©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Arterivirus Nsp2 Protease
AN UNUSUAL CYSTEINE PROTEASE WITH PRIMARY STRUCTURE SIMILARITIES TO BOTH PAPAIN-LIKE AND CHYMOTRYPSIN-LIKE PROTEASES (*)

Eric J. Snijder (1)(§), Alfred L. M. Wassenaar (1), Willy J. M. Spaan (1), Alexander E. Gorbalenya(§) (2) (3)(¶)

From the (1)Department of Virology, Institute of Medical Microbiology, Faculty of Medicine, Leiden University, Postbus 320, 2300 AH Leiden, The Netherlands, the (2)Institute of Poliomyelitis and Viral Encephalitides, Academy of Medical Sciences, 142782 Moscow Region, Russia, and the (3)Department of Biological Sciences, Purdue University, W. Lafayette, Indiana 47907-1392

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The replicase ORF1a polyprotein of equine arteritis virus, a positive-stranded RNA virus, is proteolytically processed into (at least) six nonstructural proteins (Nsp). A papain-like Cys protease in Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this process. In this paper we demonstrate that the Nsp2/3 junction is not cleaved by either of these previously described proteases. Comparative sequence analysis suggested that an additional Cys protease resided in the N-terminal Nsp2 domain. For equine arteritis virus, this domain was shown to induce Nsp2/3 cleavage in a trans-cleavage assay. Processing was abolished when the putative active site residues, Cys-270 and His-332, were replaced. Other Nsp2 domains and three other conserved Cys residues were also shown to be essential. The Nsp2 Cys protease displays sequence similarity with viral papain-like proteases. However, the presumed catalytic Cys-270 is followed by a conserved Gly rather than the characteristic Trp. Replacement of Gly-271 by Trp abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a hallmark of viral chymotrypsin-like Cys proteases. Thus, the arterivirus Nsp2 protease is an unusual Cys protease with amino acid sequence similarities to both papain-like and chymotrypsin-like proteases.


INTRODUCTION

Proteolytic processing of viral proteins fulfills a key role in the life cycle of the majority of viruses(1, 2, 3) . Positive-stranded RNA viruses usually synthesize polyproteins composed of structural and/or replicative subunits. The processing of nonstructural precursors is almost exclusively conducted by specific virus-encoded proteases. The majority of these enzymes are assumed to be distant relatives of papain-like or chymotrypsin-like cellular proteases(4) .

Between positive-stranded RNA viral and cellular papain-like proteases the only conserved residues appear to be: a catalytic Cys, followed by a bulky hydrophobic residue (usually Trp), and a more downstream catalytic His(5) . The distance between Cys and His tends to be smaller and more variable in viral papain-like proteases. It remains to be determined whether viral and cellular papain-like proteases are structurally similar.

Only a fraction of the viral chymotrypsin-like proteases contains the ``canonical'' catalytic triad of Ser, His, and Asp, which is found in cellular chymotrypsin-like enzymes(6, 7) . Several viral chymotrypsin-like proteases employ an active site Cys instead of Ser or a Glu instead of Asp(8, 9) . It was argued that viral chymotrypsin-like Cys proteases (also known as picornavirus 3C-like Cys proteases) might be direct descendants of an ancestral chymotrypsin-like protease(10, 11) . Recently, the structures of proteases belonging to the serine (12) and cysteine (13, 14) branches of the viral chymotrypsin-like group have been solved. It is now clear that, within the common framework of the two -barrel domain fold, the divergent evolution of catalytic systems and structures of positive-stranded RNA virus proteases has generated a set of unique proteolytic enzymes.

