Alternative Proteolytic Processing of the ... - Journal of Virology

JOURNAL OF VIROLOGY, Dec. 1997, p. 9313–9322 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 71, No. 12

Alternative Proteolytic Processing of the Arterivirus Replicase ORF1a Polyprotein: Evidence that NSP2 Acts as a Cofactor for the NSP4 Serine Protease ALFRED L. M. WASSENAAR,1 WILLY J. M. SPAAN,1 ALEXANDER E. GORBALENYA,2,3 1 AND ERIC J. SNIJDER * Department of Virology, Leiden University Medical Center, The Netherlands,1 and M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, 142782 Moscow Region,2 and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow,3 Russia Received 7 July 1997/Accepted 16 September 1997

The C-terminal half of the replicase ORF1a polyprotein of the arterivirus equine arteritis virus is processed by a chymotrypsinlike serine protease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864–4871, 1996) located in nonstructural protein 4 (nsp4). Three probable SP cleavage sites had previously been identified in the ORF1a protein. Their proteolysis explained the main processing products generated from the C-terminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755–5764, 1994). By using sequence comparison, ORF1a expression systems, and site-directed mutagenesis, we have now identified two additional SP cleavage sites: Glu-14302Gly and Glu-14522Ser. This means that the ORF1a protein can be cleaved into eight processing end products: nsp1 to nsp8. By microsequence analysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu2(Gly/Ser) substrates. Importantly, our studies revealed that the C-terminal half of the ORF1a protein (nsp3-8) can be processed by the SP following two alternative pathways, which appear to be mutually exclusive. In the majority of the nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 and nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 site only, whereas the newly identified nsp5/6 and nsp6/7 sites appear to be inaccessible to the protease. In the alternative proteolytic cascade, which is used at a low but significant level in infected cells, it is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp6/7 sites are processed to yield a set of previously unnoticed processing products. Coexpression studies revealed that nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, the first step of the major processing pathway. When the nsp2 cofactor is absent, the nsp4/5 site cannot be processed and nsp3-8 is processed following the alternative, minor pathway. Equine arteritis virus (EAV) is the prototype of the Arteriviridae, a recently established family of positive-stranded RNA viruses. Other arteriviruses are lactate dehydrogenase-elevating virus (LDV), simian hemorrhagic fever virus, and porcine reproductive and respiratory syndrome virus (PRRSV) (28, 30). The arterivirus replicase gene, which covers about three quarters of the genome, is related to that of coronaviruses (9, 31). Despite a considerable size difference, their common ancestry is evident from the presence of a number of highly conserved domains in comparable relative positions and from the use of identical genome expression strategies, including the discontinuous transcription of a nested set of subgenomic mRNAs. Because of these similarities, the two virus families were recently united in the order of the Nidovirales (4). The EAV replicase gene consists of two large open reading frames (ORF1a and ORF1b) which encode two precursor proteins that are proteolytically processed into smaller functional units: the 1,727-amino-acid (aa) ORF1a protein (187 kDa) and the 3,175-aa ORF1ab protein (345 kDa), the latter being produced by ribosomal frameshifting (9). Processing of the ORF1b-encoded polyprotein, which is assumed to contain functions essential for viral RNA replication and mRNA transcription (9, 39), yields four cleavage products, including those that carry the putative viral polymerase and helicase activities

(40, 41). The EAV ORF1a protein contains three protease domains and a number of potential transmembrane regions, which have been implicated in the membrane association of the viral replication complex (38, 41). Previously, we have reported that the ORF1a-encoded polypeptide is cleaved at least five times (Fig. 1A) (34). The first two processing steps lead to the autoproteolytic release of nonstructural proteins 1 and 2 (nsp1 and nsp2) from the Nterminal half of the ORF1a precursor protein (33–35). The remaining 96-kDa C-terminal part of the ORF1a polypeptide (p96) is autoprocessed by a chymotrypsinlike serine protease (SP) which is located in nsp4. Although the nsp4 SP contains a canonical catalytic triad (His-1103, Asp-1129, and Ser-1184 [36]), its substrate-binding region shares similarities with the picornavirus 3C-like cysteine proteases (for reviews, see references 12 and 16). On the basis of this property, results derived from comparative sequence analysis of arterivirus replicase sequences, and site-directed mutagenesis, we have tentatively identified three SP cleavage sites in the ORF1a protein: Glu10642Gly, Glu-12682Ser, and Glu-16772Gly (36). Cleavage at these positions explains the main processing products generated from the C-terminal half of the ORF1a polyprotein in infected cells and eukaryotic expression systems (Fig. 1A) (34). The nsp4 SP first cleaves at its own C terminus (the Glu12682Ser site), thereby splitting p96 into two intermediates with estimated sizes of about 50 and 44 kDa. The first of these products is nsp3-4, which is slowly cleaved at the Glu10642Gly site to yield nsp3 and nsp4 (36). The 44-kDa product (p44; Fig. 1A) is cleaved at the Glu-16772Gly site close to

* Corresponding author. Mailing address: Department of Virology, Leiden University Medical Center, AZL P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: [email protected]. 9313

