Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

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0022-538X/94/$04.00+0

Both NS3 and NS4A Are Required for Proteolytic Processing of

Hepatitis C Virus Nonstructural Proteins

CRISTINAFAILLA, LICIATOMEI,AND RAFFAELEDEFRANCESCO* Istituto di Ricerche diBiologia Molecolare "P. Angeletti"-Pomezia, 00040 Rome, Italy

Received 20 December 1993/Accepted 11 March 1994

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyproteinare effected bythe virus-encoded serineprotease containedwithinNS3. UsingtransientexpressioninHeLa cells of cDNAfragmentsthatcode forregionsof the HCVpolyprotein,we studiedwhetherviral functionsother thanNS3arerequired for proteolytic processingatthese sites.We found that,inadditiontoNS3,aC-terminal33-amino-acidsequenceof the NS4Aproteinisrequiredforcleavageat theNS3-NS4AandNS4B-NS5A sitesand thatitaccelerates therateofcleavage attheNS5A-NS5B junction. Inaddition,weshowthatNS4AcanactivatetheNS3proteasewhensuppliedintrans.Our datasuggestthat HCV NS4Amaybe thefunctionalanalogof flavivirus NS2Bandpestivirus plO proteins.

Hepatitis C virus (HCV) isthe major etiological agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis (11, 31). Inmostcases, HCVcauses a persis-tent liver infection that eventually develops into cirrhosis or hepatocellular carcinoma (10).

HCV contains a positive-stranded linear RNA genome of about 9.5 kb in length (12, 29, 41). On the basis of their genomicstructureand virionproperties, HCV, the pestivirus, andthe flavivirus have been classified as threegenera in the family Flaviviridae (19).Asin thecaseofflavi- and pestiviruses, HCV isasmall, enveloped virus, whose genomicRNAincludes asingleopenreading frame encodinga precursorpolyprotein that is cleaved co- or posttranslationally into mature viral polypeptides. The structural proteins are encoded by the N-terminal portion of thegenome,whereas the remainingpart

codes for the nonstructural proteins, whicharebelievedtobe

components of the viral replication machinery (for a recent

reviewonthe molecularbiology of HCV,seereference34). The map of the HCV polyprotein has been obtained by cell-free and cell culture expression studies (23, 24, 42). The geneorder that hasemergedfrom these studies is 5'-C-E1-E2-NS2-NS3-NS4A-NS4B-NSSA-NSSB-3'. C is a basic protein that binds RNA(39a), andit is postulated to be the nucleo-capsid protein.ElandE2areputative envelope glycoproteins. NS2throughNS5B representthe viral nonstructural proteins andcould befunctional analogsof thecorresponding proteins ofpesti-andflaviviruses (7, 13).

Processing of the polyprotein requires both host and viral proteases. The signal peptidase ofendoplasmic reticulum is responsible for generating the N termini of El, E2, and possibly NS2 (24). Whereas it has been recently established that anovel virus-encoded metalloprotease is responsible for the cleavage between NS2 and NS3 (21, 25), we and others have identified NS3as theproteaseresponsible forcleavages

at the NS3-NS4A, NS4A-NS4B, NS4B-NSSA, and NS5A-NS5B junctions (3, 15, 22, 42). Analysis of the amino acid sequence of theNS3proteinof HCVsuggestedthat this viral protein contains a trypsin-like serine protease domain that functions intheprocessing of the viralpolyprotein (35), as is

*Corresponding author. Phone: 93221. Fax:

39-6-910-93225.Electronic mail address: [email protected].

thecasefor thehomologousNS3proteinof flaviviruses(9)and p80ofpestiviruses (45).

The active siteofserineproteases invariably contains three residues, histidine, aspartate, and serine, that maintain the same relative spatial position in all the known structures of these enzymes. These residues constitute the catalytic triad: the histidine imidazolegroup abstracts the alcohol protonof the serine and transfers ittotheamine-leavinggroup,while the aspartate stabilizes the positive charge that develops on the histidine in the intermediatecomplex (36).The histidine1083,

aspartate 1107,and serine 1165 residues(numbered according

to their location in thepolyprotein of BK strain HCV [41]), found in the N-terminal domain of the NS3protein,arehighly conserved amongall HCV strainssequenced sofar and have been proposed to constitute the catalytic triad of the HCV protease (4, 7, 35).Consistent with this prediction, when the presumptive catalytic serine 1165 of the polyprotein was mutatedtoalanine, processingattheNS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites was abolished (22, 42). Cleavageatthree of the four sites of the mutatedpolyprotein could be restored by supplyingfunctional NS3 intrans,while processingatNS3-NS4A occurredonlyincis(42).Thisfinding indicated that thecleavage of the latter site isan intramolec-ular event. Interestingly, the NS3-NS4A cleavage site differs from the others in that the processingtakesplace betweena threonine and a serine residue, whereas the remainder are cleaved between acysteine anda serine(22, 37).