Both papain-like and chymotrypsin-like proteases are involved in the replication cycle of arteriviruses, a newly recognized group of animal positive-stranded RNA viruses(15) . The genome structure of three closely related arteriviruses, equine arteritis virus (EAV; Ref. 16),()porcine reproductive and respiratory syndrome virus (PRRSV; Ref. 17), and lactate dehydrogenase-elevating virus (LDV; Ref. 18), was determined recently. The 5` three-quarters of the 12.7-kilobase genome of EAV, the arterivirus prototype, contain two open reading frames (ORFs), which encode replicative proteins. The ORF1a product is 1727 amino acids (aa) long. ORF1b is expressed through ribosomal frameshifting which generates a 3175-aa ORF1ab fusion protein (16). In infected cells and expression systems, the ORF1a polyprotein is cleaved at least five times, yielding nonstructural proteins (Nsp) 1 through 6 (Fig. 1; Ref. 19). A papain-like Cys protease (PCP) in EAV Nsp1 cleaves the Nsp1/2 junction(20) . A chymotrypsin-like Ser protease was identified in the arterivirus Nsp4 protein (16) and is currently being characterized. In this paper we show that neither of these proteases mediates the cleavage of the Nsp2/3 site. Instead, a Cys protease in the N-terminal domain of Nsp2 was shown to be involved in this process. To a certain extent, this new protease resembles both papain-like and chymotrypsin-like Cys proteases. In our opinion, this unusual Cys protease occupies a unique position among the different protease families.


Figure 1: Expression of EAV ORF1a and ORF1a deletion mutants using the vaccinia virus/T7 system (33). The upper part of the figure shows a schematic representation of the expressed proteins. The EAV PCP and SP domains, corresponding cleavage sites, and Nsp1-6 are indicated for construct pM1a. Deletions made in pM1a to obtain other expression vectors are indicated. The sizes of the full-length expression products were calculated from their known amino acid sequences. The sizes of the pMX8 and pM cleavage products are estimates, which are based on the assumption that the Nsp2/3 cleavage site is located close to residue 825. Expression products were immunoprecipitated using an anti-Nsp2 serum (19) and analyzed by SDS-polyacrylamide gel electrophoresis. Lysates from mock- (M) and EAV-infected (E) cells and from mock-transfected (MT) cells were included as controls. Note that the vaccinia virus infection induces a set of background bands (especially in the 30-50-kDa region) which is absent from the M and E lanes. The precipitation of the C-terminal cleavage products of the pMX8 and pM proteins by the anti-Nsp2 serum is due to the previously described association between Nsp2 and Nsp3 (19).




MATERIALS AND METHODS

Comparative Sequence Analysis

EAV nucleotide (nt) and aa numbers refer to previously published sequences(16) . Pairwise sequence comparisons were performed using the DotHelix program(21) . Multiple sequence alignments were produced employing the OPTAL (22) or CLUSTAL V programs(23) . Different scoring tables, including those of the PAM (24) and BLOSSUM (25) families, were utilized. Protein sequences were compared with the nonredundant database maintained by the National Center for Biotechnological Information and the SwissProt data base using the BLAST (26) and BLITZ (27) programs, respectively. Profile analysis (28) was supported by the GCG package(29) . An improved version of the profile analysis (30) was employed for comparison of profiles with other profiles and sequences.

Expression Construct pM1a

Recombinant plasmids were constructed using standard techniques. Expression vector pM1a, a pBS (Stratagene) derivative, contained the complete EAV ORF1a sequence downstream of a T7 promoter, a copy of the encephalomyocarditis virus internal ribosomal entry site, which was used to enhance translation(31) , and 12 nt encoding the N-terminal extension Met-Ala-Thr-Thr(19) . The ORF1a sequence in pM1a was modified to facilitate mutagenesis; translationally silent mutations were introduced to create unique restriction sites. A detailed description of pM1a will be published elsewhere. The new restriction sites used here were NcoI (nt 224, containing the ORF1a initiation codon) and SphI (nt 1975).

Other Expression Vectors

Constructs pMX6 and pMX8 (Fig. 1) were derived from pM1a by insertion of a termination codon into SalI (nt 2608) and ApaI (nt 3688) restriction sites, respectively. To create pM (Fig. 1), two deletions were made in pM1a. First, a translation initiation codon was engineered close to the Nsp1/2 border and the Nsp1-encoding sequence was deleted. The N-terminal sequence of the pM protein was Met-Ala-Thr-Thr-Met-Val/Gly-261. The second deletion comprised the sequence encoding Nsp4 and a part of Nsp5. To this end, SmaI restriction sites were generated at nt 3415 and nt 4513 and an in-frame SmaI deletion was made. The Nsp2 deletion mutants (Fig. 3) were created by digestion of pairs of unique pM restriction sites and subsequent religation. The following sites were used: NcoI (nt 997), HindIII (nt 1501), KpnI (nt 1802), SphI (nt 1875), ClaI (nt 2296), and SalI (nt 2608). Bicistronic construct pBC (Fig. 4) was generated by insertion of the following sequence elements into the pM HindIII site at nt 1501: a termination codon for the first cistron, a second encephalomyocarditis virus internal ribosomal entry site, and an NcoI site containing the initiation codon for the second cistron.


