JOURNAL OF VIROLOGY, May 1991, p. 2467-24750022-538X/91/052467-09$02.00/0Copyright C) 1991, American Society for Microbiology

Both Nonstructural Proteins NS2B and NS3 Are Required for theProteolytic Processing of Dengue Virus Nonstructural Proteins

BARRY FALGOUT, MICHtLE PETHEL, YI-MING ZHANG,t AND CHING-JUH LAI*Molecular Viral Biology Section, Laboratory ofInfectious Diseases, National Institute ofAllergy and

Infectious Diseases, Bethesda, Maryland 20892

Received 9 August 1990/Accepted 4 February 1991

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3,NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a

virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NSregion of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates ofrecombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. Apolyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at theNS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acidsof NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2Bwere not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2Bcomplemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus,NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experimentsshowed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavagesin NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a

two-component protease activity requiring both NS2B and NS3.

The four serotypes of dengue virus (DEN) constitute asubgroup of the Flaviviridae, a family of about 70 serologi-cally related viruses (15, 55). Many members of this familyare the etiologic agents of a variety of human diseases, suchas dengue fever, yellow fever, Japanese encephalitis, andtick-borne encephalitis. Most flaviviruses, including themedically important ones, are transmitted to humans bymosquito or tick bite.Like other flaviviruses, DEN is composed of the envelope

(E) and membrane (M) proteins, embedded in a host-derivedmembrane, and the capsid (C) protein, which is complexedwith the single-stranded genomic RNA of approximately 11kb (35). The genomic RNA is of the positive sense and iscapped but not polyadenylated (12, 54). Subgenomic RNAwas not detected in flavivirus-infected cells (5, 36). Thecomplete sequences of several flavivirus genomes have beendetermined (6, 7, 13, 14, 16, 17, 24, 25, 27, 29, 30-32, 38-40,43, 50, 53, 57). Analysis of these sequences revealed that theflavivirus genome contains one open reading frame spanningmore than 10 kb. Individual flavivirus proteins are evidentlyexpressed by translation of the genomic RNA into a poly-protein followed by proteolytic cleavages. For the prototypeyellow fever virus, the order of the flavivirus polyproteinwas established to be NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH, where prM is the precursorto M and NS are nonstructural proteins (2, 43, 44). Severalcleavage sites have been reassigned as the N-terminal aminoacid sequences for almost all Kunjin virus proteins havebecome available (13, 47, 49). The N-terminal amino acidsequence data obtained for other flavivirus systems are inagreement with the cleavage sites predicted by sequence

* Corresponding author.t Present address: Laboratory of Molecular Retrovirology,

Georgetown University, Washington, DC 20007.

homology to Kunjin virus (3, 4, 6, 7, 9, 10, 22, 29, 34, 38, 50,53, 56), except for two reports of an N-terminally shortenedform of C (7, 38). The C-terminal amino acid sequences ofmost of the flavivirus proteins have also been determined(37, 48, 56). These results show that for all proteins exceptC, the C terminus exactly abuts the N terminus of thedownstream protein, such that no amino acids are excisedduring proteolytic processing.

Analysis of the amino acid sequences near the cleavagesites of the flavivirus polyprotein suggests that there areseveral distinct classes of cleavage mechanisms. The cleav-ages at the C/prM, prM/E, E/NS1, and NS4A/NS4B junc-tions are thought to be mediated by signalase (43, 47).Support for this for all but the NS4A/NS4B cleavage hasbeen obtained from in vitro translation and processing stud-ies (33, 37, 45, 51). The protease that cleaves the NS1/NS2Ajunction has not been identified, although it has been shownthat the 8 C-terminal amino acids of NS1 and the N-terminal70% of NS2A are required for this cleavage (19, 20, 26). TheprM/M cleavage apparently represents another processingmechanism, in which cleavage occurs in an acidified vesic-ular compartment at a late step during virion maturation (42).The remaining cleavages at the NS2A/NS2B, NS2B/NS3,NS3/NS4A, and NS4B/NS5 junctions share a short sequencemotif near the cleavage sites (43, 47). These cleavages followa pair of basic amino acids (RR or KR or RK), or exception-ally QR at the NS2B/NS3 junction in DEN, and precedeeither G, S, or A. The cleavage site for processing the Cterminus of anchored C also shares this motif (37, 48). It hasbeen speculated that this class of processing events occurs inthe cytoplasm and is mediated by a virus-encoded proteinase(43, 47). Recently, two laboratories have proposed thatflavivirus NS3 is a protease on the basis of a limitedsequence homology to serine proteases (1, 21). Four sepa-rate regions of homology were identified within the N-termi-nal 180 amino acids ofNS3; three encompassed the catalytic

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2468 FALGOUT ET AL.

triad D, H, S, and the fourth was designated as a substrate-binding domain.We have initiated studies to analyze the DEN type 4

(DEN4) polyprotein cleavages and to identify viral functionsthat are required for these proteolytic processing events.Our approach has been in vivo expression ofDNA fragmentsthat code for regions of the polyprotein of interest, usingvaccinia virus as a vector. We previously used this approachto show that NS2A is required for NS1INS2A cleavage (19).In this report, we describe evidence that NS2B and theproposed protease domain of NS3 are both required for theNS2A/NS2B and NS2B/NS3 cleavages. We show that NS2Bis capable of acting in trans to effect cleavage at these sites.Indirect evidence is also provided that NS3 can act in trans.In addition, we demonstrate that NS2B is necessary forNS3/NS4A cleavage, as well as for several apparently spe-cific cleavages within NS3.

