus of NS3. Similarly,

polyprotein

NS4A-NS4B-NS5 containedaMetresidue

preceding

the

predicted

N-terminal

sequenceof NS4A.TheNS4B-NS5

polyprotein

specified

by

vNS4B-NS5 contained the initiating Met

plus

the last 17

amino acids ofNS4A

preceding

the Nterminusof NS4B.All

four

polyproteins

containedthe

full-length

NS5 sequenceat

the C terminus. These recombinants were used to infect cells, and the labeled

lysates

were

analyzed

by

immunopre-cipitation using

HMAF or the

NS5-specific

antiserum. As

shown in

Fig.

3B

(lane 2),

vNS2B-NS3-NS4A-NS4B-NS5

expressed a

protein

with an apparent molecular size of73

kDa,as

predicted

fortheNS3

protein. Previously,

we have shown thatan

analogous

recombinant,

vNS2B-NS3-NS4A-84%NS4B,

expressesauthentic

NS3,

as identified

by

comi-grationwith theNS3

product

ofDEN4 virus

(14).

Inlane

2,

two

protein

bands,

migrating

at

approximately

52 and 54 kDa,

appeared

to beDEN4

specific

since

they

were

immu-noprecipitated by

HMAF. These bands were

previously

observed

(14)

and

probably

derivedfrom

internal

cleavages

of NS3. Severalother

large

polyproteins ranging

from 85to VOL. 66,1992

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J. VIROL. 1538 CAHOUR ET AL.

N 2 NNj533 N54A NS4B

lI

\55

77

__I_-ra1-

:

F

U

'I

B 1 2 3 4 5 6 7

210-W.

to i^; _ ..:.

210- 1' -NS3-NS4A-NS4B-NS5

_ -NS4A-NS4B-NS5

- --NS4B-N55

- NS5

694- ..1

46 * ,

46

-+ + + HMAF

+ + + + antiNS5

FIG. 3. Processingof various lengths of the NS2B-NS3-NS4A-NS4B-NS5domain ofthedengueviruspolyprotein.(A)Diagramsof various lengthsof dengue virus polyproteins expressed by

recom-binantvaccinia viruses. Theputative signalsequencethatprecedes theNS4A/NS4B cleavage junction is marked withafilled box. (B)

Analysis of dengue virus proteins expressed by recombinant

vac-cinia viruses. Radiolabeled lysates of CV-1 cells infected with recombinantvacciniaviruses shown in panel A were

immunopre-cipitated with polyvalent HMAF or the NS5-specific serum. The

labeledprecipitates wereanalyzed bySDS-8% polyacrylamidegel

electrophoresis. Recombinant vaccinia viruses used forinfection:

lane 1, vSC8control; lanes2and 3, vNS2B-NS3-NS4A-NS4B-NS5;

lanes 4 and5,vNS3-NS4A-NS4B-NS5; lane 6,vNS4A-NS4B-NS5;

lane7,vNS4B-NS5. Positionsof molecularsize markersareshown

in kilodaltonsontheleft.

210 kDa, whichwerenotseenin the control lysate,werealso

precipitated.Thesepolyproteins mostprobably represented

processingintermediatesorincompleteprocessingproducts,

for example, NS3-NS4A (86 kDa), NS2B-NS3-NS4A (100 kDa), and NS4A-NS4B-NS5 (146 kDa). Because antisera

specific for NS2B, NS4A, or NS4B were not available,

definite identificationofthese protein bands was not

possi-ble. When theanti-NS5 serum was used, alabeled band of

approximately 103 kDawas detected, aspredicted forNS5.

This finding indicates that cleavage at bothNS3/NS4A and

NS4BINS5 junctions had occurred. In contrast,

vNS3-NS4A-NS4B-NS5 did not express NS3 or NS5; rather, it

expresseduncleavedNS3-NS4A-NS4B-NS5. This result

in-dicates that NS2B is required for processing of the

down-stream nonstructural proteins, including the

NS3/NS4A

and NS4B/NS5junctions, bothofwhichcontain adibasic amino

acid sequence preceding the cleavage site.

