Mutational analysis of the active centre of coronavirus 3C-like proteases

Annette Hegyi1, Agnes Friebe1, Alexander E. Gorbalenya2 and John Ziebuhr1

Institute of Virology and Immunology, University of Würzburg, Versbacher Straße 7, 97078 Würzburg, Germany1
Advanced Biomedical Computing Center, 430 Miller Dr. Rm 228, SAIC/NCI-Frederick, Frederick, MD 21702-1201, USA2

Author for correspondence: John Ziebuhr. Fax +49 931 2013934. e-mail ziebuhr{at}vim.uni-wuerzburg.de


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Formation of the coronavirus replication–transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CLpro, was determined. Comparative sequence analyses revealed that FIPV 3CLpro and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CLpro domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CLpro catalytic system employs His41 and Cys144 as the principal catalytic residues. Second, the amino acids Tyr160 and His162, which are part of the conserved sequence signature Tyr160–Met161–His162 and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly83 and Asn64, which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn64 mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CLpro mutants in which the equivalent Asn residue (HCoV 3CLpro Asn64) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Coronaviruses are positive-stranded RNA viruses with exceptionally large genome sizes (up to 31 kb). Based on a similar polycistronic genome organization, common transcriptional and (post)-translational strategies and a conserved array of homologous domains in the viral polyprotein, the Coronaviridae have been united with the much smaller Arteriviridae in the (newly established) order Nidovirales (which is composed of these two virus families) (den Boon et al., 1991 ; Cavanagh, 1997 ). Another common hallmark of corona- and arteriviruses is the use of a unique discontinuous transcription mechanism, resulting in the production of a nested set of subgenomic mRNAs (Spaan et al., 1983 ). Available experimental data suggest that fusion of the noncontiguous 5' and 3' sequences occurs during negative-strand RNA synthesis (Sawicki & Sawicki, 1998 ; Sawicki et al., 2001 ) and involves mechanisms that resemble similarity assisted, copy-choice RNA recombination (van Marle et al., 1999 ).

Feline infectious peritonitis virus (FIPV) strain 79-1146 as well as other feline coronaviruses belong to group I coronaviruses and are related closely to both canine coronavirus and porcine transmissible gastroenteritis virus (TGEV) (Vennema et al., 1992 ). In fact, all of these viruses are antigenically so similar that they may be regarded as ‘host range mutants’ rather than as separate species (Horzinek et al., 1982 ). FIPV infects both domestic and wild cats and generally causes mild enteric diseases. However, in about 5–10% of FIPV infections, the disease progresses and results in a debilitating lethal disease characterized by an exudative fibrinous serositis in the abdominal and thoracic cavities (reviewed by Olsen, 1993 ; de Groot & Horzinek, 1995 ). Previous sequence analyses have focussed mainly on the 3'-terminal region of the FIPV genome, which encodes the structural proteins of the virus (Vennema et al., 1992 , 1998 ; Herrewegh et al., 1995 , 1998 ). Although there is nearly no sequence information on the 5'-proximal region of the FIPV genome, it is reasonable to assume that this sequence will be very similar to the TGEV replicase gene sequence reported previously (Eleouet et al., 1995 ).