MATERIALS AND METHODS

Cells and viruses. CV-1 cells and TK-141 cells were grownas monolayers in Eagle minimal essential medium supple-mented with 10% fetal bovine serum. The vaccinia virusrecombinant vSC8 has been described previously (8).Recombinant vaccinia viruses were constructed from vac-cinia virus WR and plasmid pSC11 derivatives containingDEN4 cDNA sequences to be expressed as described pre-viously (8, 19). The nomenclature of these vaccinia virusrecombinants followed previous practice: for example, avirus derived from pSC11/NS1-NS2A was named vNS1-NS2A, where NS1-NS2A is the portion of the DEN4 poly-protein encoded in the construct.

Plasmid construction. Standard recombinant DNA tech-niques were used for the construction and propagation ofplasmids. The vaccinia virus intermediate transfer vectorpSC11, the derivatives pSC11[BglII] and pSC11/NS1-NS2A,and the sources of the DEN4 cDNA have been describedpreviously (8, 19, 57). The structures of the DEN4 cDNAsegments cloned into pSC11, encoding various portions ofthe DEN4 polyprotein between NS2A and NS4B, are dia-grammed in Fig. 1. The structures of these plasmids wereverified in all cases by appropriate restriction enzyme diges-tions. Oligodeoxyribonucleotides were used to introduce aninitiation codon and a termination codon for NS2A, NS2B,or NS3 and a termination codon at the EcoRV site (nucleo-tides [nt] 4748 to 4753) or at the BstBI site (nt 5068-5073),creating NS3 C-terminally truncated after 77 amino acids(12%) or 184 amino acids (30%), respectively. Dideoxynu-cleotide sequencing (46) was used to verify the sequences inthe region of the oligodeoxyribonucleotides. More detailsabout the nucleotide and amino acid sequences at the terminiof these clones are given in Table 1. DEN4 nucleotide andamino acid numbers are from Mackow et al. (30) and Zhao etal. (57).The two internal in-frame deletions of NS2B were created

by polymerase chain reaction. These deletions, ANS2B(10)and ANS2B(20), encode only the N-terminal Ser of NS2Bfused to the last 10 or last 20 residues of NS2B, respectively.The polymerase chain reaction products (about 600 bp) were

subcloned, and the sequence of the entire amplified region(approximately nt 4460 to 5080) was determined for bothdeletion mutants and for a wild-type clone that had not beenamplified by polymerase chain reaction. The deletion mu-tants and the wild type were found to differ from thepublished sequence in this region at four locations: all hadsilent base changes at nt 4721 (G to A) and nt 4925 (A to T)

NS2A NS2B NS3 NS4A NS4B

vNS2A-NS2B

vNS2A-NS2B-1 2%NS3

vNS2A-NS2B-30%NS3

vNS2A-ANS2B(1 0)-30%NS3

vNS2A-ANS2B(20)-30%NS3

vNS2B

vNS2B-NS3

mI l

I I 1E

I I I I

E11C1)

IEI Z I

vNS2B-NS3-NS4A-84%NS4B

v30%NS3

vNS3*

vNS3

vNS3-NS4A-84%NS4B

FIG. 1. Diagrams of DEN4 proteins encoded by recombinantvaccinia viruses. The diagram on the top depicts the linear map ofDEN4 nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B(N-terminal to C-terminal) in the form of a polyprotein. The cleav-age site between neighboring proteins is shown by a vertical line.The shaded area at the NS4A/NS4B junction represents a sequenceof 19 hydrophobic amino acids which is thought to direct signalase-mediated cleavage between NS4A and NS4B (43, 47). The otherdiagrams depict the DEN4 proteins encoded by the vaccinia virusrecombinants listed at the left (see also Table 1). The two deletionsin NS2B are indicated by lines connecting discontinuous regions ofthe polyprotein. The NS3* protein has a single-base deletion result-ing in a frameshift after approximately 90% of NS3, resulting in a17-amino-acid C-terminal tail translated from the new frame, repre-sented by the short horizontal line.

and nonsilent changes at nt 4827 (A to G; resulting in Arg toGly) and nt 4860 (A to G; Ser to Gly). In addition,ANS2B(20) had a base change at nt 5050 (A to T; Glu to Val),which seems to have been introduced by polymerase chainreaction. Note that the NS3 residue changed lies outside ofsequences in NS3 homologous to serine proteases (1, 21).

Analysis of DEN-specific proteins. The procedures forinfection of CV-1 cells with recombinant vaccinia virus,preparation of [35S]methionine-labeled lysates, immunopre-cipitation with DEN4 hyperimmune mouse ascitic fluid,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and fluorography have been described previ-ously (19). 35S-labeled lysate of DEN4-infected LLC-MK2cells was prepared as described previously (18) and was thegift of Michael Bray.