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vations on SP-mediated processing have been made which required further analysis. First, two additional potential SP cleavage sites (Glu-14302Gly and Glu-14522Ser) have been identified in the region of the EAV ORF1a protein from which p44 and p41 are derived. Both sites are conserved in two other arteriviruses (LDV [15, 27] and PRRSV [25]) (Fig. 1B). However, mutagenesis of one of these sites (Glu-14302Gly) in EAV had been shown not to affect the generation of any of the processing products shown in Fig. 1A (36). Nevertheless, their conserved nature strongly suggested both sites to be important, either in proteolytic processing or in another function of the ORF1a protein. A second question which remained to be addressed was the location of the N-terminal border of p44 (Fig. 1A). This C-terminal cleavage product could result from cleavage at the Glu-12682Ser site, but its apparent 44-kDa size in sodium dodecyl sulfate (SDS)-polyacrylamide gels was considerably smaller than the 51 kDa (aa 1269 to 1727) which can be calculated from the ORF1a protein sequence. Thus, the existence of an additional processing product, which would be derived from the region between the nsp4 C terminus and the p44 N terminus, could not be excluded (36). In this study, we demonstrate that p44 migrates anomalously during SDS-polyacrylamide gel electrophoresis (PAGE) and that this cleavage product is adjacent to nsp4 in the ORF1a polyprotein. Furthermore, we show that the Glu-14302Gly and Glu-14522Ser sites can indeed be processed by the nsp4 SP and that they are part of an alternative processing pathway in which the Glu-12682Ser site directly downstream of nsp4 appears to remain uncleaved. This secondary proteolytic pathway, which may yield up to nine novel processing products, is only used to a limited extent in infected cells. When the major processing pathway is used, cleavage of the Glu-12682Ser site does occur, but this requires the association of cleaved nsp2 with the C-terminal part of the ORF1a protein. MATERIALS AND METHODS

FIG. 1. Proteolytic processing of the EAV replicase ORF1a protein. (A) Modification of the previously published processing scheme for the ORF1a protein (34). The three EAV protease domains (boxes) and corresponding cleavage sites (arrows with P1 and P19 residues indicated) are depicted. The dashed left side of the p44 and p41 boxes indicates the uncertainty about the N terminus of these products (see text). Abbreviations: PCP, papainlike cysteine protease; CP, cysteine protease; SP, serine protease. (B) Alignments of (putative) cleavage sites for the nsp4 SP in the ORF1a proteins of the arteriviruses EAV, LDV, and PRRSV.

its C terminus (aa 1727). This yields a small C-terminal product, which has not been detected due to its 5-kDa size, and a product with an apparent size of 41 kDa (p41), which was shown to be N coterminal with p44 (34). These results strongly suggested that the arterivirus nsp4 SP is the major proteolytic enzyme residing in the ORF1a protein and that its cleavage site specificity resembles that of the picornavirus 3C-like cysteine proteases: Glu is the preferred residue at the P1 position, whereas the P19 position is occupied by a small residue (Ser or Gly). In addition, two other obser-

Cells and viruses. The Bucyrus strain of EAV (11) was grown in baby hamster kidney (BHK-21) or rabbit kidney (RK-13) cells as described before (10). Vaccinia virus recombinant vTF7-3 (13), which produces the T7 RNA polymerase, was also propagated in RK-13 cells. EAV ORF1a expression plasmids. Recombinant plasmids were constructed by using standard techniques. All ORF1a T7 expression constructs used in this study were based on plasmid pL1a (36), which contains EAV ORF1a downstream of the T7 promoter and a copy of the encephalomyocarditis virus internal ribosomal entry site, which is used to enhance translation (17). Mutations were introduced into shuttle plasmids by oligonucleotide-directed PCR mutagenesis (20). The generation of the mutations specifying the following single amino acid substitutions in the EAV ORF1a protein was described previously: Cys-1643Ser (33); Cys-2703Ser and His-3323Tyr (35); and Glu-10643Pro, Ser-11843Ile, Glu12683Pro, Glu-14303Pro, and Glu-16773Pro (36). A Glu-14523Pro-encoding mutation (and a HindIII restriction site) was engineered by changing nucleotide 4579 to 4587 from GAGAGTCTC to CCAAGCTTG. We have previously described the expression of N- and/or C-terminally truncated ORF1a proteins (synthetic nsps) from constructs in which initiation and termination codons had been introduced at appropriate positions (36). The nsp4 and nsp3-4 expression constructs pL(1065-1268) and pL(832-1268) were extended to create pL(1065-1727) (see Fig. 3A) and pL(832-1727), respectively (numbers indicate the first and last ORF1a-derived residues of the expression product). To create nsp2 expression vector pL(261-831), an NcoI restriction site (CCATGG), containing a translation initiation codon, was introduced upstream of the Gly-261 codon and the nsp1-encoding region was deleted. Subsequently, a termination codon derived from construct pL(1-831) (36) was inserted downstream of nsp2. Finally, a set of expression vectors was made to express various parts of the C-terminal region of the ORF1a protein (Fig. 2A). In each of these constructs [pL(1269-1727), pL(1300-1727), pL(1310-1727), and pL(1342-1727)] a translation initiation codon was engineered upstream of the sequence encoding the indicated ORF1a-specific residues. To achieve the in-frame fusion of nsp1 to nsp5 in the protein expressed from pL1aDGS (Fig. 2A), the region encoding residues 261 to 1268 was deleted by PCR mutagenesis. In the pL1aDGG protein, a Gly was introduced at Ser-1269 to create the naturally occurring Gly-2602Gly cleavage site for the nsp1 protease. Labeling and analysis of proteins from transfected or EAV-infected cells. Transient T7 expression using vaccinia virus recombinant vTF7-3, RK-13 cells,