Figure 3: Expression of Nsp2 deletion mutants (see also Fig. 1). Six in-frame deletions (indicated as A through F) were made in the Nsp2-encoding region of construct pM. The deleted residues are indicated on the left. The hatched box represents the central hydrophobic domain in Nsp2. Immunoprecipitations were carried out using anti-Nsp2 (2) and anti-Nsp5 (5) sera (19). The position in the gel of the 58-kDa C-terminal pM cleavage product, which is indicative of the Nsp2/3 cleavage, is shown.




Figure 4: Construction and expression of bicistronic construct pBC. The putative Nsp2 CP (pBC protease) and its substrate (pBC precursor) were expressed as two separate proteins and immunoprecipitated using the anti-Nsp2 (2) and anti-Nsp5 (5) sera (19). The results obtained with pM and control constructs expressing either the first (pMX3) or the second (pM(d261-426)) cistron are shown. The N- and C-terminal pBC cleavage products, which are indicative of processing of the Nsp2/3 site in the precursor, are indicated. Replacement of Cys-270 (C270S) and His-332 (H332Y) and two in-frame Nsp2 deletions (d427-794 and d427-691) abolished cleavage. IRES, internal ribosomal entry site.



Mutagenesis

Nucleotide changes were introduced using oligonucleotide-directed mutagenesis as described by Kunkel et al.(32) . After complete sequence analysis, restriction fragments carrying mutations were transferred back to pM1a or pM1a-derived vectors.

Expression and Protein Analysis

Transient T7 expression using vaccinia virus recombinant vTF7-3(33) , RK-13 cells, and cationic liposomes was carried out as described(19) . Proteins were labeled from 4 to 7 h postinfection using [S]methionine. The 2 and 5 antisera, and the methods for cell lysis, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis have been described previously(19) .


RESULTS

Processing of the EAV Nsp2/3 Site Is Not Mediated by the Nsp1 Papain-like Protease or the Nsp4 Chymotrypsin-like Protease

When full-length EAV ORF1a construct pM1a was expressed, the 61-kDa Nsp2 protein was detected (Fig. 1), indicating that the Nsp2/3 junction was cleaved efficiently. The slightly smaller size (58-kDa; Fig. 1) of the pMX6 product indicated that the Nsp2/3 cleavage site is located approximately 30 aa downstream of Val-795. Cleavage at this site was observed with truncated constructs pM and pMX8 (Fig. 1). The 120-kDa pM product lacked both the Nsp1 and Nsp4 protease domains, indicating that the Nsp2/3 site was cleaved by a third protease. The Nsp56 region could be excluded as the possible location of this new protease, since it was absent from the pMX8 product.

The pM construct was used for a more detailed experimental analysis of the processing of the Nsp2/3 site (see below). Its N-terminal cleavage product (62 kDa) consists of Nsp2 and a six-residue N-terminal extension. Its C-terminal cleavage product, which was estimated at 58 kDa but consistently migrated in the 50-kDa region of our gels, was comprised of Nsp3 and the C-terminal 297 residues of the ORF1a protein.

Computer-assisted Identification of a Novel Type of Cysteine Protease in Nsp2

With a few exceptions, positive-stranded RNA viral nonstructural polyproteins are processed by virus-encoded proteases. Hence, we assumed that the protease which cleaves the EAV Nsp2/3 site resided in Nsp2 or Nsp3. Comparison of arterivirus Nsp2 and Nsp3 sequences with sequence data bases did not produce any significant matches. Assuming that the unknown protease could be conserved among arteriviruses, we scanned the EAV, LDV, and PRRSV sequences for conserved residues that might be involved in proteolytic catalysis (Ser, Cys, His, Asp, Glu, or Asp/Glu).