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PROTEINASE ACTIVITY OF FLAVIVIRUS NS2B PLUS NS3

TABLE 1. Terminal nucleotide sequences of recombinant DNA constructs and the predictedN and C termini of expressed polyproteins

Protein Nucleotide sequencea Structure of terminusb

5' end N terminusNS2A . . . agatotccaccATGGCCGGA,3479. . . MAG1126.. -NS2B . . . agatcccatATGGGATCT4133. . . MGS1344.NS3 . . . agatccATGTCA4523. MS1474.. -

3' end C terminusNS2B . . . 4515CAAAGAtgaggatct . . . . 1472QR12%NS3 . . . 4746ATGATCtagctaactagggatct. . . . .1549mi30%NS3 . . 5067TTTCGCtagctagctagcgaa. . . . 1656FRNS3*c . .6180GGCA11T-TT. . .TTTtag6240. . . . . .2027GIFTKIGNGASQGKEITKFNS3 . . 6369AGGAAGtaaggatct . . . 209ORK84%NS4B . . . 7437AACCCGGGAATTCTGtga. . . . 2446NPGIL

a Lowercase letters are noncoding sequences, either 5' of the initiating ATG or 3' of the last codon. Capital letters are coding sequences. Underlined codingsequences are the 5'- or 3'-most codons of the relevant DEN4 protein. Stop codons are double underlined. DEN4 nucleotide numbers are those of Mackow etal. (30) and Zhao et al. (57).

b Underlined amino acids are derived from translation of vector or oligodeoxyribonucleotide sequences or out-of-frame translation ofDEN4 sequences. Aminoacid numbers are those of Mackow et al. (30) and Zhao et al. (57).

c The C terminus of NS3* results from a frameshift in the original NS3 clone. DEN4 nt 6187 is deleted, indicated by -. The resulting protein is colinear withNS3 through amino acid 2028 (555 residues, or 90%o of NS3), at which point translation in a new reading frame adds 17 residues unrelated to NS3 to the C terminusbefore a termination codon is encountered.

RESULTS

DEN4 NS proteins predicted from alignment with KuDjinNS proteins. Processing of most DEN NS proteins from thepolyprotein has been little studied. The cleavages that definethe DEN4 NS proteins have been predicted (30, 40) on thebasis of alignment of the deduced DEN4 polyprotein se-quence (30) with that of the flavivirus Kunjin (13), for whichthe N-terminal amino acid sequences of most NS proteinshave been determined (13, 47, 49). This allows prediction ofthe sizes of the DEN4 NS proteins. These data are summa-rized in Table 2.

Identification of NS2B and NS3. To study the proteolyticprocessing of DEN4 NS proteins, we expressed varioussegments of the DEN4 polyprotein in vivo from cloned DNAsequences, using vaccinia virus as a vector. The DEN4

TABLE 2. Predicted NS proteins of DEN4

Amino Molecular N-terminalProtein Nucleotidesa acidSa b cleavage site'acldsa (kDa)b cevg le

NS1 2421-3476 774-1125 39.6 FTVQA/DMGCVNS2A 3477-4130 1126-1343 24.1 SQVTA/GQGTSNS2B 4131-4520 1344-1473 14.0 GASRR/SWPLNNS3 4521-6374 1474-2091 69.5 VKTQR/SGALWNS4A 6375-6824 2092-2241 16.4 ASGRK/SITLDNS4B 6825-7560 2242-2486 26.5 GLIAA/NEMGLNS5 7561-10262 2487-3386 103 QTPRR/GTGTT

a Nucleotide numbers and amino acid numbers are from Mackow et al. (30)and Zhao et al. (57).

b Predicted molecular masses are for the protein backbone only; contribu-tions from modifications (such as glycosylation of NS1) have not been takeninto account.

c Arbitrarily, five amino acids on either side of each cleavage site have beenlisted. Cleavage occurs between the residues separated by a slash. The aminoacid following the slash is the N-terminal residue of the protein listed on thatline. The first amino acid to the left of each slash on any given line is the Cterminus of the preceding protein, which is found on the line above. TheFTVQA sequence on the NS1 line is the C terminus of E. The C terminus ofNS5 is defined by a stop codon.