Inflavivirus,NS3 isnecessary,butnotsufficient,for

process-ing at the nonstructural cleavage sites: in addition to the catalyticNS3domain,NS2B isrequiredforproteolytic activity (1, 6, 18, 33, 38). A stable physical interaction between NS3 and NS2B has been demonstrated (1, 18). Inthe caseof the othertwo genera of the family Flavivinidae, pestiviruses and HCV, it has been observed that the respective viral serine proteases,p80andNS3,canfunctionindependentlyof all the

regions of the polyprotein that are present upstream of the proteases themselves (22, 42, 45). However, in the case of bovine viral diarrhea virus (BVDV),amember of the

pestivi-rus genus, a region of the polyprotein outside p80 may be

necessarytoeffectcleavage bythe viral serineproteaseat the

p58-p75 site(45). Thisregionhastentativelybeenmappedto

aportionof thepolyprotein (13, 45)thatcorrespondstoHCV NS4B.

We initiated a study aimed to elucidate whether viral 3753

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functions other than NS3 are required for the proteolytic processingof thenonstructuralregion of theHCVpolyprotein. Using transient expression in HeLa cells of cDNAfragments that encoderegions of the HCVpolyprotein,we analyzed the cleavageactivityoftheNS3serineprotease oneach individual cleavage site. Inthispaper, we reportthat,inadditiontoNS3, a C-terminal domain of the NS4Aprotein is also required for efficient cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5Bjunctions. In addition, we also show that NS4Acan act in trans to affectcleavage atthese sites.

MATERLALSAND METHODS

Cells and virus. HeLa cells, originally obtained from the AmericanType CultureCollection,were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS). Vaccinia virus vTF7-3 (20) was grown in RK13 cells cultured in minimum essential medium containing 10% FCS. Construction of recombinantplasmids. HCV cDNAclones of the HCV-BK isolatewereusedfor allplasmid constructions (41). DNAfragments derived from HCVcDNA were inserted downstream of the 5' untranslated region of

encephalomyo-carditis virus and under a T7 promoter in the pCite-1 vector

(Novagen), in the appropriate translationalreading frame and followed by a termination codon. All the plasmids described belowwere obtained with standard recombinant DNA tech-nology (39).

Plasmid pCite(NS3) contains the portion of the HCV ge-nome between nucleotides 3351 and 5177 and therefore en-codestheportion of the viralpolyproteinbetweenamino acids 1007 and 1615. Construction of this plasmid has been de-scribed earlier(42).

pCite(NS4AB) bears a cDNA fragment corresponding to

nucleotides 5276 to 6225 of the HCV genome (amino acid residues 1649 to 1964 of the polyprotein).

pCite(NS5AB) contains the portion of the HCV genome

between nucleotides 6224and 9400(aminoacid residues 1965 to3010of the polyprotein).

pCite(NS4B/5A)wasobtainedbycloninginpCite-1 acDNA

fragment containing nucleotides 5653 to 7472.This construct

encodes the portion of the polyprotein between amino acid residues 1775 and2380.

pCite(NS3-AP) is a derivative of pCite(SX) (42) which contains HCVcDNA from nucleotides 3305 to 5373 (amino

acid residues 992 to 1680). A fragment derived from the

pBC12/RSV/APvectorandcodingforplacentalalkaline

phos-phatase (5)wasinserted downstream ofthis region,withcare

taken tomaintain thecorrect readingframe.

pCite(NS3/4A) contains a cDNA fragment comprising the

region between nucleotides3305and5465 ofHCV cDNA.The resultingconstructcodes for theportion of the HCV

polypro-tein between amino acids992 and 1711.

pCite(NS3/4A:S,

165-A)

encodes the same polypeptide,

ex-ceptthat thecatalyticserine(S-1165)of NS3 has beenreplaced withan alanine.

pCite(NS3/4AAC) contains a cDNA fragment comprising

theregionbetween nucleotides 3305 and 5373 ofHCVcDNA.

The resultingconstructcodes therefore for theportion ofthe HCV polyproteinbetween amino acids 992 and 1680.

pCite(NS4A) bearsacDNAfragment spanningnucleotides

5276 to 5465 of the HCV genome(aminoacid residues 1649 to 1711).

pCite(NS4ABAN) contains the HCV sequence from nucle-otide 5367 to nucleotide 6225 (amino acid residues 1679 to

1964).

pCite(NS3AC)

is a 3-deletion product of

pCite(NS3) (42)

thatcontainsHCV cDNA from nucleotide3351 to nucleotide

4043 (aminoacid residues 1007to 1237).

Preparation of labelled extracts. HeLa cells seeded at a

density of 4 x 105 cells per 6-cm plate were infected with

vaccinia virus vTF7-3 at a multiplicity of 5 PFU per cell as

previouslydescribed

(42).

Afteradsorptionfor30 minat

37°C,

3 ml of Dulbecco's modified Eagle's medium

supplemented

with 10% FCS was added. Cellswere incubated foran addi-tional30 minat

37°C.