The protein functions encoded by the human coronavirus 229E (HCoV) replicase gene have been shown recently to suffice for both genome replication and transcription (Thiel et al., 2001b ) and it is likely that the same applies to other coronaviruses, including FIPV. The replicase gene occupies the 5'-proximal two-thirds of the genome and comprises two open reading frames (ORFs), ORFs 1a and 1b, which are connected by a ribosomal frameshift site (Brierley et al., 1987 ). Thus, two overlapping polyproteins are translated from the genome RNA: an ORF1a-encoded protein, ~450-kDa (pp1a), and a C-terminally extended frameshift protein. The latter protein, called pp1ab, is encoded by ORFs 1a and 1b and has a calculated molecular mass of ~750 kDa. The replicative polyproteins are processed extensively by viral proteases to produce the functional subunits of the virus replication/transcription machinery. The N-proximal region of pp1a/pp1ab is cleaved by zinc finger-containing papain-like proteases (so called accessory proteases) at two or three sites (Baker et al., 1989 ; Gorbalenya et al., 1991 ; Bonilla et al., 1997 ; Herold et al., 1998 , 1999 ; Kanjanahaluethai & Baker, 2000 ; Lim et al., 2000 ; Ziebuhr et al., 2001 ). In contrast, the C-proximal region is processed by a 3C-like cysteine protease (3CLpro), which is encoded in the 3'-proximal region of ORF1a and derives its name from a remote similarity to the picornavirus 3C proteases (Gorbalenya et al., 1989b ). 3CLpro cleaves the viral polyprotein at 11 conserved interdomain junctions (Ziebuhr et al., 2000 ), including those that release the core replicative domains, such as the putative RNA-dependent RNA polymerase (RdRp) and the helicase (Gorbalenya et al., 1989b ; Liu et al., 1994 ; Grötzinger et al., 1996 ; Heusipp et al., 1997b ; Denison et al., 1999 ; Seybert et al., 2000a ). Because of its key role in replicase gene expression, 3CLpro has been termed coronavirus ‘main protease’ (Ziebuhr et al., 2000 ). Previous studies using recombinant main proteases of avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and HCoV, representing all three major groups of coronaviruses, provided initial insight into the functional and structural properties of this enzyme (reviewed by Ziebuhr et al., 2000 ). First, the coronavirus main protease was shown to possess a well-defined substrate specificity; all cleavage sites contain bulky hydrophobic residues at the P2 position, Gln at the P1 position and small aliphatic residues at the P1' position (Liu et al., 1994 , 1997 , 1998 ; Lu et al., 1995 , 1998 ; Ziebuhr et al., 1995 ; Grötzinger et al., 1996 ; Heusipp et al., 1997a , b ; Ziebuhr & Siddell, 1999 ; Denison et al., 1999 ). Second, both sequence comparisons and mutation analyses suggested that coronavirus main proteases may employ a catalytic dyad of conserved His and Cys residues. Thus, in contrast to most other viral proteases with (predicted or proven) chymotrypsin-like folds, no clear evidence has been obtained for the involvement of a third (acidic) residue in catalysis (Liu & Brown, 1995 ; Lu & Denison 1997 ; Ziebuhr et al., 1997 ). Third, coronavirus main proteases possess an additional, C-terminal domain with unknown function(s) (Gorbalenya et al., 1989b ). Although truncations of this domain reduced significantly or abolished completely the proteolytic activities of the IBV, MHV and HCoV main proteases in both in vivo and in vitro experiments (Lu & Denison, 1997 ; Ziebuhr et al., 1997 ; Ng & Liu, 2000 ), a direct involvement of the C-terminal domain in catalysis or substrate binding appears unlikely. Thus, it was shown recently for IBV 3CLpro that the removal of as much as 67 amino acids from the C terminus was partially tolerated (Ng & Liu, 2000 ).

To gain additional insight into the biochemistry of coronavirus 3CLpro-mediated proteolysis, we have cloned and sequenced FIPV 3CLpro and compared the deduced amino acid sequence with those of other coronavirus main proteases and the more distantly related potyviral 3C-like proteases. Functional predictions derived from the sequence comparison were tested subsequently. To this end, a series of recombinant proteins carrying single amino acid replacements was expressed in bacteria and the proteolytic activities of the purified proteins were analysed using a peptide-based assay. Using this approach, we have been able to confirm and extend the results of previous studies on the active site of coronavirus main proteases. The data suggest that coronavirus main proteases have evolved a catalytic system that only resembles very distantly that of the canonical chymotrypsin-like enzymes. Furthermore, we have identified specific amino acid replacements that enhanced significantly the activity of 3CLpro, indicating that the proteolytic activities of coronavirus main proteases may be tuned to meet specific functional constraints.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Virus and cells.
FIPV strain 79-1146 was propagated in monolayers of Felis catus whole foetus (FCWF) cells (Jacobse-Geels & Horzinek, 1983 ) maintained in Dulbecco’s modified Eagle medium containing 10% foetal bovine serum, nonessential amino acids (11140-035; Life Technologies), glutamine and antibiotics.