Cleavage at the NS4A/NS4Bjunction. By

alignment

with

the established N-terminal sequence ofKunjin virus NS4B

(39), the N-terminal amino acid of DEN4NS4B is placedat Asn-2242. In the flavivirus sequence, the cleavage site between NS4A and NS4B is preceded by a runof

hydropho-bic amino acids that could serve as a signal for

cotransla-tional cleavage mediated by asignal peptidase. Inthecaseof

DEN4, this putative signal sequence includes 19 hydropho-bic amino acids. Since antisera specific forDEN4 NS4Aor NS4B were not available, we used the NS5-specific sera to determine whether the NS4B-NS5 cleavage product was made from the polyprotein NS4A-NS4B-NS5 precursor. Cells were infected with vNS4A-NS4B-NS5, and the labeled lysate was analyzed. Figure 3B (lane 6) shows that the NS5-specific antiserum precipitated two bands, both of which were larger than NS5; one was identified as uncleaved NS4A-NS4B-NS5 polyprotein on the basis of molecular size, and the other was identified as NS4B-NS5. This finding indicates that cleavage at the

NS4A/NS4B

junction had apparently occurred in the absence of the previously identi-fied NS2B-NS3 viral protease. This cleavage is probably mediated by a signal peptidase and may occur cotranslation-ally. This notion is further supported by a pulse-chase study which indicates that the relative amounts of NS4A-NS4B-NS5 and NS4B-NS4A-NS4B-NS5 did not change during the chase period (data not shown). The observation that NS4B-NS5 (lane 6) comigrated with the product of vNS4B-NS5 suggests that the signal sequence was cleaved from the vNS4B-NS5 product or that the presence of the signal did not affect the mobility. It should be noted that cleavage at

NS4A/NS4B

did not occur when polyprotein NS3-NS4A-NS4B-NS5 was expressed, since NS4B-NS5 could not be seen. However, in a previous study we observed that cleavage at the

NS4A/

NS4B junction did occur when vNS3-NS4A-84%NS4B was expressed (14). We speculate that uncleaved NS3 plus NS5 prevented NS4A/NS4B cleavage from taking place, perhaps because the putative signal sequence in this polyprotein is not accessible for recognition by the responsible protease.

Cleavage at the NS4B/NS5 junction by NS2B and NS3 in trans. Recombinants that expressed an uncleaved NS4B-NS5 polyprotein such as vNS4A-NS4B-NS4B-NS5 or vNS4B-NS4B-NS5 were used for coinfection of cells with vNS2B alone, v30%NS3 alone, or vNS2B plus

v30%NS3.

Lysates of infected cells were analyzed by immunoprecipitation with the NS5-specific serum. The results (Fig. 4) show that a labeled band of the predicted size for NS5 was detected during coinfection with vNS2B plus

v30%NS3

but not with vNS2B orv30%NS3 alone. Note that the shift in mobility of NS4B-NS5 in lane 11 was probably an artifact since it was not observed in other experiments. As estimated by the intensity of the labeled protein bands, more than 50% of each polyprotein precursor was converted to the NS5 product. Interestingly, the NS4A-NS4B-NS5 polyprotein (lane 7) appeared to be completely processed, whereas a significant portion of NS4B-NS5 persisted. This finding suggests that NS4A-NS5 is more efficiently processed than NS4B-NS5 at the NS4B/NS5junction. These results indicate that cleavage at the NS4B/NS5 junction in the polyprotein was mediated by both NS2B and NS3 in trans. Additional evidence was sought to confirm that the appearance of an NS5 band in Fig. 4 resulted from proper processing at the NS4B/NS5 junction rather than from a spurious cleavage

A

v(NS2B-5)

v(NS3-5)

v(NS4A-5)

vf(NS4B-5)

92

-.7

* jib. .":."

46

-,- 4m

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PROCESSING OF DENGUE VIRUS NONSTRUCTURAL PROTEINS

vNS4A-NS4B-NS5 vNS4B-NS5

2B 2B coin

fecting

_ 2B 3 3 - 2B 3 3 virus

210-_

92

-_0 j&j4S -N54A-NS48-N55

m

~_

_i _ _-NS4B-NS5 NS4B-50%NS5 -69

-N54B-2 2%N55- _ 4 6 -_W

[image:5.612.64.304.79.234.2]

1 2 3 4 5 6 7 8 9 10 11

FIG. 4. Demonstration that cleavage oftheNS4B/NS5junction requires both NS2B and NS3. CV-1 cells were infected with

vNS4A-NS4B-NS5orwithvNS4B-NS5,producingthe DEN4

poly-proteintobe tested for cleavage.Lanes 4 and 8, control showing the uncleaved polyproteins; lanes 5 and 9, coinfection with vNS2B alone; lanes 6 and10,coinfection with v30%NS3alone; lanes7and 11, coinfection with vNS2B plusv30%NS3. Other controls: lane 2, infection with vSC8; lane 3, infection with vNS5 showing the authentic NS5. All radiolabeled cell lysates were

immunoprecipi-tatedby theanti-NS5serum.Protein sizemarkersareshown in lane 1 andindicated inkilodaltonsonthe left.