Recombinant

plasmid

DNAwas

precip-itated by the calcium phosphate method (39) and added

directly to each plate. When required, pUC19

plasmid

DNA

wasused ascarrierDNAfortransfection.At 4h

posttransfec-tion, the medium was replaced with minimum essential

me-dium

lacking

methionine

(GIBCO)

and thecellswerestarved for 1hat

37°C.

Cellswerethen radiolabelled for 3 hwith 400

jLCi of Tran35S-label

(ICN)

in 2 ml of minimum essential medium

lacking

methionine and

supplemented

with 2%

dia-lyzed FCS. Cells were harvested and

resuspended

in IPB150

(20 mM Tris-HCl

[pH 8],

150 mM

NaCl,

1%

Triton)

supple-mented with 1 mM

phenylmethylsulfonyl

fluoride,

1 mM

EDTA, and 1 mMdithiothreitol.

Immunoprecipitations

were

conducted on denatured extracts with HCV

region-specific

rabbit

polyclonal

antisera as described

previously

(37, 42).

Analytical gel

electrophoresis

of the

immunoprecipitated

products was

performed

as described

previously (37).

When

necessary,

quantitation

of radioactive

protein

bandswas

car-riedoutbydensitometric

scanning

of the

autoradiograms

with the program Image

(version 1.22)

on a Macintosh LC475

personal computer equippedwith a

Sony

CCD videocamera.

RESULTS

Expression plasmids and method of

analysis.

polyprotein processing

indicatedthat NS3isa

serine proteaseinvolved in the maturation of HCV

nonstruc-tural

proteins (3,

22,

42). Although

it has been demonstrated that NS3 is

absolutely

required

for the

cleavage

at the

NS3-NS4A, NS4A-NS4B, NS4B-NSSA, and NS5A-NS5B

sites,

no

evidence that the same

polypeptide

is per se sufficient for

cleavage,

either in vitroor in

vivo,

has been

provided.

Inorderto addressthismatter,weconstructedthe series of

expression

plasmids

describedin

Fig.

1.

Appropriate fragments

of the HCV genome were cloned downstream of the T7

bacteriophage

promoterin the

pCite-1

vector.This

expression

vector contains the

encephalomyocarditis

virus internal

ribo-some entry site

(27),

which promotes efficient and

CAP-independent

translation of mRNA transcribed from the T7 promoter (16). In orderto obtain transient

expression

ofthe various portions of the HCV

polyprotein,

HeLa cells were

infected with vTF7-3, a recombinant vaccinia virus that sup-ports the

synthesis

of

bacteriophage

T7

polymerase

in the

cytoplasmof infected cells

(20),

andwerethentransfectedwith

the

appropriate plasmids.

Cell

proteins

were

metabolically

labelled with

[35S]methionine

as described in Materials and Methods. Viral

proteins

and

processing products

were then identified

by

immunoprecipitation

of denatured whole-cell

extracts with

region-specific polyclonal

antibodies to HCV

NS3, NS4, and NSSA (37,

42)

and on the basis of their

molecularweights.Itmustbe

pointed

out,

however,

that in the absence of N-terminal sequence

analysis

of each

processing

productwe cannot ruleoutthatnew

cleavage

siteshave been

generated

inthe

genetically

altered NS3 proteasesubstrates. NS4A isrequiredfor theNS3-NS4Acleavage.NS3generates the N terminus ofNS4A

by cleaving

itselfoff the

polyprotein

viaanintermolecularreaction

(in cis) (42).

We

initially

wanted

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5' c

pCite(NS3) pCite(NS3AC) pCite(NS3/4AAC) pCite(NS3/4A)

pCite(NS3-AP) pCite(NS4A) pCite(NS4AB) pCite(NS4ABAN)

pCite(NS4B/5A) pCite(NS5AB)

pCite(NS3/4A:Scat-A)

E1 E2 NS2

190 383 810 1027

NS4

16581712 1973

Icat.A

FIG. 1. Schematic representation of the recombinant expression plasmids used in this study. The organization of viral polyprotein isshownat thetop.The vertical bars indicate theboundaries between the different proteins. The numbersatthetopof each bar indicate the position of the N-terminalamino acid of the following protein within the viral polyprotein.The HCV polyprotein portions expressed by the differentconstructs

areshownbelow; thenamesgiventothe corresponding plasmidsareshownonthe left. The rectangle marked AP indicatesaportion of placental

alkalinephosphatase.

tostudy whatviralstructuresarerequired for this intramolec-ularcleavagetooccur.

As a first step towards this goal, we analyzed the protein products of pCite(NS3/4A), a plasmid predicted to drive the expression of a polypeptide spanning the active protease

domain ofNS3 and endingatthe C terminus of NS4A (Fig. 1). As shown in Fig. 2A, transient expression of this plasmid in HeLa cellsyielded proteinsof70 kDa(lane 1) and 6 kDa (lane 2) that immunoprecipitated with NS3 and NS4 anti-sera,respectively. The NS3 protein generated by thisconstruct

migrates anomalously in comparison with wild-type NS3 be-cause it contains ashort stretchofunprocessed NS2at itsN terminus (42). The result of the experiment just described indicates that the NS3-dependent cleavage between NS3 and NS4A occurs efficiently in the absence of any other viral proteins.