{blacksquare} Determination of the FIPV 3CLpro-coding sequence.
Polyadenylated RNA was prepared from FIPV-infected FCWF cells and reverse transcribed using oligonucleotide AH10 [5' (A/C)ACAGC(A/T)CGTGCTTCTTT(A/G)TACAT 3']. The sequences of both AH10, which was also used as the downstream primer in the subsequent PCR, and the upstream PCR primer AH11 [5' AATCTTTT(C/T)GAAGGTGA(C/T)AAATTTG 3'] were derived from replicase gene regions that are highly conserved among group I coronaviruses. The nucleotide sequences of three independently obtained 2·5 kb PCR products were determined and the putative FIPV 3CLpro domain was identified by comparison of the deduced amino acid sequence with coronavirus replicase polyprotein sequences analysed previously (Boursnell et al., 1987 ; Lee et al., 1991 ; Herold et al., 1993 ; Eleouet et al., 1995 ). The nucleotide sequence of FIPV 3CLpro, together with the flanking sequences, was deposited in GenBank under accession number AF326575.

{blacksquare} Construction of plasmid pMALc2-FIPV 3CLpro.
The coding sequence of the putative FIPV 3CLpro domain was amplified by PCR from the cDNA template described above using oligonucleotides AH12 (5' TCCGGATTGAGAAAAATGGCA 3') and AH17 (5' CGCGGATCCTTACTGAAGATTAACACCATACATTTG 3'). The 918 bp PCR product was digested with BamHI and ligated with XmnI/BamHI-digested pMAL-c2 DNA (New England Biolabs). The resulting plasmid, pMALc2-FIPV 3CLpro, encodes a 75 kDa fusion protein consisting of the Escherichia coli maltose-binding protein (MBP) and the putative FIPV 3CLpro domain.

{blacksquare} Bacterial expression and purification of FIPV 3CLpro.
Expression of recombinant FIPV 3CLpro in E. coli TB1(pMALc2-FIPV 3CLpro) and purification of the recombinant fusion protein were carried out essentially as described previously for HCoV 3CLpro (Herold et al., 1996 ; Ziebuhr et al., 1997 ). The MBP–FIPV 3CLpro fusion protein was purified by amylose-affinity chromatography and cleaved with factor Xa to release the FIPV 3CLpro domain. The protein mixture was then loaded onto a phenyl–Sepharose HP column (Pharmacia Biotech) that had been preequilibrated with a solution containing 15 mM Bis–Tris–HCl (pH 7·0), 600 mM NaCl, 1 mM DTT and 0·1 mM EDTA. Recombinant FIPV 3CLpro was eluted with 15 mM Bis–Tris–HCl (pH 7·0), 1 mM DTT and 0·1 mM EDTA. The protein was concentrated (Centricon-3, Millipore) and dialysed with buffer I containing 20 mM Tris–HCl (pH 8·8), 55 mM NaCl, 1 mM DTT and 0·1 mM EDTA. Then, the protein solution was loaded onto an anion-exchange column (UNO Q-1, Bio-Rad Laboratories) that had been preequilibrated with buffer I. The flow-through fractions containing 3CLpro were pooled, concentrated and loaded onto a Superdex 75 column (Pharmacia Biotech) run under isocratic conditions with 11 mM Tris–HCl (pH 7·5), 100 mM NaCl, 1 mM DTT and 0·1 mM EDTA. The purified protein was concentrated to 15 mg/ml and stored at -80 °C.

{blacksquare} Peptide synthesis.
The synthetic 15-mer peptides F1, SP7 and SP8 were prepared by solid-phase chemistry (Merrifield, 1965 ) and purified by high-performance liquid chromatography (HPLC) on a reverse-phase C18 silica column (Jerini Bio-Tools). Identity and homogeneity of the peptides were confirmed by mass spectrometry and analytical reverse-phase chromatography. Peptide F1 represents the FIPV replicase polyprotein sequence H2N–VSVNSTLQSGLRKMA–COOH (boldface indicates the cleaved dipeptide bond). Peptides P7 and P8 have the sequences H2N–VDYGSDTVTYKSTAC–COOH and H2N–NKDASFIGKNLKSNC–COOH, respectively.

{blacksquare} Peptide cleavage.
The enzymatic activity of mutant and wild-type FIPV 3CLpro (1 µg total protein) was determined by incubation with 0·5 mM substrate peptide F1 at 25 °C in 20 mM Tris–HCl (pH 7·5), 200 mM NaCl, 1 mM DTT and 1 mM EDTA. Reaction aliquots were mixed with equal volumes of 2% trifluoroacetic acid and stored at -20 °C prior to analysis by reverse-phase HPLC on a Delta Pak C18 column (3·9x150 mm; Waters). Cleavage products were resolved using a 5–90% linear gradient of acetonitrile in 0·1% trifluoroacetic acid, as described previously (Ziebuhr et al., 1997 ). Quantification of peak areas was used to determine the extent of substrate conversion.