within NS5. Recombinants that expressedNS4B-NS5 trun-cated atthe C terminus tothe size of50%NS5or 22%NS5

were constructed and used for coinfection with

vNS2B-30%NS3, whichwasshown toprovide the NS2B-NS3

pro-tease activity (14). Figure 5 shows that coinfection yielded the predicted shortened NS5 band of50%NS5 or22%NS5

resulting frompropercleavageattheNS4B/NS5junction of the truncated NS4B-NS5 polyprotein.

Functionalsimilarity between NS2B-NS3 proteases of

den-gue and yellow fever viruses. Comparison of sequences

between DEN4 and yellow fever virus indicates that the amino acid homology is 37% for NS2B and 50% for the protease domain ofNS3 (consisting of the N-terminal 180 amino acids). We wereinterested in comparing the

NS2B-NS3 protease activities of dengue virus and yellow fever virus for cleavage at the DEN4 NS4B/NS5 junction. A recombinant vaccinia virus expressing yellow fever virus

NS2B-32%NS3,corresponding approximately in sizeto

den-gue virus NS2B-30%NS3, was constructed and used for

coinfection with vNS4B-NS5. The results (Fig. 6) indicate that both theNS2B-NS3 proteaseofyellowfever virus and that ofdengue virus cleaved the DEN4NS4B-NS5 polypro-teinwithhigh efficiency, sincethepolyproteinprecursorwas

mostly processed. This resultsuggests thatbothviral

prote-ases cleaved the NS4BINS5junction, presumably through the recognition ofthe cleavagesequence RR/G, despitethe significant sequence divergence that exists betweendengue virus andyellowfever virus proteases.

DISCUSSION

The current study examined the requirements for the

processing of DEN4 nonstructural proteins NS4A, NS4B, and NS5 from the polyprotein precursor. The sequences at

thecleavagesites that define theseproteins predictthattwo different proteolytic events are involved: the viral

NS2B-NS3 protease isresponsible forNS3/NS4A and NS4BINS5 cleavages, and asignal peptidase-like protease cleaves

be-

30-22°,oN55

[image:5.612.358.532.83.332.2]

coinfection - + +

FIG. 5. Verification ofpropercleavageatNS4B/NS5. CV-1cells

were infected with recombinant vaccinia viruses that expressed C-terminally truncated NS4B-NS5. Cleavage at the NS4B/NS5 junctionwastestedby coinfection withvNS2B-30%NS3. Anti-NS5 serum wasused forimmunoprecipitation of radiolabeledlysatesof infected cells. Lanes: 1, vNS4B-22%NS5; 2, vNS4B-22%NS5 plus vNS2B-30%NS3; 3, vNS4B-50%NS5; 4, vNS4B-50oNS5 plus vNS2B-30%NS3. Protein size markers areindicated in kilodaltons onthe left.

tween NS4A and NS4B. Processing of the

NS2B-NS3-NS4A-NS4B-NS5 polyproteinappears tobe efficient in the recombinant vaccinia virus expression system. Incontrast,

recombinant vaccinia virus vNS3-NS4A-NS4B-NS5

ex-pressed only the uncleaved polyprotein. In an experiment

not shown, the addition of NS2B in trans, achieved by coinfection of cells with vNS3-NS4A-NS4B-NS5 and vNS2BorvNS2B-30%NS3, restored cleavage atthe NS4B/

NS5 junction because properly cleaved NS5 was readily

detected.However, cleavageattheNS3/NS4Ajunctionwas

notobserved in the same experiment, as indicated by the absence of NS3. In contrast, NS3 was detected when vNS2A-NS2B was used for coinfection. In this regard,

NS2A-NS2B may be more efficient than NS2B alone at

pairing with NS3 to produce the viral protease activity. Cleavage at NS3/NS4A apparently takes place efficiently when NS2B ispresentin cis butinefficientlywhen NS2B is provided in trans. These observations suggest that the functional requirement for components of the NS2B-NS3 viralproteasemaydifferforcleavagesatthe NS3/NS4Aand NS4B/NS5 junctions, althoughboth sitesappeartoshare the

consensus cleavage sequence. The requirements for

cleav-age atthe NS4B/NS5 junction were studied by expressing

polyproteinNS4A-NS4B-NS5 orNS4B-NS5 inacoinfection

procedure. EfficientprocessingofNS5 fromeither polypro-teinwas observed when both NS2Band NS3 weresupplied

intransbutnotwith either alone. Thisobservationprovides directevidence that the twocomponents ofthe viral

prote-ase canboth actintrans.