Our computer analysis showed that the arrangement of the conserved Cys-270 and His-332 residues (Fig. 2A) in the N-terminal Nsp2 domain resembles the catalytic dyad of papain-like proteases of arteriviruses and other positive-stranded RNA viruses. The sequences surrounding the LDV/PRRSV counterpart of EAV Cys-270 produced a match with the region around the catalytic Cys-164 of the EAV Nsp1 papain-like protease (PCP; Fig. 2B). A close inspection of the Cys-270 region revealed that it also resembles the context of the catalytic nucleophile of chymotrypsin-like Cys proteases (Fig. 2C). Furthermore, it was noticed that His-332 is the only conserved His residue in the sequences of Nsp2 and Nsp3. This type of residue fulfills a catalytic role in cysteine, serine, and metalloproteases(34) . Its presence was an additional indication for a possible proteolytic function of the Nsp2 N-terminal domain. Therefore, we speculated that Cys-270 and His-332 form the catalytic dyad of an unusual type of Cys protease (CP), which mediates the Nsp2/3 cleavage.


Figure 2: Delineation and comparison of the arterivirus Nsp2 cysteine protease (CP) domain. A, alignment of the N-terminal Nsp2 domain of the arteriviruses LDV, PRRSV, and EAV. asterisk, putative catalytic residues; +, residues which were mutated (Fig. 5). B, dot plot comparison of the N-terminal regions of EAV and LDV/PRRSV ORF1a proteins. An LDV/PRRSV alignment profile was made and used for comparison in the Proplot program (30) using the Blossum 62 table, a 21-aa window, and a 0.05 cut-off level. Processing schemes of EAV (left) and LDV/PRRSV (bottom) are shown along the axes. The location of protease domains and selected (catalytic) residues are shown at the opposite axes. The inactivated EAV PCP* domain (39) contains a Lys at the position of the catalytic Cys. The importance of the various PCP Cys and His residues has been documented elsewhere (20, 39). Matches revealing similarity between presumed orthologous or paralogous domains are indicated by fat diagonals. C, comparison of the context of the putative Nsp2 CP active site residues (asterisk) with that of catalytic residues in arterivirus Nsp1 papain-like proteases and a number of viral chymotrypsin-like (picornavirus 3C-like) Cys proteases. Abbreviations: BaYMV, barley yellow mosaic virus; FCV, feline calicivirus; RHDV, rabbit hemorrhagic disease virus (42). Swissprot (SW) or GenBank (GB) data base accession numbers are shown.



The Entire Nsp2 Protein Appears to Be Essential for Proteolytic Activity

Four domains could be discriminated in arterivirus Nsp2 proteins. The N- and C-terminal regions (about 100 and 200 aa, respectively) are highly conserved and rich in Cys residues. A 130-aa hydrophobic domain precedes the conserved C-terminal region. The remaining part of Nsp2 is hydrophilic and extremely variable, both in size (160-600 aa) and in sequence(18, 19) . To test the involvement of the putative Nsp2 CP domain in the Nsp2/3 cleavage, we deleted the sequence encoding the Nsp2 N-terminal region from construct pM (Fig. 3, deletion A). As expected, processing of the Nsp2/3 site was completely abolished. In addition to the pM precursor protein, an indistinct band in the 90-kDa region was observed. Similar bands were also seen when some of the other pM deletion mutants were expressed. Their origin is unclear, although there appears to be a correlation between their size and the size of the corresponding precursor protein.

Subsequently, we deleted part of the Nsp2 sequence which separates the putative CP and its cleavage site (Fig. 3, deletions B and C). Also these deletions inhibited proteolysis, since the 58-kDa C-terminal cleavage product was not produced in detectable amounts. Even smaller deletions in the hydrophilic, hydrophobic, and C-terminal Cys-rich Nsp2 regions (Fig. 3, deletions D, E, and F) were not tolerated, suggesting that an intact Nsp2 is required for processing of the Nsp2/3 site. Again some of the constructs yielded some unexplained minor bands (e.g. two bands in the 55-60-kDa region in the case of deletion B; Fig. 3), which we assume to be unrelated to the processing of the Nsp2/3 junction.

The N-terminal Nsp2 Domain Can Induce the Nsp2/3 Cleavage in trans

To test trans-cleavage activity, the putative Nsp2 CP and the rest of the pM protein were separately expressed from bicistronic construct pBC (Fig. 4). The product of the first cistron, containing the N-terminal 165 aa of Nsp2, was seen as a double band of approximately 18 kDa. This doublet is explained by the presence of two translation initiation codons, separated by 9 nt, at the 5` end of the ORF(35) . A substantial part of the pBC substrate encoded by the second cistron was cleaved, apparently at the authentic Nsp2/3 site (Fig. 4). This showed that the N-terminal Nsp2 domain can induce processing in trans. When Cys-270 or His-332 were mutated, processing was completely abolished. The same result was obtained when the central and/or C-terminal domains of Nsp2 were deleted from the substrate (Fig. 4).