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nsp2-7 (residues 261 to 1677) was engineered (see Fig. 4A) by using the 59 end of the nsp2 reading frame from pL(261-831) and a PCR-generated 39 end encoding the ORF1a protein sequence up to residue 1676, followed by the sequence Pro, Gly, six His residues, and a termination codon. This reading frame was cloned downstream of the 26S subgenomic mRNA promoter of Sindbis virus (Sin) expression vector pSinRep5 (3). RNA transcripts were generated in vitro from linearized pSinEAV(2611677)His plasmid DNA and from the Sin helper construct pTdSsH-59tRNA (3). A mixture of the two RNAs was electroporated into BHK-21 cells as described before (39), after which the cells were seeded and incubated at 37°C. Recombinant Sin particles [vSinEAV(261-1677)His] were harvested after 48 h and titrated on fresh BHK-21 cells. To produce labeled His-tagged EAV protein, 107 BHK-21 cells were infected with vSinEAV(261-1677)His at a multiplicity of infection of 20 and protein synthesis was labeled from 5 to 10 h postinfection using [3H]leucine (120 mCi/ml) or [35S]methionine (150 mCi/ml). Cells were lysed as described above. Purification of His-tagged proteins and radiosequence analysis. [35S]methionine- or [3H]leucine-labeled lysates from vSinEAV(261-1677)His-infected cells were 10-fold diluted in NiC buffer (20 mM HEPES (N-2-hydroxyethylpiperazineN9-2-ethanesulfonic acid), pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100, 15% glycerol). The His-tagged proteins were allowed to bind to nickelnitrilotriacetic acid (Ni21-NTA)-agarose (Qiagen), washed with NiC buffer, and eluted in 25 mM MES (morpholineethanesulfonic acid), pH 5.0, 50 mM EDTA, 1% SDS, 2% b-mercaptoethanol, and 10% glycerol. After SDS-PAGE and electroblotting to polyvinylidene difluoride membrane, the bands representing the His-tagged proteins of interest (nsp5-7/His and nsp7/His) were cut from the membrane. The N-terminal amino acid fractions of these proteins were collected from a protein sequenator (Applied Biosystems model 475A), and the amount of radioactivity which they contained was determined by liquid scintillation counting.

RESULTS

FIG. 2. Tentative mapping of the p44 N terminus. (A) Overview of the expression plasmids used to determine the approximate location of the N terminus of p44. (B) Translation initiation codons were introduced to allow expression from residues 1342, 1310, and 1300 to the end of the C terminus of the ORF1a protein. The migration of these expression products in SDS-polyacrylamide gels was compared with that of p44, which was produced by expression and processing of the full-length ORF1a protein. (C) Comigration analysis of p44 with the C-terminal cleavage product of the proteins produced from pL1aDGS and pL1aDGG and with the pL(1269-1727) expression product.

and cationic liposomes was carried out as described previously (34). Proteins were labeled from 5 to 8 h postinfection, using Expre35S35S label (NEN). The EAV replicase-specific antisera and methods for pulse-chase experiments have been described previously (34). Cell lysis was performed in lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.5% deoxycholic acid [DOC], 1% Nonidet P-40 [NP-40], 0.1% SDS, and 0.4 mM PMSF). The nuclear fraction was removed by centrifugation, and immunoprecipitations were carried out in IP buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 5 mM EDTA, 0.1% DOC, 0.5% NP-40, and 0.5% SDS). Antiserum a4 was a 1:1 mixture of a4M and a4C (34), and serum a78 was previously named a5 (34). Intracellular membranes were isolated from EAV-infected and mock-infected cells essentially as described by Fujiki et al. (14). After metabolic labeling, cells were washed in phosphate-buffered saline, scraped from the dish, and washed again. Cells were lysed in a hypotonic buffer (1 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, 15 mM NaCl) containing leupeptin (2 mg/ml) and PMSF (0.4 mM) using 20 strokes of a Dounce homogenizer. A postnuclear supernatant was prepared, and membranes were pelleted through a 6% sucrose cushion by ultracentrifugation (Beckman SW55 rotor) for 30 min at 150,000 3 g and 4°C. EAV-specific proteins were immunoprecipitated from the pellet and supernatant fractions and analyzed as described above. Construction and use of Sindbis virus expression vector pSinEAV(2611677)His. A reading frame encoding a hexahistidine-tagged version of EAV

Tentative identification of Ser-1269 as the p44/p41 N-terminal residue. Previously, we have shown that the EAV ORF1a protein is cleaved at least five times (Fig. 1A) (34, 36) and that its processing in the vaccinia virus/T7 expression system is identical to that of the native protein in infected cells. We have shown that the SP probably generates the nsp4 C terminus by cleavage at the Glu-12682Ser-1269 site (36). Mutagenesis of Glu-1268 to Pro abolished the generation of cleavage products from regions both upstream (nsp3-4 and nsp4) and downstream (p44 and p41) of the Glu-12682Ser-1269 site (Fig. 1A) (36). Although this implied that Ser-1269 was the N-terminal residue of p44 and p41, the sizes calculated for these cleavage products (residues 1269 to 1727 and 1269 to 1677) are 51 and 46 kDa, respectively, which are substantially larger than their apparent sizes during SDS-PAGE. As in the case of nsp4 (36), severe aberrant migration in polyacrylamide gels could explain this difference. On the other hand, it could not be excluded that a small additional cleavage product might be generated from the region between Glu-1268 and the p44/p41 N terminus. To determine the p44/p41 N terminus, we first engineered a number of translation initiation codons in the region downstream of the Ser-1269 codon (Fig. 2A). Expression of this set of constructs yielded proteins migrating faster than p44 in SDS-PAGE (Fig. 2B), and it was concluded that the p44 N terminus is located upstream of Phe-1300. All three constructs yielded additional products, which were explained by partial translocation across the endoplasmic reticulum membrane and subsequent processing of the polypeptide. Each of the expression products contained an extremely hydrophobic N-terminal domain, which could apparently function as an artificial signal sequence. The 39-kDa product which was generated from the three constructs was explained by removal of this signal sequence. By endoglycosidase F treatment (data not shown), the higher molecular weight bands were shown to be N-glycosylated forms of the translocated proteins. On the basis of the amino acid sequence, residues Asn-1501 and Asn-1577 were identified as potential N-glycosylation sites. It should be stressed that the partial translocation of these proteins is an