cDNA sequences coding for the predicted amino acid se-quences of NS2B and NS3 were used to construct therecombinant vaccinia viruses vNS2B and vNS3 (Fig. 1;Table 1). Cells were infected with one of these recombinantsor with vSC8, a negative control vaccinia virus recombinantthat does not contain DEN4 sequences. The DEN-specificproteins expressed by these cells are shown in Fig. 2. ThevNS2B lane contained a faint doublet band with a molecularmass of about 12 kDa and a few background bands that werealso seen in the vSC8 lane. The larger member of the doubletcomigrated with a band seen in the DEN4 lane. (Note thatthe vNS2B lane is overexposed compared with the DEN4lane.) This 12-kDa band was identified as NS2B, eventhough the molecular mass of NS2B was predicted to be 14kDa (Table 2). It is noteworthy that Speight et al. (47, 48)found that Kunjin virus NS2B had an observed molecularmass of 10 kDa despite a predicted molecular mass of 14kDa. These investigators concluded that small hydrophobicproteins such as NS2B can have anomalous mobilities inSDS-PAGE. The origin of the smaller member of the doubletis not known, but it could be due to internal initiation oftranslation at the ninth amino acid of NS2B, which wouldreduce the predicted size of NS2B by 1 kDa. NS3 of DEN4was predicted to have molecular mass of 69.5 kDa (Table 2).A protein was observed in the DEN4 lane with an apparentmolecular mass of 73 kDa. The recombinant vNS3 (lane 5)expressed a protein that comigrated with this 73-kDa pro-tein, and this was assigned as NS3. Similarly, Speight et al.found that Kunjin virus NS3 migrated slightly slower thanexpected (47). Recombinant vNS3 also expressed a secondprotein slightly smaller than NS3. This smaller protein couldhave arisen from internal initiation of translation, perhaps atamino acid 26 or 42 of NS3, which would reduce the size ofNS3 by 3 or 4.5 kDa, respectively. The sequence aroundamino acid 42 is particularly favorable for translation initia-tion.To obtain further evidence that the 73-kDa band was NS3,

we analyzed the proteins produced by vNS3* and v30%oNS3.The C-terminally truncated NS3 products of these viruses

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2470 FALGOUT ET AL.

1 2 3 4 5 6 DM

E

30%NS3

-prM

18

-NS2B

FIG. 2. Expression of DEN4 NS2B and NS3. Aliquots of

[Simethionine-labeled lysates of virus-infected cells were immu-

noprecipitated with DEN4-specific hyperimmune mouse ascitic

fluid, and the precipitates were resolved by SDS-PAGE and de-

tected by fluorography. The positions of DEN4-specific protein

bands are indicated, as are the sizes (in kilodaltons) of the 14C-

labeled marker proteins in lane M In lanes 1 to 6, recombinant

vaccinia viruses were used to infect CV-1 cells 1 vSC8 (negative

control); 2, vNS2B; 3, v30%NS3; 4, vNS3*; 5, vNS3; 6, vNS2B-

NS3. Lane D contains DEN4-infected LLC-MK2 cell lysate. Lanes

1 to 3 were exposed to film for 9 days; the other lanes were exposed

for 1 day.

were expected to have molecular masses of 64 and 20 kDa,

respectively. The vNS3* lane contained two closely spaced

bands, at estimated molecular masses of 71 and 68 kDa. The

v30%NS3 lane contained faint bands at 23 and 19 kDa (note

that this lane is greatly overexposed compared with the

vNS3* and vNS3 lanes). Neither vNS3* nor v30%NS3

produced the 73-kDa band that was seen in the vNS3 lane.

Our interpretation of these data was that the larger band in

each case was the correctly initiated N53 product, while the

smaller band. was aberrant and was perhaps due to internal

initiation of translation. In all cases, the mobilities of the

full-length and truncated N53 species were slightly lower

than expected. Taken together, these results provide genetic

evidence that the 73-kDa band made by DEN4 and vNS3

was NS3.The viral protease activity lies within NS2B-NS3. Having

identified the DEN4 proteins NS2B and NS3, we wished to

characterize the viral sequences required for the cleavages

that define these proteins. In particular, we wanted to map

the location of the hypothetical viral proteinase. As a first

step toward this goal, the products of recombinant vNS2B-

Nt 3 (Fig. 1; Table 1) were analyzed. vNS2B-NS3 made

NS2B and Ni3 (Fig. 2, lane 6). This result mapped thehypothetical virally encoded proteinase responsible for the

NS2B/NS3 cleavage to the NS2B-NS3 region. Such a pro-

teinase is presumably also responsible for other cleavages

such as NS2AINS2B, N53/NS4A, and NS4BINS5, which

contain the same cleavage site motif as NS2BlNS3.In addition to NS2B and NS3, several other specific bands

were seen in the vNS2B-NS3 lane. The largest of these

bands, with an apparent molecular mass of 86 kDa, had a

mobility consistent with that expected for uncleaved NS2B-

NS3. Recombinant vNS2B-NS3* expressed a protein whichran slightly faster than this 86-kDa species, as would beexpected for an uncleaved NS2B-90%NS3 product (data notshown). This supports the identification of the 86-kDa spe-cies as uncleaved NS2B-NS3. The other specific bands,marked on the side of the figure with asterisks, were smallerthan NS3 but larger than NS2B. These products must havebeen derived from internal cleavages within NS3. A compar-ison of the products made by vNS2B-NS3 infected cells withthose made by cells coinfected with vNS2B plus vNS3indicated that the internal cleavages within NS3 are greatlyfacilitated when NS2B is in cis (not shown). Since many ofthese bands were not present in DEN4-infected cells, theymay represent adventitious cleavages within NS3 in thevaccinia virus recombinant expression system. However,the two prominent bands near 50 kDa in lane 6 comigratedwith two faint bands in the DEN4 lane, and these speciesmnay represent authentic products not previously predictedfrom the N-terminal amino acid sequence data. These 50-kDa bands were faintly visible in the vNS3 and vNS3* lanesupon long exposure, suggesting that they were composedentirely of NS3 sequences. We speculate that the internalcleavages in NS3 also occur at sites which share the se-quence motif found at the previously predicted cleavagesites. Six such sites are found in DEN4 NS3, after aminoacids 1488, 1501, 1616, 1850, 1931, and 2012. Partial cleav-ages at these sites could generate many possible products.NS3 is required for the NS2A/NS2B and NS2B/NS3 cleav-