Probing the Putative Protease Domain by Site-directed Mutagenesis

To characterize the putative CP in more detail, 30 substitutions at 12 different positions in the Nsp2 N-terminal region were tested (Fig. 5). Substitution of Cys-270 and His-332, the putative catalytic residues, abolished proteolytic activity. This was also the case when three other conserved Cys residues (aa 319, 349, and 354; Fig. 2A) were replaced. Previously, similar results were obtained during a mutagenesis study of conserved Cys residues of another Cys protease, the poliovirus 2A protein(36) . Even the mutagenesis of nonconserved Cys residues in EAV Nsp2 was not without consequences: although a Cys-344 Ala substitution was tolerated, the Cys-344 His mutant was severely impaired. Conversely, a His, but not an Ala, substitution was tolerated at the position of Cys-356 (Fig. 5). Conservative replacements within a cluster of acidic residues (aa positions 291 and 295-297) did not influence proteolysis. Finally, a Gly-271 Trp substitution completely disabled the protease. When tested in bicistronic construct pBC, a selection of these mutations produced results identical to those obtained with the monocistronic pM (data not shown).


Figure 5: Site-directed mutagenesis of the putative Nsp2 CP (see also Fig. 2A). Amino acid substitutions, indicated in single-letter codes, were introduced into reporter construct pM and tested by expression in the vaccinia/T7 system (33). The position in the gel of the pM precursor (P) and the N-terminal (N) and C-terminal (C) cleavage products is indicated.




DISCUSSION

We have shown that the proteolysis of the Nsp2/3 junction in the EAV ORF1a protein is not mediated by the previously described Nsp1 and Nsp4 proteases. Instead, our theoretical and experimental data strongly suggest that Nsp2 contains a novel type of Cys protease, which cleaves the Nsp2/3 site. A putative catalytic dyad was identified by comparative sequence analysis and shown to be essential for proteolytic activity (Fig. 5). The context of Cys-270 resembles that of the catalytic nucleophiles of known proteases from both the papain-like and chymotrypsin-like protease families (Fig. 2C and ). Furthermore, it was shown that the Nsp2 CP forms a distinct domain which can induce cleavage of the Nsp2/3 site in trans (Fig. 4). Formally, our results do not exclude the possibility that the Nsp2 N-terminal domain serves as a co-factor for an unidentified host protease. It should be noticed, however, that to date many proteolytic cleavages in viral nonstructural polyproteins were attributed to viral proteases, whereas only two flavivirus cleavages were shown to be mediated by host proteases(37, 38) .

Immunofluorescence experiments with infected cells have revealed that Nsp2 is associated with intracellular membranes (data not shown). This interaction may be mediated by the hydrophobic domain of Nsp2. Likewise, the extremely hydrophobic Nsp3 may be membrane-bound. Both Nsp2 and Nsp3 contain clusters of conserved Cys residues and remain associated after cleavage of the Nsp2/3 site(19) . Together with the data from our Nsp2 deletion and site-directed mutagenesis ( Fig. 3and 5), these observations suggest that the formation of a specific Nsp2-Nsp3 complex, possibly involving host components like membranes, is required for proteolysis of the Nsp2/3 junction. This could explain the deleterious effects of our Nsp2 deletions and some amino acid substitutions. Our failure to demonstrate the Nsp2/3 cleavage in bacterial cells and reticulocyte lysates()may be attributed to the absence of certain host components. Apparently, a complex with a cleavable Nsp2/3 site can be formed when the Nsp2 CP domain is expressed separately and the remaining part of Nsp23 is intact (Fig. 4). It is also remarkable that the previously described insertion of a 10-kDa heterologous sequence into Nsp2 (at aa 427) did not affect cleavage at the Nsp2/3 site(19) .