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artefact of the expression constructs. There are no indications whatsoever that translocation, signal sequence cleavage, or glycosylation can occur when this protein sequence is expressed as an internal part of the full-length ORF1a polyprotein. Subsequently pL(1269-1727) was created (Fig. 2A); it encoded the entire ORF1a sequence downstream of Ser-1269 (calculated size, 51 kDa), extended with a single N-terminal Met residue. Furthermore, we employed the nsp1 papainlike cysteine protease (PCP [33]) to generate a cleavage product with Ser-1269 as the N-terminal residue. To this end, we deleted the region encoding nsp2 to nsp4 from ORF1a (construct pL1aDGS; Fig. 2A), thereby creating a Gly-2602Ser-1269 cleavage site for the nsp1 PCP. As a control, pL1aDGG was created in which the usual Gly-Gly cleavage site for the nsp1 PCP (33) was maintained by substituting Ser-1269 with Gly. Although a preference for Gly at the P19 position was noticed, the artificial nsp1/5 site in both expression products was cleaved relatively efficiently. The C-terminal pL1aDGS and pL1aDGG cleavage products and also the pL(1269-1727) protein comigrated perfectly with p44 (Fig. 2C). This strongly suggested that Ser-1269 is indeed the N-terminal residue of p44, and that the actual size of this C-terminal processing product is 51 kDa. Identification of two novel SP cleavage sites and a set of novel processing products. On the basis of the nsp4 SP cleavage site specificity (Glu2(Gly/Ser)) and their conservation in other arteriviruses, the closely spaced EAV dipeptides Glu14302Gly and Glu-14522Ser (Fig. 1B) had previously been identified as potential additional substrates for the SP (36). To study processing at these sites, we expressed residues 1065 to 1727 of the EAV ORF1a protein (Fig. 3A). The pL(1065-1727) protein contained the nsp4 SP domain at its N terminus and all downstream sequences normally present in the ORF1a polypeptide. Remarkably, an immunoprecipitation analysis revealed that the SP in this protein was unable to process the Glu-12682Ser site. This was concluded from the absence of nsp4 and the C-terminal p44/p41 cleavage products, which are normally generated abundantly (Fig. 3B and C; also see below). Instead, the anti-nsp4 serum precipitated two apparently C-terminally extended versions of nsp4 of approximately 45 and 46 kDa (Fig. 3B, a4 panel). The a78 serum, which recognizes the C-terminal 220 residues of the ORF1a protein, precipitated four prominent products of between 25 and 33 kDa, which had not been observed before (Fig. 3B, a78 panel). Inactivation of the SP (Ser-11843Ile) completely abolished the generation of these novel cleavage products (Fig. 3B, a78 panel). In view of the specificity of the a78 serum, the novel 25- to 33-kDa products had to be derived from the C-terminal part of the ORF1a protein. Thus, they had to overlap with the previously described p44 and p41 products, which were not produced upon pL(1065-1727) expression. The apparent sizes of the 25- to 33-kDa products suggested that their N termini could originate from processing of the Glu-14302Gly or Glu14522Ser site, whereas their C-terminal residue could be either Asn-1727 (the C-terminal residue of the ORF1a protein) or Glu-1677 (derived from processing of the previously identified Glu-16772Gly-1678 junction). The nsp4-containing 45and 46-kDa proteins (Fig. 3B, a4 panel) were assumed to represent the N-terminal cleavage products from processing at the two novel SP sites (Fig. 3A): residues 1065 to 1430 (from cleavage at Glu-14302Gly) and 1065 to 1452 (from cleavage at Glu-14522Ser), respectively. Although the calculated sizes for these products are 39 and 41 kDa, the previously described aberrant migration in SDS-PAGE of nsp4 (calculated size, 21

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FIG. 3. Expression of the C-terminal part of the EAV replicase ORF1a protein. (A) Schematic representation of the protein expressed from expression vector pL(1065-1727). The products derived from processing at the two novel cleavage sites (see panels B and C) and their calculated sizes are depicted below. Polypeptide p72 is the full-length pL(1065-1727) protein. The p67 cleavage product is derived from processing of p72 at the Glu-16772Gly site only. (B) Immunoprecipitation analysis of pL(1065-1727) expression in the vaccinia virus/T7 system. In the leftmost two lanes a comparison with the full-length ORF1a expression vector pL1a is made using the anti-nsp4 serum. The two other lanes show the products recognized by the anti-nsp78 serum and the effect of replacement of the catalytic serine (Ser-1184) of the nsp4 SP. (C) Mutagenesis of the two novel cleavage sites (Glu-14302Gly and Glu-14522Ser) and the previously described Glu-16772Gly site by replacing the P1 Glu residue with Pro. Each mutation prevents the production of two of the four pL(1065-1727) products in the 25- to 33-kDa range, and the Glu-16772Gly mutation also prevents the generation of p67.

kDa; observed size, 31 kDa) (36) can readily explain this size difference. To gain further insight into the generation of the novel 25to 33-kDa products, the putative P1 residues Glu-1430, Glu1452, and Glu-1677 were replaced by Pro, a substitution which had previously been shown to abolish processing at other SP sites (36). As expected, each mutation prevented the generation of two of the four novel C-terminal cleavage products (Fig. 3C). These observations strongly suggest that, in the context of the pL(1065-1727) expression product, Glu-14302Gly and Glu-14522Ser were processed efficiently. They also support the map locations for the 25- to 33-kDa products outlined in Fig. 3A. As a consequence of the results presented above, the numbering of the processing products derived from the C-terminal region of the ORF1a protein was revised (Fig. 4A): residues

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FIG. 4. N-terminal microsequencing of His-tagged EAV cleavage products generated by a SIN expression vector. (A) Revised ORF1a protein processing scheme and schematic representation of the nsp2-7 reading frame engineered for expression from the SIN subgenomic mRNA promoter. The nsp5-7/His and nsp7/His cleavage products, which were used for sequence analysis, are depicted. (B) Purification of His-tagged cleavage products after vSinEAV(261-1677)His infection of BHK-21 cells. The total 35S-labeled cell lysate, the Ni21-NTA-agarose eluate, and an immunoprecipitation (lane i.p.) with the a78 serum were analyzed by SDS-PAGE. For comparison, an immunoprecipitation of pL1a expression products was included, showing the positions of the native nsp5-8 and nsp5-7 products. The arrowheads point toward the nsp5-7/His and nsp7/His bands, which were excised from a Western blot and used for microsequencing. (C through E) Microsequencing profiles of [35S]methionine-labeled (C) and [3H]leucine-labeled (D) nsp5-7/His and [3H]leucine-labeled nsp7/His (E), respectively. The amount of radioactivity in the fractions collected from a protein sequenator was determined by liquid scintillation counting. The amino acid sequences of the corresponding regions of the EAV ORF1a protein are depicted below the graphs.