ages. The finding that a hypothetical viral protease maps toNS2B-NS3 is consistent with the notion that NS3 is aprotease (1, 21). Since the postulated protease domainincludes approximately the N-terminal 180 amino acids ofNS3, we investigated the processing of vNS2A-NS2B-30%NS3 (Fig. 1; Table 1), which terminates translation after184 amino acids of NS3. This recombinant expressed NS2Band 30%NS3 (Fig. 3, lane 2). Note that vNS2A-NS2B-30%NS3 produced much more 30%NS3 and NS2B than thecorresponding products expressed by v30%NS3 and vNS2B;the reason for this is not known. A third band with anapparent molecular mass of 38 kDa was also seen in lane 2.This size was consistent with either uncleaved NS2A-NS2Bor uncleaved NS2B-30%NS3. However, the recombinantvNS2A-NS2B made a product with an apparent molecularmass of 32 kDa (see Fig. 5), which is presumably uncleavedNS2A-NS2B. The 38-kDa species was thus identified asuncleaved NS2B-30%NS3. Since vNS2A-NS2B-30%NS3made 30%NS3, the NS2B/NS3 cleavage occurred. Similarly,the production of NS2B by this recombinant indicated thatcleavage between NS2A and NS2B occurred. This impliedthat NS2A ought to have been made, although it was notdetected. These results are consistent with the hypothesisthat the N-terminal 180 residues of NS3 contains a proteasedomain.To determine whether the putative NS3 protease domain

is necessary for the NS2A/NS2B and NS2B/NS3 cleavages,we constructed and analyzed recombinant vNS2A-NS2B-12%NS3 (Fig. 1; Table 1). This virus contains a DEN cDNAsegment that encodes an NS3 product truncated in themiddle of the protease domain, removing two of the pro-posed regions of homology to serine proteases, including thedomain containing the proposed catalytic serine residue;therefore, vNS2A-NS2B-12%NS3 should lack NS3 proteaseactivity. vNS2A-NS2B-12%NS3 made neither NS2B nor12%NS3, but did make a protein with an apparent molecularmass of 45 kDa, which was assigned as uncleaved NS2A-NS2B-12%NS3 (Fig. 3, lane 4). Recombinant v12%NS3,

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PROTEINASE ACTIVITY OF FLAVIVIRUS NS2B PLUS NS3

1 2 M M 3 4 1 2 3 D

-0 -NS2A-NS2B-.... .... 12%NS3

NS2B- 30Ow30%NS3-

m. i. 22/1i../ 22S

30%NS3- 5

* / 3

NS2B- _

FIG. 3. The putative protease domain of NS3 is required for theNS2A/NS2B and NS2B/NS3 cleavages. Samples were treated asdescribed in the legend to Fig. 2. The positions of DEN4-specificbands are indicated, as are the sizes of the marker proteins in lanesM. In lanes 1 to 4, recombinant vaccinia viruses were used to infectCV-1 cells: 1, vSC8; 2, vNS2A-NS2B-30%oNS3; 3, vSC8; 4, vNS2A-NS2B-12%NS3. The gel on the left was 12% acrylamide; the one onthe right was 17% acrylamide.

encoding only the truncated NS3 protein, made no detect-able product (data not shown). The failure to detect 12%NS3could have been due to the lack of specific antibodies orinstability of the protein. Although the failure to detect a12%NS3 product from vNS2A-NS2B-12%NS3 was not in-formative, the finding that this virus failed to make NS2Bsuggested that both NS2AINS2B and NS2BINS3 junctionswere not cleaved. Furthermore, the uncleaved productsNS2A-NS2B and NS2B-12%NS3 were not detected, sug-gesting that no cleavage had taken place. These results areconsistent with the hypothesis that the protease domain ofNS3 is required for both the NS2A/NS2B and NS2B/NS3cleavages.NS2B is required for NS3/NS4A cleavage. Evidence that

NS2B is also required for the proteolytic activity is presentin Fig. 4, which shows analysis of the recombinants vNS2B-NS3-NS4A-84%NS4B and vNS3-NS4A-84%NS4B (Fig. 1;Table 1). Recombinant vNS2B-NS3-NS4A-84%NS4B madeboth NS2B and NS3, indicating that the NS2B/NS3 andNS3/NS4A cleavages occurred. In addition, there was aseries of other bands in this lane. Many of these bands(asterisks) were smaller than NS3, and their identities arediscussed above (Fig. 2). Three bands, designated c, d, ande, were larger than NS3. Band e was previously assigned asuncleaved NS2B-NS3. The sizes of bands c and d wereconsistent with uncleaved NS2B-NS3-NS4A-84%NS4B andNS2B-NS3-NS4A, respectively. The existence of the latterspecies would imply that NS4A/NS4B cleavage had oc-curred. In contrast, the vNS3-NS4A-84%NS4B lane con-tained neither mature-sized NS3 nor any of the other bandsseen in the vNS2B-NS3-NS4A-84%NS4B lane. These obser-vations indicate that NS2B has a role in the NS3/NS4Acleavage and suggests a role for NS2B in the NS2A/NS2B,NS2B/NS3, and NS4BINS5 cleavages, since these all sharethe same amino acid sequence motif at the cleavage site. ThevNS3-NS4A-84%NS4B lane had doublet bands at 85 kDa(labeled b) and at 105 kDa (labeled a). These sizes wereconsistent with uncleaved NS3-NS4A and NS3-NS4A-84%NS4B, respectively. This assignment would suggest that