{blacksquare} Site-directed mutagenesis of FIPV 3CLpro.
Site-directed mutagenesis was done by a recombination PCR method, as described by Yao et al. (1992) . The nucleotide sequences of the primers used for site-directed mutagenesis are given in Table 1. The pMALc2-FIPV 3CLpro-derived plasmids encoding mutant forms of FIPV 3CLpro were then transformed into E. coli TB1 cells and the recombinant proteins were synthesized, affinity purified and cleaved with factor Xa, as described above. The purity and structural integrity of the mutant proteins were analysed by SDS–PAGE. The control protein for this experiment, wild-type FIPV 3CLpro, was purified in an identical manner.


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Table 1. Oligonucleotides used for the amplification or mutagenesis of FIPV and HCoV sequences

 
{blacksquare} Computer-aided comparative sequence analyses.
Coronavirus amino acid sequences were derived from the Genpeptides database. 3CLpro sequence alignments were produced using CLUSTAL X, version 1.81 (Thompson et al., 1997 ) and the Blossum series of scoring interresidue tables (Henikoff & Henikoff, 1994 ). The virus interfamily alignments were generated in the profile mode separately for the N- and C-terminal halves and then fused in an overall alignment. The alignments obtained were sent as input for the PhD program (Rost et al., 1995 ; Rost, 1996 ) to predict secondary structures and used also to build profiles using PROFILEWEIGHT (Thompson et al., 1994 ). These profiles were compared in pairs using PROPLOT (Thompson et al., 1994 ). Two profiles were compared by sliding windows of variable sizes along each possible register. Matches between two profiles that were within the top 0·1% and between the top 0·1 and 0·05% were marked by two different types of dots. Cluster phylogenetic trees were reconstructed using the neighbour-joining (NJ) algorithm (Saitou & Nei, 1987 ) with the Kimura correction (Kimura, 1983 ) and evaluated with 1000 bootstrap trials, as implemented in CLUSTAL X, version 1.81. Parsimonious trees were generated through exhaustive search and evaluated with bootstrap analysis using a UNIX version of PAUP* (Swofford, 2000 ) that is included in the GCG-Wisconsin Package of programs (Genetics Computer Group). Trees were prepared and modified using TREEVIEW (Page, 1996 ).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Comparative sequence analysis of FIPV 3CLpro and related coronavirus proteases
Using RT–PCR, we have amplified the complete coding region of FIPV 3CLpro. The sequences of the primers used in these experiments were derived from 3CLpro flanking regions that are highly conserved among group I coronaviruses. The nucleotide sequences of three independent RT–PCR products were determined and the deduced amino acid sequence was compared to the replicase polyprotein sequences of other coronaviruses (Figs 1 and 2). On the basis of the overall sequence similarity with other coronavirus main proteases and the flanking sites of the MHV, IBV, and HCoV main proteases characterized previously, it is safe to predict that mature FIPV 3CLpro is released from the replicase polyproteins at flanking Gln–Ser cleavage sites and encompasses 302 amino acids (Fig. 2). With 94% amino acid sequence identity, the 3CLpro domain of FIPV is related most closely to the TGEV main protease, which, as yet, remains to be characterized. Overall, the phylogeny of the 3CLpro domains (Fig. 1) closely parallels that obtained for the structural proteins (Siddell, 1995 ), the core of the RdRp (Stephensen et al., 1999 ) and the ORF1b-encoded region of pp1ab (Chouljenko et al., 2001 ), indicating a concerted evolution of all coronavirus components.



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Fig. 1. An unrooted tree of coronavirus 3C-like proteases. The tree was generated using a multiple alignment of coronavirus 3C-like proteases (see Fig. 2B) by the NJ algorithm, as implemented in the CLUSTAL X, version 1.81 program. The same topology was inferred by exhaustive tree searches using parsimony criteria (data not shown). The two bovine coronavirus (BCoV) branches collapsed to a single branch. The number of trees, in which a particular bifurcation sustained in the course of 1000 bootstrap simulations, is given at each node. The scale indicates 10% amino acid replacements.