1065 to 1268, 1269 to 1430, 1431 to 1452, 1453 to 1677, and 1678 to 1727 will from now on be referred to as nsp4 to nsp8, respectively. All other products described above can be considered as processing intermediates consisting of two or more of these subunits (e.g., p44 and p41 are nsp5-8 and nsp5-7, respectively). Radiosequence analysis of the nsp5-7 and nsp7 N termini confirms the cleavage site specificity of the SP. To prove the cleavage site map in Fig. 4A and to confirm for the first time the presumed cleavage site specificity of the arterivirus nsp4 SP, the N termini of nsp5-7 and nsp7 were determined by protein microsequencing. A reading frame encoding a C-terminally hexahistidine-tagged polyprotein consisting of EAV nsp2 to nsp7 was engineered (Fig. 4A) and expressed from the 26S subgenomic mRNA promoter of the Sin expression system (3). The His tag facilitated the purification of cleavage products from the C-terminal region of the polyprotein. Autoprocessing of the SinEAV(261-1677)His expression product by the internal EAV nsp2 and nsp4 proteases was tested (data not shown), and cleavage of all known sites was observed. The His-tagged equivalents of nsp5-7 and nsp7 could readily be identified (Fig. 4B). Subsequently, recombinant Sin particles containing SinEAV (261-1677)His RNA were prepared and used to infect fresh cells with a multiplicity of infection of 20. Protein synthesis was

radiolabeled with either [35S]methionine or [3H]leucine. Histagged proteins were purified from the cell lysate using Ni21NTA-agarose (Fig. 4B) and used for radiosequencing. From the data presented in Fig. 2, it was clear that the nsp5 N terminus had to be located upstream of Phe-1300 and probably was identical to Ser-1269. This was indeed confirmed by Nterminal radiosequence analysis of the nsp5-7/His cleavage product (Fig. 4C and D). Also the position of Ser-1453 at the N terminus of nsp7/His was corroborated (Fig. 4E). These results are ultimate proof of the cleavage site specificity of the nsp4 SP, which had only been established using indirect methods thus far (36). Detection of novel processing products in EAV-infected cell lysates. The data presented in Fig. 3 indicated that different sets of (partially) overlapping cleavage products can be generated from the C-terminal half of the EAV ORF1a protein. In infected cells (Fig. 1A), nsp3-8 (p96) had been reported to be cleaved into nsp3-4 and nsp5-8 (p44) and subsequently into nsp3, nsp4, nsp5-7 (p41), and nsp8. There was no evidence that this cascade resulted in processing at the newly identified nsp5/6 and nsp6/7 sites. Upon nsp4-8 expression [pL(10651727)] (Fig. 3), these novel sites were processed efficiently (yielding nsp4-5, nsp4-6, nsp6-8, nsp6-7, nsp7-8, and nsp7), but now processing of the nsp4/5 junction did not occur. Since a number of the cleavage products generated by the two pro-

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FIG. 5. Identification of novel ORF1a-derived processing products in EAV-infected cells. (A) Immunoblot analysis of EAV-infected (lane E) and mock-infected (lane M) RK-13 cell lysates using the a78 serum. Prolonged exposure revealed the presence of a set of bands in the 25- to 33-kDa size range, in addition to the previously described nsp5-8 and nsp5-7 processing products. (B and C) Pulse-chase analyses of EAV ORF1a protein processing using an alternative method for cell lysis, followed by separation of the cellular proteins into a cytoplasmic (lanes c) and membrane-associated (lanes m) fraction. For details, see Materials and Methods and Results. EAV-specific proteins were immunoprecipitated using the a78 (B) and a4 (C) sera.

cessing pathways are mutually exclusive, our data suggested that alternative proteolytic cascades can be used in which either the nsp4/5 site or the nsp5/6 and nsp6/7 junctions are cleaved. The novel C-terminal processing products described in Fig. 3 had not been noticed during previous immunoprecipitation and Western blot analyses of EAV-infected cell lysates (34). However, prolonged exposure of Western blots stained with the a78 serum revealed small quantities of nsp6-8, nsp6-7, nsp7-8, and nsp7 (Fig. 5A). A comparison with the amounts of nsp5-8 and nsp5-7 (Fig. 5A, lane E) underlines the fact that there is a large quantitative difference between these two sets of products in EAV-infected cells. Our standard cell lysis method includes the use of a mixture of detergents (see Materials and Methods). For an analysis of the membrane association of the EAV replication complex (38, 41), we recently used an alternative protocol. A hypotonic buffer and a Dounce homogenizer were employed to gently break the cells and prepare a postnuclear supernatant which was used for immunoprecipitation analyses. Due to reduced background in the 30-kDa range of the gels, the set of 25- to 33-kDa bands was more readily detected when the latter procedure was followed. Therefore, we applied this method in a pulse-chase experiment to study the generation of nsp6-8, nsp6-7, nsp7-8, and nsp7 (Fig. 5B). EAV-infected cells were pulse-labeled for 20 min and chased for up to 90 min. Subsequently, cytoplasmic as well as membrane-bound protein fractions were prepared from the postnuclear supernatant, and both samples were immunoprecipitated using the a78 serum. It was clear that the major fraction of the nsp3-8 precursor molecules was rapidly processed at the nsp4/5 site to generate nsp3-4 and nsp5-8. However, relatively small amounts of nsp6-8 and nsp7-8 could also be detected immediately after the pulse-labeling (compare the intensity of the nsp5-8 band with that of the nsp6-8 and nsp7-8 bands in Fig. 5B). Subsequently, the nsp7/8 sites in all three primary cleavage products were processed at comparable, relatively slow rates, yielding large amounts of nsp5-7 and small amounts of nsp6-7 and nsp7. The expected N-terminal products of nsp3-8 cleavage at the nsp5/6 and nsp6/7 sites are nsp3-5 and nsp3-6 (calculated sizes, 64 and 66 kDa, respectively). These could subsequently be processed into nsp3 and nsp4-5/nsp4-6 for example. Unfortunately, we have not been able to detect nsp3-5, nsp3-6, or any