a-b-

-c-d-e

-NS3-E

-NS1

-prM

_-NS2B

FIG. 4. NS2B is required for NS3/NS4A cleavage. Samples weretreated as described in the legend to Fig. 2. The positions of someDEN4-specific bands are indicated. The asterisks and lettered bandsindicate other specific products discussed in the text. In lanes 1 to 3,recombinant viruses were used to infect CV-1 cells: 1, vSC8; 2,vNS3-NS4A-84%NS4B; 3, vNS2B-NS3-NS4A-84%NS4B. Lane Dcontains DEN4-infected LLC-MK2 cells lysate.

NS4A/NS4B cleavage did not require NS2B, which agreeswith the proposal that this cleavage is mediated by signalpeptidase (43, 47).

Deletions of NS2B are defective for cleavage at the NS2A/NS2B and NS2B/NS3 junctions. To test whether NS2B isrequired for the NS2A/NS2B and NS2B/NS3 cleavages, weconstructed the recombinants vNS2A-ANS2B(10)-30%NS3and vNS2A-ANS2B(20)-30%NS3 (Fig. -1; Table 1). Thesetwo viruses were similar to vNS2A-NS2B-30%NS3, exceptthat each contains a large in-frame deletion in NS2B, retain-ing only the N-terminal serine residue and the C-terminal 10or 20 amino acids of NS2B. Both vNS2A-ANS2B(10)-30%NS3 (Fig. 5, lane 1) and vNS2A-ANS2B(20)-30%oNS3(Fig. 5, lanes 2, left and right) made no 30%NS3 butexpressed products with molecular masses of approximately45 kDa, as predicted for uncleaved NS2A-ANS2B-30%NS3.Consistent with this assignment is the observation that theproduct of vNS2A-ANS2B(20)-30%oNS3 was slightly largerthan that of vNS2A-ANS2B(10)-30oNS3. In lane 2 (leftside), the band at approximately 60 kDa (unlabeled tickmark) might not be DEN specific, since this band was notseen in other experiments in which the same virus stock wasused (see, for example, lane 2, right side). The failure toobserve products at or near the position of 30%NS3 indi-cated that neither NS2B/NS3 nor NS2A/NS2B cleavageoccurred with these NS2B deletion mutants.NS2B exhibits a trans-acting function. The NS2B deletions

described above contained the last 10 or 20 residues at the Cterminus of NS2B, whose lengths most probably kept theNS2B/NS3 cleavage recognition sequence intact. The failureof these NS2B deletions to cleave NS2B/NS3 was thereforeprobably due to the loss of a specific NS2B function. Thesame might not be true for the NS2A/NS2B cleavage, since

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1 2 1+3 2+3 3 1+6 2+6 1+5 2+5 4 5

-NS3

-NS2A- ANS2B30%NS3

-NS2A-NS2B

-30%NS3

-NS2B

30

22

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FIG. 5. Demonstration that NS2B acts in trans. Samples were treated as described in the legend to Fig. 2. The positions of someDEN4-specific bands are indicated, as are the sizes of the marker proteins in lane M. The unlabeled tick mark and the asterisk indicate bandsdiscussed further in the text. In lanes 1 to 6, recombinant viruses were used to infect CV-1 cells: 1, vNS2A-ANS2B(10)-30%NS3; 2,vNS2A-ANS2B(20)-30%NS3; 3, vNS3; 4, vSC8; 5, vNS2A-NS2B; 6, vNS2B. Lanes with two numbers were coinfections with both indicatedviruses; for example, lane 1+3 was infected with vNS2A-ANS2B(10)-30%NS3 plus vNS3. The gel on the left was 12% acrylamide; the gelon the right was 17% acrylamide.