Extensive site-directed mutagenesis of Cys-270 and His-332 showed that they are indispensable for proteolytic activity. The relative position of these putative catalytic residues is typical for viral papain-like proteases. Two papain-like protease domains have been recognized in the Nsp1 region of arteriviruses (Refs. 20 and 39; Fig. 2B). In LDV and PRRSV both proteases, named PCP and PCP, are cis-acting Cys proteases which produce Nsp1 and Nsp1 (39). The PRRSV/LDV PCP domain and Nsp1/2 cleavage correspond to the EAV Nsp1 PCP and Nsp1/2 cleavage, respectively. The EAV counterpart of LDV/PRRSV PCP (PCP*; Fig. 2B) has lost the catalytic Cys(39) . The Nsp1 PCPs are clearly more diverged than the Nsp2 CPs (Fig. 2B), but a number of similarities can be detected and the Nsp2 CP appears to be more closely related to the viral papain-like protease group than to other currently known groups of proteases. Thus, the Nsp2 CP is likely to be another descendant of an ancestral papain-like protease.

Still, two important differences between the arterivirus Nsp2 CP and viral papain-like proteases can be observed. In the first place, most viral papain-like enzymes cleave just downstream of the catalytic His residue(5) . In the case of the EAV Nsp2 CP approximately 500 residues separate the putative protease and its cleavage site, and this distance may even be up to 450 residues larger in other arteriviruses(19, 39) . Most of all, however, the conserved Gly-271 which follows catalytic Cys-270 is a special feature of the Nsp2 CP. In arterivirus Nsp1 PCPs, and other papain-like proteases, this position is typically occupied by Trp, or sometimes another bulky amino acid, but never by Gly(5) . Remarkably, conservation of an active site Cys-Gly dipeptide is a hallmark of chymotrypsin-like Cys proteases (Fig. 2C), in which the linear order of catalytic Cys and His is reversed in comparison with papain-like proteases(4) . When EAV Gly-271 was replaced by Trp, the Nsp2/3 cleavage was abolished (Fig. 5). This underlines its importance and indicates that the Trp Gly replacement is probably coupled to additional substitutions. Chymotrypsin-like proteases contain a catalytic Asp/Glu in the region separating the active site His and Ser/Cys residues. In EAV Nsp2, the region between Cys-270 and His-332 contains a conserved Asp, but mutagenesis of this and other acidic residues did not affect the Nsp2/3 cleavage (Fig. 5). However, this result may be inconclusive, since mutagenesis proved to be an inappropriate tool for the identification of a catalytic Asp in at least one viral protease(40) . Together, the characteristics described above distinguish the Nsp2 CP from previously described papain-like and chymotrypsin-like Cys proteases.

The sequence similarities observed for the arterivirus Nsp2 Cys protease can be analyzed in the context of the structural properties of the papain-like and chymotrypsin-like enzymes. The fold and the linear order, context, and type of active site residues are different for both these groups of cellular proteases. The only structural resemblance between them, which was noticed previously by Garavito et al.(41) , is the apparently similar organization of the catalytic triads of papain and chymotrypsin, Cys/His/Asn and Ser/His/Asp, respectively. However, these triads cannot be spatially superimposed, since they are of opposite hand. The resemblance was therefore concluded to be the result of convergent evolution(41) .

The recent identification of different RNA viral Cys proteases has greatly expanded the diversity of both papain-like and chymotrypsin-like proteases. Similarities within and between the two groups were highlighted (), and comprehensive evidence was reported for the common ancestry of a part of the RNA viral Cys proteases and the cellular and viral chymotrypsin-like Ser proteases (13, 14, 42). Due to the lack of structural data, the evolutionary relationships between cellular and viral papain-like proteases, including the arterivirus Nsp2 CP, are less clear, but the available amino acid sequence information favors divergent evolution. On the basis of the analysis of the triplets encoding a variety of catalytic Ser residues, Brenner has postulated previously that an ancestral Cys protease may have preceded the chymotrypsin-like Ser proteases(10) . The analysis of present day chymotrypsin-like Cys proteases points toward the conserved Gly-X-Cys-Gly sequence () as the most plausible context of the catalytic Cys residue in such an ancestral chymotrypsin-like protease. Remarkably, this characteristic Gly-X-Cys-Gly sequence is also conserved in the catalytic center of the arterivirus Nsp2 Cys proteases that belong to the papain-like group (). Although divergent evolution could obviously provide the most parsimonious explanation for this similarity between chymotrypsin-like and Nsp2 Cys proteases, this interpretation cannot be reconciled with the postulated convergent evolution of the prototypes of the two families, chymotrypsin and papain(41) . Future studies of RNA virus proteases, particularly the arterivirus Nsp2 CP, may resolve this puzzle and increase our insight in a possible evolutionary relationship between papain-like and chymotrypsin-like proteases.