of their possible cleavage products in infected cells (Fig. 5C). Obviously, the amounts of these products in infected cells will be small. Our analysis was also hampered by the relatively poor quality of our anti-nsp4 serum and the lack of antiserum recognizing nsp3, nsp5, or nsp6. Finally, the detection of nsp3-5 and nsp3-6 may also have been prevented by the large amount of nsp2 (61 kDa), which coprecipitates with nsp3-containing processing intermediates (34). In spite of our failure to identify any of the upstream cleavage products, we feel that the data presented in Fig. 5 provide convincing evidence that the nsp5/6 and nsp6/7 cleavages, which were initially observed upon nsp4-8 expression, do indeed occur at a low but significant level in EAV-infected cells. Thus, we conclude that the C-terminal half of the ORF1a protein can be processed along two alternative pathways: a major pathway which is probably initiated by cleavage of the nsp4/5 junction and a minor pathway in which the nsp5/6 and nsp6/7 sites can be processed. Our data suggested that the two pathways are mutually exclusive. For example, the pulse-chase experiment in Fig. 5B, and similar experiments using chase times up to 6 h (data not shown), showed that nsp5-7 is a processing end product in which the two novel sites (nsp5/6 and nsp6/7) are not accessible to the SP. This finding is compatible with our observation that the amount of C-terminal products generated from the minor pathway (nsp6-8, nsp7-8, nsp6-7, and nsp7) did not increase significantly during a pulsechase experiment (Fig. 5B), despite the presence of large amounts of nsp5-8 and nsp5-7, which could in principle be used to generate these products. Therefore, the choice between the major and the minor proteolytic cascade appears to be an early and irreversible event. Cleaved nsp2 is required as a cofactor for processing of the nsp4/5 site. In EAV-infected cells the nsp4/5 site is processed efficiently in the majority of the ORF1a polyproteins (Fig. 5). In contrast, cleavage at this site was completely absent upon expression of recombinant nsp4-8 (Fig. 3). This strongly suggested that protein sequences upstream of nsp4 (nsp1, nsp2, and/or nsp3) play an essential role in processing of the nsp4/5 junction by the nsp4 SP. In principle, this cofactor could thereby determine whether nsp3-8 is processed via the major or the minor proteolytic pathway. Deletion of nsp1 from the ORF1a polyprotein (e.g., in the Sin expression vector used in this study; Fig. 4) or inactivation

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and nsp2 1 nsp3-8). Interestingly, the balance between the two processing pathways could be shifted by changing the molar ratio of the two expression plasmids in the transfection experiment. When a fourfold molar excess of the nsp2 expression construct was used, the relative processing of the nsp4/5 site in coexpressed nsp3-8 was considerably enhanced and closely resembled the situation observed with the native ORF1a protein (Fig. 6B and 6C). To rule out once more the involvement of the nsp2 protease activity in the cleavage of the nsp4/5 site, the experiment was repeated with two nsp2 proteins that contained mutations which have previously been shown to completely inactivate the nsp2 cysteine protease (35). The putative activesite residues (Cys-270 and His-332) were replaced, and these nsp2 mutants were found to induce the nsp4/5 cleavage in nsp3-8 with the same efficiency as the wild-type protein. These data, and the previously described interaction between nsp2 and nsp3-containing proteins (34), strongly suggest (cleaved) nsp2 to be the cofactor which enables the nsp4 SP to cleave the nsp4/5 site in nsp3-8. DISCUSSION

FIG. 6. Coexpression of EAV nsp2 and nsp3-8. (A) ORF1a protein processing scheme and an overview of the expression plasmids used in cotransfection experiments. (B) Immunoprecipitation analysis (using the a78 serum) of the processing products derived from nsp3-8 in the absence (lane 2) or presence of nsp2. nsp2 was produced either from an nsp1-2 or from an nsp2 expression vector. For the lanes indicated with 1:1 and 4:1, different molar ratios of nsp2 and nsp3-8 expression plasmids were transfected. nsp2(C*) and nsp2(H*) are proteins from mutant pL(261-831) expression vectors, in which the active-site residues of the nsp2 protease have been replaced (Cys-270 3 Ser and His-332 3 Tyr, respectively) (35). pL1a and pL(1065-1727) were included as controls. A control immunoprecipitation with the anti-nsp2 serum revealed comparable nsp2 levels in all cotransfections (data not shown). (C) Immunoprecipitation analysis using the a4 serum for a selection of the cotransfections from panel B.