only the first residue ofNS2B was retained. Failure to cleaveat NS2A/NS2B could therefore have been due to destructionof the cleavage site; however, see below.To further investigate the role ofNS2B in these cleavages,

we performed complementation experiments by coinfectionof cells with two recombinant viruses. Vaccinia virus recom-binant vNS2A-ANS2B(10)-30%NS3 or vNS2A-ANS2B(20)-30%NS3 was used as the source of uncleaved precursor, andvNS2B, vNS3, or vNS2A-NS2B was used as a possiblesource of complementing activity. Both deletion mutantswere partially complemented for NS2B/NS3 cleavage uponcoinfection with vNS2B (Fig. 5, lane 1+6 and lanes 2+6, leftand right) or vNS2A-NS2B (lanes 1+5 and 2+5), as shownby the appearance of 30%NS3 and the reduction in theamount of uncleaved precursor. No such complementingactivity was seen upon coinfection with vNS3 (lanes 1+3and 2+3). Thus, NS2B can act in trans to complement thedefect in NS2B/NS3 cleavage caused by deletion of NS2B.These results showed that the target site for NS2B/NS3cleavage does not require more than the 10 C-terminalresidues of NS2B. Furthermore, this demonstrated thatproviding the putative NS3 protease in trans (assuming thatthe NS3 made by vNS3 is an active protease) failed tocorrect the cleavage-defective phenotype of these NS2Bdeletion mutants. Since a multiplicity of infection of up to 10PFU of each infecting virus per cell was used, it was highlyunlikely that the observed incomplete complementation wasdue to a significant fraction of cells that were not coinfected.Rather, the incomplete complementation was perhaps due tofailure of the DEN protein products to reach the sameintracellular site quantitatively. The 30%NS3 bands in lanes2+5 and 2+6 migrated slightly more slowly than the corre-sponding bands in lanes 1+5 and 1+6. The difference inapparent molecular mass was only 0.25 kDa. We attributedthis small difference to the Glu-to-Val change at amino acid1650 in the 30%NS3 derived from vNS2A-ANS2B(20)-

30%NS3. However, we cannot rule out other possible ex-planations, such as slightly imprecise NS2B/NS3 cleavage.There appeared to be more 30%NS3 product upon comple-mentation than uncleaved precursor in the absence of com-plementation. We assume that this was due to greaterstability or more efficient immune precipitation of the prod-uct than the precursor. Also note that more NS2B waspresent in the coinfected lanes than with vNS2B alone (lane6). We have not investigated this phenomenon further.

In addition to the 30%NS3 band seen when each of theNS2B deletion mutants was complemented by vNS2B orvNS2A-NS2B, another band slightly larger than 30%NS3was seen in each of these lanes (asterisk). The second bandin the vNS2A-ANS2B(20)-30%NS3 coinfections was largerthan that in the vNS2A-ANS2B(10)-30%NS3 coinfections.This suggested that the second bands were either uncleavedANS2B-30%NS3 or NS2A-ANS2B. The use of monospecificsera for either NS2A or NS3 should resolve the identity ofthese second bands. Although we could not be certain of theidentity of these species, the fact that we have neverdetected NS2A suggested that these were probably /NS2B-30%NS3. If this is the case, it implies that the NS2A/NS2Bcleavage also was complemented in trans by NS2B in thecoinfections and that the NS2A/NS2B cleavage site se-quence does not extend beyond the N-terminal serine ofNS2B.NS3 may act in trans to cleave NS2A/NS2B. The results in

Fig. 5 also provided indirect evidence that NS3 could act intrans. A band was seen in the vNS2A-NS2B lane with amolecular mass of 32 kDa, which was presumably uncleavedNS2A-NS2B. This precursor was expected to have a molec-ular mass of 38 kDa, but as discussed above, the mobility ofNS2B was known to be aberrant. Note also that no NS2Bwas seen in this lane. However, upon coinfection withvNS2A-ANS2B(10)-30%NS3 (lane 1+5) or vNS2A-ANS2B(20)-30%NS3 (lane 2+5), a strong NS2B signal was pro-

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duced. This NS2B must have been derived from cleavage ofthe NS2A-NS2B product of vNS2A-NS2B, since the otherviruses in the coinfections did not encode NS2B. Thisprovides evidence that the 30%NS3 encoded by vNS2A-ANS2B(10)-30%NS3 and vNS2A-ANS2B(20)-30oNS3 wasable to cleave the NS2A/NS2B junction in trans. The appar-ent excess production of NS2B product compared withNS2A-NS2B precursor was presumed to be due to differen-tial stability or immunoprecipitability. In contrast, attemptsto complement vNS2A-NS2B with vNS3 or v30%NS3 re-sulted in little or no NS2B production (data not shown). Onepossible explanation for the lack of complementation is thatthe products of vNS2A-NS2B and of vNS3 or v30%NS3failed to reach the same specific intracellular site. Finally, itwas noteworthy that uncleaved NS2A-NS2B was not ex-pressed by vNS2A-NS2B-30%NS3 (Fig. 3), although a bandcorresponding to NS2B-30%NS3 was seen. This observationsuggests that NS2A/NS2B cleavage preceded NS2B/NS3cleavage. Whether this implies a highly regulated cleavagemechanism or is an adventitious result remains to be deter-mined.