Processing between NS4A and NS4B occurred in the

92-I

1 2 3 4

210-_00 -NS5

OM-- 50%NS5

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1540 CAHOUR ET AL.

vNS4B-NS5

I ., I

(vNS2B-NS3)

210--NS4B-NS5

_ _0M _Wm* -NS5 92

-69- *

1 2 3 4 5 6

FIG. 6. ComparisonofcleavagesatNS4B/NS5 junction by

den-guevirus NS2B-NS3andby yellowfevervirus NS2B-NS3. Exper-iments were carried out essentially as described forFig. 4 and 5. Complementation was carried out by coinfection of cells with vNS4B-NS5 plus vNS2B-30%NS3 of DEN4orvNS2B-32%NS3 of yellow fever virus. Lane 4, without coinfection; lane 5, with vNS2B-30%NS3 of dengue virus; lane6, with vNS2B-32%NS3 of yellow fever virus; lane 1, protein size markers, indicated in kilodaltonson the left. Other controls: lane2, vSC8;lane 3,vNS5. The bandmigratingbetween NS4B-NS5and NS5 isbackground.

absence of the NS2B-NS3 viral protease, as expression of

NS4A-NS4B-NS5 polyprotein yielded NS4B-NS5. The

un-cleaved precursor polyprotein NS4A-NS4B-NS5 was also

detected, and in some gels the level of the precursor was

equaltothe level of the cleaved NS4B-NS5 product (Fig. 3, lane 6). This finding indicates that cleavage atNS4A/NS4B

wasapparently less efficient thanthatoccurring duringother

signal-directed cleavages of the DEN4 virus polyprotein, such as at the pre-M/E and E/NS1 junctions, for which uncleaved precursors were notdetected (48). Experiments to demonstrate the requirement of the putative signal se-quencefor processing ofNS4A-NS4B-NS5 bv using an in

vitro translation procedure in the presence of microsomal membranes have notyieldedadefinitive result. A

recombi-nant vaccinia virus expressing NS4A-NS4B-NS5 polypro-teinthat lacks 17 aminoacids of theputative signal preceding the NS4A/NS4B junction was analyzed. Unexpectedly,

cleavageatNS4A/NS4Bwasnotcompletely abrogated (data

not shown). Since NS4A consists of mostly hydrophobic aminoacids, it is temptingto speculate that another portion ofNS4A, upstreamof the deletion, could serveas a

surro-gate signal and allow a low level of NS4A/NS4B cleavage. Interestingly, cleavage atNS4A/NS4B did notoccurwhen polyprotein NS3-NS4A-NS4B-NS5 was expressed. On the

other hand, in a previous study the results showed that

NS4A/NS4B cleavage appearedto occurwhen

NS3-NS4A-84%NS4B wasexpressed (14). One interpretation for these

findings is that thepresence of uncleaved NS3 and NS5 in

the molecule prevents proper recognition of the putative

signal sequence by a signal peptidase-like protease. This observation suggests that cleavage at the NS3/NS4A or

NS4B/NS5 junction takes place priorto the processing of

NS4A/NS4B.

Thus, in general, the NS2B-NS3 protease is responsible for processing at cleavage sites that consist of two basic amino acids followed by G, A, or S in the nonstructural

protein region. However, the cleavage efficiencies vary

accordingtothe individualcleavage sites. Variations of the amino acidsequenceatthe cleavage site mayinfluence the

cleavage

efficiency.

A deviation from this motif is the

QR/G

sequence at the

NS2B/NS3

junction

found among all four

d%,ngue

virus serotypes.

Previously,

we obtained evidence

suggesting

that

cleavage

at

NS2B/NS3

containing

the

QR/G

sequence followed

cleavage

at

NS2A/NS2B,

which utilizes

theRRISsequence

(14).