of the nsp1 autoprotease (34) had shown that this protease is not required for processing at the nsp4/5 site. However, we have previously reported that cleavage at the nsp4/5 site in a full-length ORF1a expression product was abolished when the processing of the nsp2/3 junction was blocked by mutagenesis of the nsp2/3 cleavage site (36). Identical results were obtained when the nsp2 autoprotease was inactivated (32). To assess the role of nsp3 in more detail, the nsp4-8 expression product was N-terminally extended with nsp3 [construct pL(832-1727)], thereby creating the naturally occurring nsp3-8 processing intermediate. Although the relative processing of the nsp5/6, nsp6/7, and nsp7/8 sites in this nsp3-8 expression product was clearly influenced by the presence of nsp3 (compare the two rightmost lanes of Fig. 6B), the nsp4/5 site was not cleaved at all. To test whether nsp2 could induce processing of the nsp4/5 site, nsp2 and nsp3-8 were coexpressed from two different T7 expression vectors (Fig. 6B and 6C). Processing of the nsp4/5 site in nsp3-8 was indeed observed when nsp2 was coexpressed, although processing of the nsp5/6 and nsp6/7 sites still occurred at a rate which was considerably higher than that in the wild-type ORF1a protein (Fig. 6B and 6C; compare lanes pL1a

Alternative proteolytic processing of the EAV replicase. The proteolytic processing of precursor proteins containing structural and/or replicative subunits is a crucial step in the life cycle of most positive-stranded RNA viruses (12, 19, 37). The nidovirus replicase is one of the more complex examples of such a viral precursor protein, due to its size and due to the number of protease domains it contains (31). In the case of arteriviruses, the nidovirus subgroup with the smallest replicase gene, four ORF1a-encoded protease domains have now been identified. Although one of these has been inactivated in EAV (8), the analysis of the EAV replicase processing cascade has proven to be complex. The remaining three proteolytic activities cleave the EAV ORF1a polypeptide 7 times (Fig. 7) (34) and the 345-kDa ORF1ab protein 10 times (40, 41). In addition to the processing end products, a large number of processing intermediates has been detected, which may perform specific functions in viral replication. They are produced due to the fact that different cleavages occur at different rates, that ORF1b expression is downregulated by a ribosomal frameshift, and that—as described in this paper—alternative proteolytic processing is employed to generate different sets of products from the same region of the polyprotein. Although many positive-stranded RNA viruses are known to use a combination of rapid and slow cleavages to regulate their gene expression, there is—to our knowledge—only one other documented example of genuine alternative processing. The poliovirus polyprotein was shown to contain two primary cleavage sites (2A/2B and 2C/3A) that are recognized by the viral 3C protease. Cleavage of either one of these junctions initiates an alternative proteolytic cascade that may be typical of the early or late phase of poliovirus reproduction, respectively (21). As illustrated by the pulse-chase experiment in Fig. 5B, the EAV nsp4 SP appears to cleave (probably in cis) either the nsp4/5, the nsp5/6, or the nsp6/7 junction (Fig. 7). In infected cells, there is a clear preference for the nsp4/5 site which flanks the substrate-binding region of the nsp4 SP (36). Processing of the other two sites was especially prominent under certain conditions in our expression system, when the nsp4/5 site was not cleaved. In infected cells, proteolysis of the nsp5/6 and nsp6/7 junctions appears to be part of a minor, alternative processing pathway (Fig. 7). Each of the primary C-terminal cleavage products (nsp5-8, nsp6-8, and nsp7-8), can subsequently be processed at the nsp7/8 junction, apparently at comparable rates (Fig. 5B) and probably in trans (32).

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FIG. 7. Alternative processing pathways for the EAV replicase ORF1a protein. The previously described processing cascade and its products (34) are indicated as the major pathway. The novel processing steps and cleavage sites described in this paper are part of the minor pathway, which is used at a low but significant level in EAV-infected cells.

It appears that the two proteolytic cascades outlined in Fig. 7 are mutually exclusive, and therefore truly alternative. The 41-kDa nsp5-7 product accumulated during long pulse-chase experiments (32, 34), indicating that its internal nsp5/6 and nsp6/7 sites are not available for processing. Furthermore, in the pulse-chase experiment only small amounts of nsp6-7 and nsp7 were generated (Fig. 5B), suggesting that these products can only be produced from nsp6-8 and nsp7-8 and not from the abundant nsp5-8 and nsp5-7 species generated via the major pathway (see below). However, in addition to the unique products of both pathways, at least two common products (nsp3 and nsp8) are generated. Unfortunately, in infected cell lysates we were unable to detect the complementary nsp4-5-containing products (e.g., nsp3-5, nsp3-6, nsp4-5, and nsp4-6) which should be generated from the region upstream of the nsp5/6 and nsp6/7 sites when the minor pathway is used. Therefore, our assumption that the nsp4/5 site in these products remains uncleaved is only based on the results obtained using the vaccinia virus/T7 expression system. We have shown that in the absence of nsp2, a condition under which only the minor processing pathway seems to be operational, the nsp4/5 site of nsp3-8 and nsp4-8 is not processed (Fig. 3 and Fig. 6). Likewise, mutant full-length ORF1a expression products in which the nsp2/3 cleavage was blocked were not detectably processed at the nsp4/5 site (32, 36), as judged from the absence of nsp5-8 and nsp5-7 and the generation of substantial amounts of nsp4-5 and nsp4-6. Assuming that the data obtained in our expression system reflect the in vivo situation, we can assume