DISCUSSION

We have constructed recombinant vaccinia viruses ex-pressing various portions of the DEN4 genome to studyproteolytic processing of the NS region of the DEN4 poly-protein. Our results indicate that the N-terminal 30% of NS3is required for the NS2A/NS2B and NS2B/NS3 cleavages.Indirect evidence that NS3 can act in trans at these cleavagesites was obtained from the results of the coinfections ofvNS2A-NS2B plus vNS2A-ANS2B(10)-30%NS3 or vNS2A-ANS2B(20)-30%NS3 (Fig. 5), where NS2B was produced.However, attempts to directly demonstrate trans activity ofNS3 by coinfections with vNS2A-NS2B or vNS2A-NS2B-12%NS3 and vNS3 or v30%NS3 have failed. Nevertheless,these findings are consistent with the proposed model thatNS3 has protease activity (1, 21). Two groups have recentlydescribed results of in vitro translation of transcripts ofcloned DNA that suggest that the putative protease domainof NS3 is required for both the NS2A/NS2B and NS2B/NS3cleavages (11, 41). In addition, our results demonstrate thatNS2B is also required for the NS2A/NS2B and NS2B/NS3cleavages. Complementation experiments show that NS2Bcan act in trans to facilitate NS2B/NS3 cleavage and alsoprobably NS2A/NS2B cleavage. The possibility that homol-ogous recombination efficiently generated NS2B in cis inthese experiments is unlikely because of the short overlap of30 or 60 nucleotides between NS2B and the NS2B deletionsand the short duration of the experiments. Furthermore,other studies in our laboratory have shown that both NS2Band NS3 can act in trans to facilitate NS4B/NS5 cleavage, inan experiment in which homologous recombination was nota possibility (5a). Also, Chambers et al. have recentlyreported observations that NS2B is required for NS4B/NS5cleavage in vitro (11). Thus, both NS2B and NS3 togetherappear to be required for a protease activity that cleavesNS2A/NS2B, NS2B/NS3, and NS4BINS5. The flavivirusprotease activity is presumably also responsible for theNS3/NS4A cleavage, and perhaps the C-terminal processingof C (43, 47). We have no data regarding the requirement forNS3 for either of these processing events, but our resultsshow that NS2B is needed for the NS3/NS4A cleavage. Thisactivity of NS2B is readily demonstrated when NS2B ispresent in cis, but this cleavage activity is absent or veryinefficient when NS2B is supplied in trans (data not shown).

NS4A/NS4B cleavage seems to occur in the absence ofNS2B, consistent with the suggestion that this cleavage ismediated by signalase (43, 47). Finally, the recombinantvNS2B-30%NS3 made a product of a size consistent with anuncleaved NS2B-30oNS3 precursor, but did not produceuncleaved NS2A-NS2B. This suggests that NS2A/NS2Bcleavage preceded NS2B/NS3 cleavage.

It is interesting that NS3 alone appears to exhibit somelimited proteolytic activity, as vNS3 makes minor amountsof some of the NS3 internal cleavage products. Coupled withthe finding that both NS2B and NS3 are required and eachcan apparently act in trans to mediate the NS2A/NS2B andNS2B/NS3 cleavages, this suggests that NS2B interacts withNS3 in some fashion to promote the protease activityinherent in NS3. Such a two-component proteinase is appar-ently required for at least one of the processing events in thecomovirus M segment polyprotein (28). One possibility isthat these two proteins associate as a complex to form theactive proteinase. The observation that both NS2B and NS3together seem to immunoprecipitate better than when theyare present alone is consistent with the presence of such anNS2B/NS3 complex. A related possibility is that NS2B isneeded to target NS3 to the correct cellular location formaximal proteolytic activity. NS3 is known to be membraneassociated (23, 52), despite lacking any obvious membrane-spanning hydrophobic segment. It is conceivable that thecorrect membrane association of NS3 depends on an inter-action between NS3 and the highly hydrophobic NS2B.Consistent with this idea are the preliminary results of cellfractionation studies which show that NS3 made by vNS2B-NS3 is entirely membrane associated, while about one-quarter of the NS3 made by vNS3 is soluble (17a). It is alsopossible that NS2B acts in trans to modify NS3 in somemanner to increase the proteolytic activity inherent in NS3.An alternate explanation for the observed trans activity isthat NS2B is a protease. We consider this unlikely for thefollowing reasons. First, we detected no cleavage activity inrecombinants, such as vNS2A-NS2B and vNS2A-NS2B-12%NS3, that contain intact NS2B in the absence of the NS3protease domain. Second, it would seem genetically uneco-nomical for DEN to code for functionally redundant prote-ases. Third, sequence homology to proteases has not beenfound in NS2B. Finally, it is possible that the role of NS2Bis to maintain the polyprotein precursor in a conformationcleavable by NS3. One would expect NS2B to be cis-actingin this case, which is consistent with the data for NS3/NS4Acleavage. However, trans activity is possible in such amodel. For example, the NS2A-ANS2B-NS3 precursorcould be defective for cleavage as a result of misfoldingcaused by deletion of NS2B, and providing NS2B in transmight allow cleavage to occur if NS2B can interact with theincorrectly folded molecule and restore it to a cleavableconformation. Although we cannot rule out this possibility,it seems less likely than the simpler two-component proteasemodel, whereby NS2B functions to activate the proteaseactivity of NS3. Experiments are in progress to furthercharacterize the role of NS2B in the flavivirus proteaseactivity.

ACKNOWLEDGMENTS

We thank Myron Hill and Peter Collins for the synthesis of theoligodeoxyribonucleotides, Robert Chanock for a critical reading ofthe manuscript, and Todd Heishman and Sandra Chang for editorialassistance.

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