We thenwished to determine what the minimalportionof NS4Arequiredforthisciscleavagetooccuris. To this end,we constructedpCite(NS3/4AAC),inwhich atermination codon wasinserted after the 23 N-terminalaminoacids of NS4A(Fig. 1). This latter plasmid yielded a protein, recognized by the anti-NS3antisera, thatcorresponds inmolecular weightto an uncleavedprecursor(datanotshown). However,theverysmall difference between the molecularweightof this latterprotein

precursor and that of mature NS3 made it very difficult to

establish whetherproteolytic cleavagewascompletelyoronly partially abolished. In order to resolve the possible cleavage products from theunprocessed precursor on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we decided toappendaheterologoussequence tothe N-terminal 23 residues ofNS4A. A cDNAfragment encodinga soluble form ofaplacentalalkaline phosphatase (5)wasthus fused in frame at the 3' end ofthe coding region of the pCite(NS3/ 4AAC) construct (Fig. 1). Transfection with the resulting plasmid, pCite(NS3-AP), resulted in the accumulation of a 130-kDa uncleaved proteinprecursor that wasrecognized by the anti-NS3 antiserum (Fig. 2B,lane 1).

NS3/4A

A

MW

(kDA) ct3A4

90_ ..

6 9- S NS3 45

-3

0-14

-NS4A

114~~~~+

A+/-K

N53-AP NS4A

B

;DA} a3 a4 a3

(kDA)

a_-

NS3-AP

90-69- d NS3

45-

30-

14-NS4A

[image:3.612.139.493.76.286.2]

1 2 1 2 3

FIG. 2. Processingin cisatthe NS3-NS4Ajunction.Vaccinia virus vT7F-3-infected HeLa cellsweretransfectedasdescribed in Materials and Methods. Cellswere labelled with [5S]methioninefor 3h, and SDS-denatured lysates were immunoprecipitated with antisera to specific regions of HCV polyprotein. The positions of the relevant HCV proteins immunoprecipitated with anti-HCV antibodies are

indicated. a3 and a4 indicate antiseraagainstNS3 andNS4, respec-tively. MW,molecularweightstandards.(A) Lysatesfrom cells trans-fected withpCite(NS3/4A) wereimmunoprecipitated with anti-NS3 (lane 1)and anti-NS4(lane 2).Theimmunoprecipitated productswere

resolvedon anSDS-14% PAGE. (B) Lysates from cells transfected withpCite(NS3-AP)alone(lane 1)orcotransfectedwithpCite(NS4A) (lanes2and3)wereimmunoprecipitatedwith anti-NS3(lanes1 and3) and anti-NS4 (lane 2). The immunoprecipitated products were

re-solvedon anSDS-14% PAGE. NS5

2420

XI& XI& m m

il...'.. 3'

A B A B

NS3

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A

2 \SS,:;S V NS4ABAN

NS4ABAN _ +/- E

NS3 NS4A

MW ax4 (kDA)

9 0

-a3 a4

69- NS3

-aY3 a4 MW a5A

(kDA)

9g0-NS3

B

VS4BI5A NS4B/5A

NtS4B/5A NS3 NS4A

aS3nSA

69 % N53- _ N53

-45- NS4B/5A NS4B/5A NS5A

NS5AB - +1

-NS5AB NS3 NS4A

a3a5A cx3 a aSA

-NS5AB

6 9- NS3 _ NS3

-- NS5A

45

-30- 30

-30- _ NS4ABAN - NS4ABAN

- NS4B

14 -1 4

-NS4A

2 3

1 4

-NS4A

1 2 3 4 5 6

NS4A

2 3

4 5

4 5 6

FIG. 3. Processing of the HCV polyprotein substrate intrans.(A)PlasmidpCite(NS4ABAN)wastransfected alone (lane 1), with pCite(NS3)

(lanes2and3),orwithboth pCite(NS3) and pCite(NS4A) (lanes 4 and 5). Cell lysateswereimmunoprecipitated with anti-NS3 (lanes 2 and 4)

andanti-NS4 (lanes 1, 3,and5). (B) PlasmidpCite(NS4B/5A)wastransfected alone (lane 1), with pCite(NS3) (lanes 2 and 3), orwithboth

pCite(NS3)and pCite(NS4A) (lanes 4, 5, and 6). Cell lysateswere immunoprecipitated with anti-NS3 (lanes 2 and 4), anti-NS4 (lane 5), and

anti-NS5A(lanes1and3). (C) Plasmid pCite(NS5AB)wastransfected alone (lane 1), with pCite(NS3) (lanes 2 and 3),orwithboth pCite(NS3)

andpCite(NS4A) (lanes 4, 5,and6).Cell lysateswereimmunoprecipitated with anti-NS3 (lanes 2 and 4), anti-NS4 (lane 5), and anti-NS5A (lanes

1, 3, and 6). c3, c4, and ox5A indicate antisera against NS3, NS4, and NS5A, respectively. The positions of the relevant HCV proteins

immunoprecipitated with anti-HCV antibodiesareindicated. MW,molecular weight standards.