 



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Fig. 2. Sequence conservation between coronavirus and potyvirus 3C-like proteases. (A) Dot-plot cross-comparison of corona- and potyvirus 3C-like proteases. Multiple alignments of corona- and potyvirus 3C-like proteases (see below) were converted into profiles and compared in a dot-plot fashion, as described in Methods. Shown is the dot-plot generated using a window of 37 amino acid residues. The projected positions of the catalytic residues (H46 versus H41, D81 versus G83 and C151 versus C144), as well as the substrate-binding H167 versus H162 residues, are shown at each axis. Those dots, which lay at any of the four possible crosses of projections of two functionally equivalent residues (e.g. H46 and H41) or close to a nonvisible diagonal passing these crosses, belong, or may belong, to the true matches between two profiles. The rest of the dots are background hits (false positives). (B) Sequence alignment & corona- and potyvirus 3C-like proteases. A multiple sequence alignment was produced using CLUSTAL X, version 1.81, as explained in Methods. The sequence of FIPV 3CLpro was determined in this study (AF326575) and the 3CLpro sequences of other coronaviruses, HCoV (strain 229E), TGEV (strain Purdue 115), MHV (strains JHM and A59), BCoV (isolates ENT and LUN) and IBV (strain Beaudette), were derived from the replicative polyproteins of the respective viruses whose sequences are deposited in GenBank under the following accession numbers: HCoV, X69721 (Herold et al., 1993 ), Z34093 (Eleouet et al., 1995 ), M55148 (Lee et al., 1991 ) and M95169 (Boursnell et al., 1987 ); and BCoV, AAK83364 (Chouljenko et al., 2001 ). The amino acid sequences of potyvirus 3C-like proteases were derived from polyproteins deposited in SWISS-PROT under the following accession numbers: TVMV, tobacco vein mottling virus (P09814); TUMVQ, turnip mosaic virus (strain Quebec) (Q02597); TEV, tobacco etch virus (P04517); PVY, potato virus Y (strain N) (P18247); PSBMV, pea seed-borne mosaic virus (strain DPD1) (P29152); PPVRA, plum pox virus (strain Rankovic) (P17767); PRSVH, papaya ringspot virus (strain P/mutant HA) (Q01901); PEMVC, pepper mottle virus (California isolate) (Q01500); and BSMRV, brome streak mosaic rymovirus (strain 11-Cal) (Q65730). The secondary structures of the two protease groups, as predicted by the PhD program, are shown at the top of the alignment: A, a, {alpha}-helix and B, b, {beta}-strand; predictions in upper- and lower-case letters indicate prediction reliabilities of >5 and <=5, respectively (Rost, 1996 ). The positions of the putative catalytic residues H41 and C144 (#) as well as other conserved residues characterized in this study (N64, G83, S138, Y160, M161 and H162) are highlighted by bold, italic letters.

 
The 3CLpro residues for which functional assignments have been made previously for other coronaviruses were found to be conserved also in the FIPV enzyme (Fig. 2), supporting these earlier predictions; these predictions were inferred originally from a comparison with a limited group of 3C(-like) proteases (Gorbalenya et al., 1989b ). We have decided to extend these studies and therefore compared the coronavirus main proteases with other viral chymotrypsin-like proteases in a systematic way. To this end, profiles were generated using alignments of serine and cysteine chymotrypsin-like proteases of different virus families. These profiles were then compared with the coronavirus 3CLpro profile in a dot-plot fashion. Among all proteases analysed, the potyvirus 3C-like proteases proved to be most similar to the coronavirus enzymes (data not shown), as they revealed a similarity in the regions that flank the catalytic His and Asp residues (Fig. 2A; Ziebuhr et al., 2000 ). This sequence affinity between corona- and potyvirus 3C-like proteases is compatible with other similarities between these viruses (Gorbalenya et al., 1989b ) and with the similarity between the main proteases of potyviruses and the unclassified nidovirus gill-associated virus (Cowley et al., 2000 ). When this plot (Fig. 2A) was converted to an alignment (Fig. 2B), it became evident that the catalytic Asp residue of the potyvirus proteases may be replaced by a conserved Gly residue in the coronavirus enzymes (Gly83 in FIPV 3CLpro) (Fig. 2B). This unusual substitution was also corroborated by an alignment of the predicted {beta}-strands that encompass the whole catalytic domain, including a region around the catalytic Cys residue (Fig. 2B). In the latter region, no conservation was evident in the presented (Fig. 2A) and other dot-plots (data not shown) due to unique mutations in the coronavirus proteases. To characterize the coronavirus 3C-like proteases further, some of these and other positions were probed by site-directed mutagenesis in our subsequent experiments.