  
Table: Gly -


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Mailing address (for both authors): Dept. of Virology, Postbus 320, 2300 AH Leiden, The Netherlands. Tel.: 31-71261657; Fax: 31-71263645; E-mail: azruviro@rulcri.leidenuniv.nl (E. J. S.); AEGOR@ipive.msk.su (A. E. G.).

Supported by grants from the Netherlands Organization for Scientific Research (NWO) and the International Science Foundation and by a Cooperation in Applied Science and Technology award.

The abbreviations used are: EAV, equine arteritis virus; CP, cysteine protease; Nsp, nonstructural protein; LDV, lactate dehydrogenase-elevating virus; ORF, open reading frame; PCP, papain-like cysteine protease; PRRSV, porcine reproductive and respiratory syndrome virus; aa, amino acid(s).

E. J. Snijder, A. L. M. Wassenaar, W. J. M. Spaan, and A. E. Gorbalenya, unpublished data.


ACKNOWLEDGEMENTS

We thank Johan den Boon, Leonie van Dinten, and Peter Bredenbeek for helpful discussions and critical reading of the manuscript. A. E. G. is most grateful to Michael Rossmann (supported by a Cooperation in Applied Science and Technology award and a National Science Foundation grant) for encouragement and support during his work in Purdue.


REFERENCES
  1. Kräusslich, H. G., and Wimmer, E. (1988) Annu. Rev. Biochem.57, 701-754 [CrossRef][Medline] [Order article via Infotrieve]
  2. Strauss, E. G., and Strauss, J. H. (1990) Semin. Virol.1, 347-356
  3. Dougherty, W. G., and Semler, B. L. (1993) Microbiol. Rev.57, 781-822 [Abstract]
  4. Gorbalenya, A. E., and Koonin, E. V. (1993) Sov. Sci. Rev. D. Physicochem. Biol.11, 1-84
  5. Gorbalenya, A. E., Koonin, E. V., and Lai, M. M. C. (1991) FEBS Lett.288, 201-205 [CrossRef][Medline] [Order article via Infotrieve]
  6. Bazan, J. F., and Fletterick, R. J. (1989) Virology171, 637-639 [Medline] [Order article via Infotrieve]
  7. Gorbalenya, A. E., Donchenko, A. P., Koonin, E. V., and Blinov, B. M. (1989) Nucleic Acids Res.17, 3889-3897 [Abstract]
  8. Bazan, J. F., and Fletterick, R. J. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 7872-7876 [Abstract]
  9. Gorbalenya, A. E., Donchenko, A. P., Blinov, V. M., and Koonin, E. V. (1989) FEBS Lett.243, 103-114 [CrossRef][Medline] [Order article via Infotrieve]
  10. Brenner, S. (1988) Nature334, 528-530 [CrossRef][Medline] [Order article via Infotrieve]
  11. Gorbalenya, A. E., Blinov, V. M., and Donchenko, A. P. (1986) FEBS Lett.194, 253-257 [CrossRef][Medline] [Order article via Infotrieve]
  12. Choi, H.-K., Tong, L., Minor, W., Dumas, P., Boege, U., Rossmann, M. G., and Wengler, G. (1991) Nature354, 37-43 [CrossRef][Medline] [Order article via Infotrieve]
  13. Allaire, M., Chernaia, M. M., Malcolm, B. A., and James, M. N. G. (1994) Nature369, 72-76 [CrossRef][Medline] [Order article via Infotrieve]
  14. Matthews, D. A., Smith, W. W., Ferre, R. A., Condon, B., Budahazi, G., Sisson, W., Villafranca, J. E., Janson, C. A., McElroy, H. E., Gribskov, C. L., and Worland, S. (1994) Cell77, 761-771 [Medline] [Order article via Infotrieve]
  15. Plagemann, P. G. W., and Moennig, V. (1991) Adv. Virus Res.41, 99-192
  16. Den Boon, J. A., Snijder, E. J., Chirnside, E. D., de Vries, A. A. F., Horzinek, M. C., and Spaan, W. J. M. (1991) J. Virol.65, 2910-2920 [Medline] [Order article via Infotrieve]