The data described thus far suggest that an intact NS4A protein might benecessary for theNS3 protease to cleave at theNS3-NS4A site. There areessentially two possible expla-nations for this observation: either the integrity of NS4A is required to maintain the appropriate conformation at the NS3-NS4Ajunction,or NS4A acts as a cofactor forthe NS3 protease activity. Whereas it would be difficult to prove the formerhypothesis,wereasoned that ifthe latterwerethecase,

then wemight be able to observe cleavage at the NS3-NS4A boundary of the NS3/alkalinephosphatase chimeraproviding intact NS4Aintrans. We thereforetransfected, together with thepCite(NS3-AP) construct,aplasmiddrivingtheexpression

of intact NS4A [pCite(NS4A), Fig. 1]. The cotransfection

resulted in the production of the expected 6-kDa NS4A protein(Fig. 2B, lane2). More noticeably, in addition tosome

uncleaved precursor, a protein corresponding in size to the

expectedcleavage product (70 kDa;seeabove)wasspecifically recognizedby the anti-NS3 antisera (Fig. 2B, lane 3). These

results indicate that NS3 and NS4A are both required for

cleavage at the NS3-NS4Ajunctionand thatNS4A iscapable ofactingintrans to effectcleavage at this site.

NS4A is alsoan effector ofcleavage by NS3 at the

NS4B-NS5A and NS5A-NS5B junctions. In order to establish

whether NS4A is involved as a cofactor of NS3 protease

activity on the remaining cleavage sites, we constructed pCite(NS4ABAN), pCite(NS4B/5A), andpCite(NS5AB) (Fig. 1). Eachplasmid wasthen transfected aloneor cotransfected withpCite(NS3) aloneorwith thecombination ofpCite(NS3) andpCite(NS4A).

Transfection withplasmid pCite(NS4ABAN) results in the

synthesis of a protein of 30 kDa that is recognized by the

anti-NS4 antiserum(Fig. 3A,lane 1).Thisproteincontainsthe

C-terminal 33 residues of NS4Aand most of NS4B (Fig. 1).

Coexpression of the NS3 protein (Fig. 3A, lanes 2 and 3) resulted in thepartialproteolytic conversion of sucha30-kDa protein intoasmallerproductshowing electrophoretic mobil-ity similar to thatof theauthentic NS4B(26kDa[42]). When

NS4Awascoexpressedincombinationwith NS3(Fig. 3A,lane 4),wecouldobserveareproducible increasein theefficiencyof precursorprocessing (from -65 to -90%),in additiontothe 6-kDa protein encoded by the cotransfected pCite(NS4A) plasmid (Fig. 3A, lane5).

When we transfected plasmid pCite(NS4B/5A), the ex-pected 68-kDa protein productwas detected by immunopre-cipitation with anti-NS5A antiserum (Fig. 3B, lane 1). This protein containsmostof the NS4B and NS5Asequences(Fig. 1). Interestingly, whenNS3wascoexpressedwith thisprotein (Fig. 3B,lane 2),the same 68-kDa productwas immunopre-cipitated by the anti-NS5A antiserum and we found no evi-denceofproteolytic cleavage (Fig. 3B,lane3).However,when NS3wasexpressedincombination with NS4A (Fig. 3B,lanes 4 and5),wecouldobserve theoccurrence ofpartialcleavage ofthe 68-kDaproteinandtheproductionofa46-kDaprotein product that was recognized by the anti-NS5A antisera (Fig. 3B, lane6). It should bepointedouthere thatourpolyclonal anti-NS4 antiserumrecognizesNS4A farmoreefficientlythan NS4B. In particular, the portion of NS4B encoded by the pCite(NS4B/5A) construct appears not tobe immunoprecipi-tated, either when fused to NS5A or when cleaved off the precursor (Fig.3B,lane5;seealsoFig. 4,lane4, andFig. 5A, lanes 2 and5).

Last, we analyzed the activity of the NS3 protease on an HCVpolyprotein portion containingthecleavagesite between NS5A and NS5B. Plasmid pCite(NS5AB) supported the ex-pressionofa 115-kDaproteinthatwasspecifically recognized bythe anti-NS5A antiserum (Fig. 3C, lane1). WhenNS3was

45-Y3ut4a5A MW a5A (kDA WMNS5AB

- NS5A

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coexpressed

with this latter

protein,

which contains the NS5A-NS5B

junction (Fig. 3C,

lane

2),

we observed partial

proteo-lytic cleavage and production ofa polypeptide with the

elec-trophoretic mobility and immunological properties expected

for the mature NS5A (Fig. 3C, lane 3).This finding indicates

that the cleavage between NS5A and NS5B can occur in the

absence ofNS4A.However,when NS4Awascoexpressedwith NS3 andthe NS5A-NS5B precursor (Fig. 3C, lanes4 and 5), the

cleavage efficiency

increasedsubstantially, sincean NS5A-NS5B precursorwas no longerobservable (Fig. 3C,lane 6).