Expression, purification and proteolytic activity of FIPV 3CLpro
We have reported previously the production and purification of biologically active main proteases of both MHV and HCoV using E. coli expression systems (Ziebuhr et al., 1995 , 1997 ; Seybert et al., 1997 ). In the present study, we have adapted this strategy to produce recombinant FIPV 3CLpro for biochemical analyses. FIPV 3CLpro was expressed as a fusion protein with the maltose-binding protein of E. coli and first purified using amylose-affinity chromatography (Fig. 3, lanes 1 and 2). The fusion protein was cleaved with factor Xa to release mature (302 aa) FIPV 3CLpro (Fig. 3, lane 3), which was then purified to near homogeneity by hydrophobic interaction, anion-exchange and size-exclusion chromatography (Fig. 3, lanes 4, 5, and 6).



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Fig. 3. Bacterial expression and purification of recombinant FIPV 3CLpro. Aliquots taken at each step of the purification protocol were analysed by SDS–PAGE on a 12·5% polyacrylamide gel and the proteins were stained with Coomassie brilliant blue. Lanes: M, protein molecular mass markers (kDa); 1, total cell lysate from IPTG-induced E. coli TB1(pMALc2-FIPV 3CLpro); 2, pooled peak fractions from the amylose-affinity chromatography column; 3, factor Xa-cleaved fusion protein; 4, pooled peak fractions from the phenyl–Sepharose HP column; 5, pooled peak fractions from the Uno Q column; 6, pooled peak fractions from the Superdex 75-pg column.

 
The proteolytic activity of the recombinant FIPV 3CLpro was analysed in peptide-based cleavage assays. In these experiments, 1 µg FIPV 3CLpro was incubated with a range of 15-mer peptides. Peptide F1 represented the predicted N-terminal autoprocessing site of FIPV 3CLpro and peptides P7 and P8 contained unrelated sequences. Analysis of the reaction products by reverse-phase chromatography revealed that FIPV 3CLpro hydrolysed the peptide F1 effectively, whereas the control peptides P7 and P8 remained uncleaved under the same reaction conditions, indicating the specificity of the cleavage reaction (Fig. 4). The assay thus seemed to be suitable to test the activities of FIPV 3CLpro mutants in subsequent experiments.



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Fig. 4. Peptide-based assay for the determination of the proteolytic activity of FIPV 3CLpro. Cleavage of the FIPV-specific synthetic 15-mer peptide F1 (H2N–VSVNSTLQSGLRKMA–COOH) by purified, recombinant FIPV 3CLpro was analysed by reverse-phase HPLC, as described in Methods. To assess the specificity of the reaction, the extent of proteolysis of two 15-mer control peptides, P7 (H2N–VDYGSDTVTYKSTAC–COOH) and P8 (H2N–ESGKAKPPLNRNSVC–COOH), by recombinant FIPV 3CLpro was determined under identical reaction conditions. A, Peptide F1 in the absence of FIPV 3CLpro; B, peptide P7 in the absence of FIPV 3CLpro; C, peptide P8 in the absence of FIPV 3CLpro; D, peptide F1 in the presence of FIPV 3CLpro; E, peptide P7 in the presence of FIPV 3CLpro; F, peptide P8 in the presence of FIPV 3CLpro.