  17. Meulenberg, J. J. M., Hulst, M. M., de Meijer, E. J., Moonen, P. L. J. M., den Besten, A., de Kluyver, E. P., Wensvoort, G., and Moormann, R. J. M. (1993) Virology192, 62-72 [CrossRef][Medline] [Order article via Infotrieve]
  18. Godeny, E. K., Chen, L., Kumar, S. N., Methven, S. L., Koonin, E. V., and Brinton, M. A. (1993) Virology194, 585-596 [CrossRef][Medline] [Order article via Infotrieve]
  19. Snijder, E. J., Wassenaar, A. L. M., and Spaan, W. J. M. (1994) J. Virol.68, 5755-5764 [Abstract]
  20. Snijder, E. J., Wassenaar, A. L. M., and Spaan, W. J. M. (1992) J. Virol.66, 7040-7048 [Abstract]
  21. Leontovich, A. M., Brodsky, L. I., and Gorbalenya, A. E. (1993) Biosystems30, 57-63 [Medline] [Order article via Infotrieve]
  22. Gorbalenya, A. E., Blinov, V. M., Donchenko, A. P., and Koonin, E. V. (1989) J. Mol. Evol.28, 256-268 [Medline] [Order article via Infotrieve]
  23. Higgins, D. G., Bleasby, A. J., and Fuchs, R. (1992) Comput. Appl. Biosci.8, 189-191 [Abstract]
  24. Dayhoff, M. O., Schwartz, R. M., and Orcutt, B. C. (1978) Atlas of Protein Sequence and Structure, pp. 345-352, National Biomedical Research Foundation, Washington, D. C.
  25. Henikoff, S., and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 10915-10919 [Abstract]
  26. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol.215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  27. Sturrock, S. S., and Collins, J. F. (1993) MPsrch version 1.3, Biocomputing Research Unit, University of Edinburgh, Edinburgh, United Kingdom
  28. Gribskov, M., McLachlan, A. D., and Eisenberg, D. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 4355-4358 [Abstract]
  29. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res.12, 387-395 [Abstract]
  30. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Comput. Appl. Biosci.10, 19-29 [Abstract]
  31. Jang, S. K., Kräusslich, H. G., Nicklin, M. J. H., Duke, G. M., Palmenberg, A. C., and Wimmer, E. (1988) J. Virol.62, 2636-2643 [Medline] [Order article via Infotrieve]
  32. Kunkel, T. A., Roberts, J. D., and Zakour, R. (1987) Methods Enzymol.154, 367-382 [Medline] [Order article via Infotrieve]
  33. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A.83, 8122-8126 [Abstract]
  34. Rawlings, N. D., and Barrett, A. J. (1993) Biochem. J.290, 205-218 [Medline] [Order article via Infotrieve]
  35. Kaminski, A., Howell, M. T., and Jackson, R. J. (1990) EMBO J.9, 3753-3759 [Abstract]
  36. Yu, S. F., and Lloyd, R. E. (1992) Virology186, 725-735 [Medline] [Order article via Infotrieve]
  37. Lin, C., Amberg, S. M., Chambers, T. J., and Rice, C. M. (1993) J. Virol.67, 2327-2335 [Abstract]
  38. Strauss, J. H. (ed) (1990) Semin. Virol.1, 307-384
  39. Den Boon, J. A., Faaberg, K. S., Meulenberg, J. J. M., Wassenaar, A. L. M. Plagemann, P. G. W. M., Gorbalenya, A. E., and Snijder, E. J. (1995) J. Virol.69, in press
  40. Strauss, J. H., and Strauss, E. G. (1994) Microbiol. Rev.58, 491-562 [Abstract]
  41. Garavito, R. M., Rossmann, M. G., Argos, P., and Eventoff, W. (1977) Biochemistry16, 5065-5071 [Medline] [Order article via Infotrieve]
  42. Boniotti, B., Wirblich, C., Sibilia, M., Meyers, G., Thiel, H. J., and Rossi, C. (1994) J. Virol.68, 6487-6495 [Abstract]

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