NS4Aretains itsactivityintranswhen uncleavedfrom NS3. In a

catalytic

serine of NS3 within thefull-length HCVpolyprotein

abrogated processingat the NS3-NS4A, NS4A-NS4B,

NS4B-NS5A,and NS5A-NS5B sites(42). Wild-typeNS3 providedin

trans could

effectively

restore cleavage at the NS4A-NS4B,

NS4B-NS5A, and NS5A-NS5B junctions but was unable to

cleave between NS3 and NS4A. This

finding

suggestedthat this lattercleavagecanonlyoccurincis,i.e., intramolecularly.Asa consequence, mature NS4A was not detectable by

immuno-precipitationinourtrans-complementation experiment.These

results are inapparent contradictionwith the data presented here: if the NS3proteaseneeds tobe activatedby NS4A, how could

processing

atseveral sitesberestoredby complementing

the mutated polyproteinwith NS3 alone and notwith NS4A?

A possible explanation of this paradox is that NS4A is still

capable

of

acting

asan effector of the NS3 protease in trans,

even when uncleavedfrom the C terminus ofanonfunctional NS3

protein.

To test this hypothesis, we carried out the

experiments

described below.

We inserted a mutation in plasmid

pCite(NS3/4A)

that resulted in thereplacementofthecatalyticserine of NS3 with

an alanine. The mutated construct is called pCite(NS3/4A: S1

65-A)

(Fig. 1).

When transfected in vTF7-3-infectedHeLa

cells,

this

plasmid

drives the expressionofa 75-kDa

polypep-tide

containing

both themutated NS3andNS4A(Fig. 4,lanes 3 and

4).

Wecotransfected

pCite(NS3/4A:Sl

165-A) in combi-nation with pCite(NS4B/5A) and with pCite(NS3AC). The latterplasmid

(Fig.

1)encodesaC-terminal deletionmutantof NS3 that retains full proteolytic activity (16a). The results of the

experiments

are shownin

Fig.

4.

Anti-NS3 antiserumimmunoprecipitatedtwomajor species,

of75 kDaand 29 kDa

(Fig.

4, lane3), corresponding

respec-tively

to the uncleaved NS3-NS4A precursor bearing the mutation in the

catalytic

serine of NS3 and to the active C-terminal deletion mutant of NS3. As expected, only the former

protein

was

recognized

bytheanti-NS4 antiserum(Fig.

4, lane 4). Most interestingly, immunoprecipitation with

anti-NS5A antiserum revealed that the NS4B-NS5A protein had

undergone proteolytic cleavage (Fig.

4, lane 5) nearly as

efficiently

asin the

experiment

shownin

Fig.

3B,lane 6. These results suggest that NS4Adoesnot needtobe cleaved offthe NS3

protein

in order to act as an effector of other NS3 moleculesintrans.

The C-terminal portion ofNS4A is sufficient to act as an effector ofNS3

proteolytic activity.

The results described thus far indicate that NS4A is

indispensable

for NS3cleavageatthe NS3-NS4A and NS4B-NS5A sites. In addition, NS4A

aug-ments

significantly

the rate of

proteolysis

at the NS5A-NS5B

cleavage

site.

Conversely,

the rate ofcleavage of the protein

substrate encoded by the

pCite(NS4ABAN)

plasmid, which contains the NS4A-NS4B

junction

but onlythe 33C-terminal residuesofNS4A,seems tobe littleaffectedbythe presence of NS4A. There are two possible interpretations of this

experi-mental evidence: either the rate at which the NS4A-NS4B

cleavage

site ofthe HCV

polyprotein

isprocessed byNS3 does

NS4B/5A NS4B/5A +

NS3AC ~ ;

NS3iC NS34A/Scat EA

MW (kDA)

9 0

-6 9-45

-a3 cxSA4 a3a4a54

_NS4B/5A NS34A/Scat -A _ NBSA

-w NS5A

3 0

-

14-1 2 3 4 5

FIG. 4. NS4Aretains itsactivitywhen uncleavedfrom NS3. Plas-midpCite(NS4B/5A)wastransfected withpCite(NS3AC)(lanes1 and 2)orwith bothpCite(NS3AC)and

pCite(NS3/4A:S,

65-A) (lanes3 to 5).Cell lysateswere immunoprecipitatedwith anti-NS3 (lanes 1 and 3),anti-NS4(lane 4),and anti NS5A(lanes2 and5). 03,a4, and a.5A indicate antisera against NS3, NS4, and NS5A, respectively. The positions of the relevant HCV proteins immunoprecipitated with anti-HCV antibodiesare indicated. MW, molecularweightstandards.

not depend on the presence of NS4A, or the C-terminal portion of NS4A that is contained in the protein encoded by thepCite(NS4ABAN) plasmidissufficientto act as aneffector ofthe NS3 proteolytic activity. In orderto address this issue, we performed the experiments described below.