 
Mutation analysis of the catalytic system of coronavirus main proteases
In a first set of experiments, we substituted two residues, His41 and Cys144, which, based on sequence alignments (Gorbalenya et al., 1989b ; Lee et al., 1991 ; Herold et al., 1993 ; Eleouet et al., 1995 ) (Fig. 2B) and previous mutagenesis studies with other coronavirus main proteases, are predicted to be the principal catalytic residues of FIPV 3CLpro. The structural integrity of the partially purified mutant proteins was confirmed by SDS–PAGE (Fig. 5A) and the proteolytic activities of the mutant proteins were measured in the peptide assay described above. As Table 2 shows, the proteolytic activities of the FIPV 3CLpro H41Y, H41R, C144S and C144A mutants were below the detection limit of the peptide assay. These data strongly support a catalytic function for His41 and Cys144 and are fully consistent with mutagenesis data published previously on the IBV, HCoV, MHV-A59 and MHV-JHM main proteases (Liu & Brown, 1995 ; Lu et al., 1995 ; Lu & Denison, 1997 ; Seybert et al., 1997 ; Ziebuhr et al., 1997 ). It should be noted that, in contrast to many other 3C and 3C-like proteases in which Cys->Ser substitutions retained partial or even full activities (Dessens & Lomonossoff, 1991 ; Hellen et al., 1991 ; Lawson & Semler, 1991 ; Grubman et al., 1995 ), the equivalent Cys->Ser replacement in FIPV 3CLpro reduced proteolytic activity to undetectable levels. Similar data have also been reported for recombinant MHV and HCoV main proteases (Seybert et al., 1997 ; Ziebuhr et al., 1997 ).



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Fig. 5. Purification of recombinant FIPV and HCoV 3C-like proteases. (A) Purification and factor Xa cleavage of MBP–FIPV 3CLpro fusion proteins carrying mutations of the predicted active-site residues His41 and Cys144. The indicated fusion proteins were partially purified by amylose-affinity chromatography, treated with endoprotease Xa and analysed by SDS–PAGE on a 12·5% polyacrylamide gel stained with Coomassie brilliant blue. Lanes M, protein molecular mass markers (kDa); 1, MBP–FIPV 3CLpro (wild-type); 2, MBP–FIPV 3CLpro H41Y; 3, MBP–FIPV 3CLpro H41R; 4, MBP–FIPV 3CLpro C144A; 5, MBP–FIPV 3CLpro C144S. (B) Purification and factor Xa cleavage of MBP–HCoV 3CLpro fusion proteins in which Asn64 was replaced by Ala, Glu, Gln and Asp, respectively. The indicated fusion proteins were partially purified by amylose-affinity chromatography, treated with endoprotease Xa and analysed by SDS–PAGE on a 12·5% polyacrylamide gel stained with Coomassie brilliant blue. Lanes M, protein molecular mass markers (kDa); 1, MBP–HCoV 3CLpro (wild-type); 2, MBP–HCoV 3CLpro N64A; 3, MBP–HCoV 3CLpro N64E; 4, MBP–HCoV 3CLpro N64Q; 5, MBP–HCoV 3CLpro N64D.

 

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Table 2. Enzymatic activities of mutant, recombinant FIPV and HCoV 3C-like proteases

 
The inactive phenotype of the Cys->Ser mutant may be linked to the unique active site organization of coronavirus main proteases. Obviously, these enzymes have accepted replacements in a number of key positions that are not observed in other chymotrypsin-like serine/cysteine proteases (Fig. 2) (Gorbalenya et al., 1989b ; Ziebuhr et al., 2000 ). These replacements may be incompatible with the conversion of coronavirus main proteases from a Cys- to a Ser-based protease. Specifically, our prediction that coronavirus main proteases do not possess a third catalytic residue at the canonical position may be a critical factor. This residue promotes the polarization of the catalytic serine–histidine pair in serine proteases (the charge-relay system) and ensures the correct orientation of the thiolate–imidazolium ion pair in cysteine proteases. To validate the prediction of our alignment with regard to the third catalytic residue, we have characterized a set of mutations at two positions in the FIPV and HCoV main proteases.