We also noted thatupon coinfection

of cells with

vNS3-NS4A-NS4B-NS5

and

vNS2B-30%NS3,

processing

at

NS3/NS4A,

where the

cleavage

sequence is

RK/S,

was less efficient than that at

NS4B/NS5,

which

contains the

cleavage

sequence RRIG. It is not known

whether this

temporal

orderof

cleavage

is determined

by

the

sequence variation at the

cleavage

site alone. A kinetic

analysis

of the

processing

of

dengue

virus nonstructural

proteins

similartothatconductedwith

yellow

fever virus

(9)

should

help

to address this

question.

At

present,

it is not

known whetheramino acids outside the

cleavage

sequence

motifarealsoinvolved in

influencing

the

cleavage

efficiency.

Comparison

ofthe NS4B/NS5

cleavage junctions

of DEN4

and

yellow

fever virus shows that both viruses utilize the

same

RR/G

sequence. This

finding

allowed us to

employ

DEN4 NS4B-NS5

polyprotein

tocomparethe

specificity

of

proteases from both viruses. Under the conditions ofour

analysis, yellow

fever virus protease was able to process

DEN4NS4B-NS5at an

efficiency

similartothat

occurring

in

the

homologous

dengue

virus system. This

finding

suggests

thatthe functional domains in both components of the viral

proteasemustbeconserved

despite

the fact that theoverall amino acid

homology

is

only

37% for NS2B and 50% for the

proteasedomainof NS3. Studies

using

chimeric NS2B-NS3

proteasesconstructed betweenDEN2and

yellow

fevervirus

have

yielded

an

interesting

insight

into the

enzyme-substrate

binding

and interaction between NS2B and NS3

during

proteolytic processing

(31a).

A chimeric proteasethat

con-tains the

yellow

fever virus proteasedomain of NS3

substi-tuting

for the

corresponding

sequence of DEN2 fails to

process DEN2

NS2A/NS2B

and

NS2B/NS3

cleavage

sites. On the other

hand,

another chimericconstructthat includes

a C-terminal

portion

of

yellow

fever virus NS2B and the

yellow

fever virus NS3proteasedomain isabletocleave the

DEN2

NS2A/NS2B

and

yellow

fever virus

NS2BINS3

junc-tions. This indicates that

yellow

fever virusprotease

activity

requires

proper interaction between

homologous

yellow

fever virus NS2B and

NS3, presumably through

the forma-tion of a

complex. Thus,

these results and others are

consistent with ourearlier

finding

thattheprotease domain of NS3 is necessary butnot sufficient fortheviral protease

activity.

Others have identified conserved sequences in

flavivirus NS3 with

homology

toserineproteases,

including

the

proposed

catalytic

trial and substrate

binding

domains

(1,

15).

Studiesto

identify

suchconserved sequencesin

NS2B,

andtofurther

assign

theirfunctional role in

cleavage,

remain

to be

completed.

The results in this report and our

previous study

showed that the viral NS2B-NS3 protease is

responsible

for most

intergenic cleavages

inthenonstructural

protein region (14).

Also,

this viralenzyme appearstobe involved in

cleavages

at severalsites

containing

theconsensus

cleavage

sequence within the NS3 nonstructural

protein.

Additional

cleavage

sites thatcontainthedibasic amino acidsequencemotifare

found at the anchC/C and

pre-M/M

junctions

in the struc-tural

protein region.

It was observed earlier that

pre-M

is

subsequently

cleavedtoformmatureM

during

virus

assem-bly

and release

(38).

Similarly,

cleavage

ofthe C terminus from anchC to generate mature C was also observed

(28).

Evidence is not available to suggest that

cleavage

at the

pre-M/M

junction

is also mediated

by

NS2B-NS3 viral

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PROCESSING OF DENGUE VIRUS NONSTRUCTURAL PROTEINS 1541

protease.Rather, it has been shown that cleavage ofpre-M

to generate matureMoccursinanacidic vesicular

compart-ment in the virus-infected cells (32). Consistent with the notion that viral protease is not involved in cleavage at pre-MIM is our observation that the mature M protein was not detected in the lysate of cells coinfected with vC-pre-M-E-NS1-NS2A and vNS2B-30%NS3 (4a). On the other hand, studieson processing of transientlyexpressedyellow fever virus proteins showed that yellow fever anchC/C cleavage appears to be mediated by the viral NS2B-NS3 protease (1). These studies provide evidence indicating the NS2B-NS3viral proteaseis also responsible for the delayed-type processing of the virion capsid protein.

ACKNOWLEDGMENTS

We thank L. Markofffor helpful discussions, R. Chanock for critical reading of the manuscript, and T. Heishman for expert editorial assistance.

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