As described earlier, pCite(NS4B/5A) codes for a protein precursorwhosecleavageisstrictly dependentonthepresence of the NS4Aprotein. We reasoned that if the C-terminal 33 amino acids ofNS4A were indeedsufficient to modulate the proteolytic activity of NS3, then the coexpression of the polypeptide encoded by the pCite(NS4ABAN) plasmid with NS3 should resultinthecleavageofthe NS4B-NS5Ajunction.

Thepropertiesof theproteinencodedby pCite(NS4ABAN)as

an effector of NS3 were thus compared with those of the

polypeptide encoded by plasmid pCite(NS4AB). This latter

plasmid contains, inaddition to the NS4Bregioncommonto

bothconstructs, the entire NS4Acoding region (Fig. 1). The results of an experiment in which pCite(NS4B/5A) was co-transfected with both pCite(NS3) and pCite(NS4AB) are shown inFig.SA, lanes 1 to3.Whenthe NS3protein (Fig.5A,

lane 1) is expressed in combination with the polypeptide

encodedby pCite(NS4AB), the latterwas partiallycleaved to

yield the two expected fragments of 26 and 6 kDa (Fig. SA,

lane2).Moreover,theNS4B-NS5Aproteinprecursor wasalso

efficiently processed (Fig. SA, lane3). Lanes4 to 6ofFig. 5A

show the results of similar cotransfection experiments, but in which pCite(NS4AB)wasreplaced bypCite(NS4ABzAN). The

expressionof the NS3 protease (Fig. 5A, lane4) in

combina-tion with theproductsofthepCite(NS4ABAN)construct(Fig. 5A,lane5;seealsoFig.3A,lane3)resulted in thecleavageof

thepolypeptide containingthe NS4B-NS5Ajunction (Fig. 5A,

lane 6). This last result suggests that a domain of NS4A contained within the 33 most C-terminal amino acids of the

protein may besufficient to modulate the activity of the NS3

protease.

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[image:5.612.356.533.78.288.2]

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A NS4B/5A NS4B/5A

-+_NS3i03NS\A_NS4AB _NS3S3+ _NS4ABNNS4BN

MW a3a4mLSA

(kDA)

90

-6 9

-

45-30

-NS3 _ NS4B/5A

_ NS5A

- NS4AB NS4B

a5A

NS3 _NS4B/5A

-NSSA

-14

-NS4A

1 2 3

B

Kite-Doolittle

Hydropathv plot

1660 1670 1680

4 5 6 1690 1700 1710

Hydrophobic

.:0

- , Iydrophilic

[image:6.612.72.276.67.393.2]

--3

FIG. 5. The C-terminal domain of NS4A is sufficient to activate NS3. (A) PlasmidpCite(NS4B/5A) was transfected withpCite(NS3)

and with eitherpCite(NS4AB) (lanesIto3)orwithpCite(NS4ABAN) (lanes 4 to 6). Cell lysateswere immunoprecipitated with anti-NS3 (lanes1and4),anti-NS4(lanes2 and5),and anti-NS5A(lanes3 and

6). c3, a4,and otSA indicate antiseraagainst NS3, NS4, and NS5A, respectively.Thepositionsof the relevant HCVproteins immunopre-cipitated with anti-HCV antibodies are indicated. MW, molecular

weight standards. (B) Hydropathy plotof NS4A. Theaverage hydro-phobicityindexwasobtained with thealgorithmofKyteand Doolittle withasearchlength of 8 amino acids (32).

DISCUSSION

The evidence presented here shows that, in our transient expression system,theproteasedomain of HCV NS3requires the participation of an additional virus-encoded protein, NS4A, in order to efficiently cleave the substrates contained withintheHCV polyprotein.NS4Acanactas aneffector of the NS3 proteolytic activity when supplied in trans, and further-more a 33-amino-acid domain located at the C terminus of NS4A seemstobe sufficient tocomplement the NS3 activity. Therequirement foratwo-component proteasethusseemsto

be commonto all members of the Flaviviridae(see below). In flaviviruses, NS2B is required to facilitate efficient pro-teolytic processing of the nonstructural region of the viral polyprotein by NS3 (1, 6, 18, 33, 38). In contrast to HCV NS4A,flavivirus NS2B is locatedatthe N terminus of NS3. In spite of theirdifferent topological arrangements, HCV NS4A and flavivirus NS2B share several features: both proteins are required aseffectors ofproteaseactivity,canfunction in cisor in trans, and are liberated from their respective catalytic domainsbyanintramolecularcleavage. A 40-amino-acid

seg-mentof NS2Bconservedamongallflaviviruses has been shown

tobesufficienttotrans-complement the NS3proteolytic activ-ity in dengue (17) andyellow fever (8) viruses. Ahydropathy

plot of the NS2B protein shows that this 40-amino-acid seg-mentisahydrophilic domain surrounded bytwohydrophobic

regions (17). Similarly, the 33-amino-acid region of HCV NS4A, identified as being sufficient for the activation of the NS3 protease, spansa hydrophilic, C-terminal domain and is

preceded byaveryhydrophobicN-terminalportion (Fig. 5B).