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that nsp5 and nsp5-6 are not produced during EAV infection. Thus, alternative processing may also be a mechanism which prevents the generation of certain (undesirable) viral proteins. nsp2 functions as a cofactor for processing of the nsp4/5 site. The data from various experiments in the vaccinia virus/T7 expression system indicate that cleaved nsp2 is required for processing of the nsp4/5 junction by the nsp4 SP. This cleavage is the first step of the major processing pathway (Fig. 7), and in fact the existence of the minor processing pathway only became apparent in experiments in which nsp2 could not fulfill its cofactor role. Examples of this situation are the inactivation of the putative nsp2/3 cleavage site, the inactivation of the nsp2 protease, and the complete absence of nsp2 (e.g., during expression of nsp3-8 only; Fig. 6). Cofactors that strongly influence polyprotein processing have previously been identified in several other animal positive-stranded RNA virus systems. The poliovirus 3C protease interacts with the 3D polymerase in the 3CD precursor. In this 3CD intermediate, 3D appears to be an activator of the proteolytic activity in trans of 3C toward the P1 precursor (18, 43). The 3CD precursor was shown to slowly autoconvert into 3C and 3D, a process that was efficiently stimulated by 3AB molecules (26). In the case of alphaviruses, the nsP2 protease seems to interact with domains of nsP1 and nsP3 which profoundly modulate its trans-cleavage activity (7). The resulting regulation of protease activity is an important part of the elegant mechanism the virus uses to control the switch from minus-strand to plus-strand RNA synthesis (22–24, 29, 42). In flaviviruses, NS2B is an essential cofactor for the NS3 serine protease (1, 2, 5, 6) and is required for all cleavages performed by this enzyme. In the case of EAV, however, the nsp2 cofactor role appears to be limited to just one of the eight cleavages carried out by the nsp4 SP. It may rather affect the conformation of the region containing the nsp4/5 junction than the activity of the nsp4 protease domain itself, since the SP was perfectly capable of processing other sites in the absence of nsp2 (e.g., Fig. 3 and Fig. 6). Interestingly, coexpression experiments using nsp2 and nsp3-8 revealed that nsp2 could fulfill its cofactor role in trans (Fig. 6), a property that will facilitate a future, more detailed analysis of the interaction between these two molecules. In these experiments we also ruled out the involvement of the proteolytic activity located in the N-terminal region of nsp2. Both nsp2 and nsp3-8 appear to be complex molecules containing several clusters of conserved Cys residues (34) and several hydrophobic domains, which have recently been implicated in the membrane association of the EAV replication complex (38, 41). Previously, we have described a strong interaction between nsp2 and nsp3 (or nsp3containing precursors) which may be important for the nsp2 cofactor role. Possibly, this interaction is required to obtain the membrane-associated conformation of the nsp2/nsp3-8 complex which allows the subsequent cleavage of the nsp4/5 site by the SP. When nsp3-8 does not interact with nsp2, its internal nsp4/5 site cannot be processed and consequently the molecule processes itself by following the minor pathway (Fig. 7). Conversely, after processing of the nsp4/5 junction, the conformation of nsp5-8 (later processed into nsp5-7) appears to be such that cleavage of its internal nsp5/6 and nsp6/7 sites is prevented. The minor nsp3-8 processing pathway is only used to a limited extent in infected cells. The subunits which it generates could be considered dead-end products, resulting from the failure of a small part of nsp3-8 to associate with nsp2 in time. In our opinion, however, the absolute conservation in arteriviruses of the newly discovered nsp5/6 and nsp6/7 cleavage sites strongly suggests a specific function in the EAV replication cycle for the products of the minor pathway. Together,

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these observations indicate that translation, processing, and membrane association of the nsp2 to nsp8 region are interconnected processes. The nsp4 serine protease. Three proteases and seven cleavage sites have now been identified in the EAV ORF1a protein, and extensive sequence comparisons do not suggest the presence of additional protease domains and/or cleavage sites. Various lines of circumstantial evidence (36) had suggested that the cleavage site specificity of the EAV nsp4 SP is Glu2(Ser/Gly). This has now been formally confirmed by direct protein sequence analysis. We have first determined the approximate location of the nsp5 N terminus (Fig. 2) and have subsequently purified and microsequenced a Sin-expressed equivalent of nsp5-7 (Fig. 4). The profiles obtained from Nterminal sequencing of the [35S]methionine-labeled (Fig. 4C) and [3H]leucine-labeled (Fig. 4D) nsp5 N terminus unequivocally identified Ser-1269 as the N-terminal residue. The cleavage site specificity of the SP was further corroborated by sequencing of the N terminus of the copurified His-tagged nsp7 equivalent (Fig. 4E). For a more detailed discussion of the substrate-binding properties of the nsp4 SP, we refer the reader to our previously published analysis of the arterivirus main protease (36). Finally, it is noteworthy that many forms of the EAV main protease, the nsp4 SP, appear to be present in infected cells. In addition to fully cleaved nsp4, which may be cytosolic (38), cleavage products in which the protease is associated with upstream (nsp3-4) or downstream (nsp4-5 or nsp4-6) hydrophobic domains are generated. These forms of the SP can be expected to be membrane bound, a property that will certainly influence their functioning. The recently developed infectious cDNA clone of EAV (39) will be an important tool in determining the role of the various protease forms and the alternative processing pathways in the life cycle of the virus. ACKNOWLEDGMENTS We are grateful to R. Amons (Department of Medical Biochemistry, Leiden University) for carrying out the N-terminal microsequence analysis. Protein sequencing was carried out at the gas phase sequenator facility which is supported by The Netherlands Foundation for Chemical Research (SON). We thank Jacomine Krijnse Locker and Hans van Tol for assistance in the analysis of the membrane association of the replication complex. We are grateful to Peter Bredenbeek for the Sin expression vectors and related information. We thank Leonie van Dinten and Johan den Boon for useful comments and discussions. A.E.G. was partly supported by a grant from The Netherlands Organization for Scientific Research (NWO) and by a grant from the Russian Fund for Basic Research. REFERENCES 1. Amberg, S. M., A. Nestorowicz, D. W. McCourt, and C. M. Rice. 1994. NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J. Virol. 68:3794–3802. 2. Arias, C. F., F. Preugschat, and J. H. Strauss. 1993. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology 193:888–899. 3. Bredenbeek, P. J., I. Frolov, C. M. Rice, and S. Schlesinger. 1993. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J. Virol. 67:6439–6446. 4. Cavanagh, D. 1997. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch. Virol. 142:629–633. 5. Chambers, T. J., A. Nestorowicz, S. M. Amberg, and C. M. Rice. 1993. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J. Virol. 67:6797–6807. 6. Chambers, T. J., A. Nestorowicz, and C. M. Rice. 1995. Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication. J. Virol. 69:1600–1605.

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