Thegeneticorganizationand theprocessingofthe nonstruc-tural region of the polyprotein of BVDV, a member of the

pestivirus genus, parallels closely that of the corresponding

region of HCV: thep80serine protease, which is the functional homolog of NS3, has been shown by Wiskerchen and Collett

(45) to be required for processing of the nonstructural

pro-teins. Cleavage bythis proteaseactivityattheNandC termini ofp80 itselfoccurs intramolecularly (in cis). Cleavage at the remaining nonstructural sites can occur in trans. The region

correspondingtoNS5 of flavivirus isprocessed,asinHCV,to

two distinct polypeptides, p58 and p75. The segment of the

polyprotein following p80, equivalenttoNS4,generatesasmall

N-terminal hydrophobic protein,

piO,

and possibly a 32-kDa protein that has not yet been characterized in detail. It is

currently thought that p58 and p75 are the functional

ho-mologs of HCV NS5A and NS5B, whereas plO and the

uncharacterized 32-kDa factorcorrespondtoHCV NS4A and

NS4B polypeptides, respectively. Interestingly, p80 protease

activityalonewasshowntobeinsufficient for thecleavageofa

substratecontainingthep58-p75junction. Conversely, expres-sion of a construct encoding, in addition to the entire p80

protein, plO and part of the 32-kDa protein, led to efficient

cleavage of the same site. In view of these data and of the

evidencepresented here,we suggestthat BVDVplOis

neces-sary to complement the p80 serine protease activity and

therefore is the functional homologof HCV NS4A. plO had

previouslybeendisregardedas aBVDVprotease cofactor(13,

45). This conclusion was based on the observation that

pro-cessingofapolyproteinthat hadbeen mutated in thecatalytic

serine ofp80wasrescuedatseveralsitesby

trans-complemen-tation withaprotein containinganintact protease domain.In

thisexperiment,plOwas notgenerated,but the

corresponding

sequence remained uncleaved from themutantp80

(45);

this

finding indicated that thep80-plO cleavage can onlyoccur in

cisandwastakenasanargument toruleoutthe involvement

ofplOasapossible p80cofactorrequiredforcleavage atthe

remainingsites.However, it hasemergedfrom theexperiment

described inFig.4thatHCV NS4Acan complementtheNS3 protease activity in trans even when uncleaved from an NS3 molecule that has been mutated in the catalytic serine. It is therefore alsolikely, by analogy,thatBVDVplOdoesnotneed tobe released fromamutatedp80inorderto actintrans as an

effector of functional protease molecules.

The mechanism by which HCV NS4A activates the NS3-contained serine protease is not yet clear. The two proteins

could associate inacomplex toform an activeheterodimeric protease.Indeed,astablecomplexbetween NS3anda

protein

containingNS4Aandpartof NS4B hasbeen observedin vitro

(25). A similar complex has been observed in flaviviruses

between NS3 and NS2B (1, 8). A related possibility is that NS4A is required to target NS3 to the correct cellular

com-partment for maximal protease activity. Alternatively, NS4A could act as a virus-encoded chaperonin aiding the correct

folding of the protease domain

(14).

In this regard, it is of

interestthat severalbacterial serine proteases,including

ax-lytic

protease andsubtilisin,possesspropeptidesthat share several

properties with HCV NS4A and flavivirus NS2B: they are

necessary for proteaseactivity,canactivate the proteaseeither

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in cis orin trans, and are cleaved off the protease domain by intramolecular proteolytic reactions (26, 40, 47). These propeptidesare requiredtoassist the folding of theirprotease counterpart,and whenfolding has been completed the propep-tide becomes dispensable and no longer required for

proteo-lytic activity (2, 47). Alternatively, NS4A may act as a chap-eroninthat facilitates theinteraction of NS3 with the substrate, forexample by locally unfolding the polyprotein precursor, in order tomake the cleavage site accessible to theprotease.

In other viral systems, examples in which dimerization is required for protease activity (30) or in which the protease

activity is modulated by interaction with additional polypep-tides exist. In cowpea mosaic virus, the specificity of the 24-kDa protease for trans cleavage of the capsid precursor is altered bythe virus-encoded 32-kDaprotein (43). The3Cand 3CD proteases encoded by poliovirus differ incleavage speci-ficity forcapsid precursor, the specificity being determined by the interaction of the 3D region with the substrate (28, 46). Moreover, the adenovirus cysteine protease has been recently shown tobe activated via the formation of a disulfide bridge with an11-amino-acid peptide derived from the viral protein pVI (44).

Further studieson the structures and the functions of both HCV NS3 and NS4A, aswell asof their interactions with the other components of the viral polyprotein, are required to

solve the issue of how the virus-encoded serine protease is

activated by a second viral polypeptide. These studies will

eventuallyshed lightonthe temporal and spatialregulation of

HCV gene expression.

ACKNOWLEDGMENTS

WethankR. Cortese,J.Jiricni, andN. LaMonica forcritical review, and we thank all IRBM coworkers for helpful discussion. We also thank P. Neuner for oligonucleotide synthesis and Y. Cully for graphics.

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