First, we probed both the HCoV pp1a/pp1ab Asn3029 residue (which corresponds to Asn64 of the HCoV 3CLpro sequence) and the homologous FIPV 3CLpro Asn64 residue. These residues occupy the only position between the catalytic His and Cys residues whose conservation profile (Asn, Asp and Glu) could be reconciled with the role of the third catalytic residue (Gorbalenya & Koonin, 1993 ). Although this position has a limited variability in coronavirus main proteases, a number of nonconservative substitutions were tolerated in the main proteases of MHV, HCoV and IBV, without significant loss of proteolytic activity in different in vitro assays (Liu & Brown, 1995 ; Lu & Denison, 1997 ; Ziebuhr et al., 1997 ). Only in one case, in which the HCoV Asn3029 residue was replaced by Pro, was a substantial decrease of activity measured (Ziebuhr et al., 1997 ). In the present study, we extended the list of mutations and tested Ala, Asp, Glu and Gln replacements (Fig. 2B and 5B) at this position of HCoV 3CLpro. As shown in Table 2, all these mutations resulted in proteins with significantly increased proteolytic activities (up to threefold). Notably, residues lacking acidic side chains also increased significantly the proteolytic activity of 3CLpro, with Gln being the most active replacement. Likewise, the Ala and Asp mutations at the equivalent Asn64 position of FIPV (Fig. 2B) had activities that not only retained but even surpassed the activities of the wild-type enzyme.

These data, in combination with results from previous studies (Liu & Brown, 1995 ; Lu & Denison, 1997 ; Ziebuhr et al., 1997 ), strongly contradict a catalytic function of the FIPV Asn64 residue and the equivalent residues of other coronavirus main proteases. Upon substitution of another candidate residue, Gly83, with Ala and Asp, only slight effects on the proteolytic activity of FIPV 3CLpro were observed. Even nonconservative replacements like Arg, Trp, Thr, Glu, Val and Pro resulted in active enzymes, albeit with significantly reduced activities (Table 2). These results indicate that other assays and approaches must be employed to rationalize the conservation of the Gly residue at this position and the unique evolution of the catalytic acidic residue in coronavirus main proteases.

The lack of the canonical acidic catalytic residue in coronavirus main proteases can be viewed as another manifestation of the plasticity of the catalytic system of (viral) chymotrypsin-like cysteine proteases. Thus, for example, as is also the case for the 3C-(like) proteases of picorna-like viruses, a limited variability of the third catalytic residue has been documented (Kean et al., 1991 ; Yu & Lloyd, 1991 ; Boniotti et al., 1994 ; Grubman et al., 1995 ). Furthermore, structural data have indicated that the hepatitis A virus (HAV) 3Cpro Asp84 residue, although it occupies a position in the main chain that is topologically equivalent to that of the catalytic Asp of chymotrypsin, is not, evidently, hydrogen-bonded to the catalytic His44 residue (Allaire et al., 1994 ; Bergmann et al., 1997 ). The unique Asp84 side-chain orientation contradicts the predicted catalytic role of this residue (Gorbalenya et al., 1989a ) and leaves the conservation of this residue unexplained.

Our data also provide experimental support for previous studies in which (based on sequence comparisons) a large evolutionary distance between coronaviruses and other positive-stranded RNA viruses was observed (Boursnell et al., 1987 ; Gorbalenya et al., 1989b ). In addition to 3CLpro, coronavirus RdRps, helicases and papain-like proteases also have unique structural and/or biochemical properties that separate them clearly from the homologous proteins of other positive-stranded RNA viruses, again indicating the special phylogenetic position of coronaviruses among all other positive-stranded RNA viruses (Gorbalenya et al., 1989b ; Koonin & Dolja, 1993 ; Herold et al., 1999 ; Seybert et al., 2000a , b ; Ziebuhr et al., 2001 ).

Mutation analysis of putative substrate-binding residues
An important part of the 3CLpro active site is formed by the characteristic sequence signature Gly–X–His, which plays a central role in substrate binding (Bazan & Fletterick, 1988 ; Gorbalenya et al., 1989a ; Bergmann et al., 1997 ; Mosimann et al., 1997 ). It has been predicted that coronavirus main proteases employ a deviant form of this signature, Tyr–X–His (Gorbalenya et al., 1989b ) (Fig. 2B). To date, this theoretical functional assignment has not been tested experimentally for any coronavirus 3CLpro. The need for experimental data becomes even more evident when the unusual nature of the Gly->Tyr replacement and the very low overall similarity between coronavirus main proteases and other chymotrypsin-like enzymes (see above) are taken into account. We thus decided to target the FIPV 3CLpro Tyr160 residue by site-specific mutagenesis.

First, Tyr–X–His was converted into the canonical Gly–X–His constellation by the Tyr->Gly substitution. The mutant exhibited a strongly reduced activity towards the peptide substrate, indicating that the aromatic side chain of Tyr is involved (either directly or indirectly) in proteolytic activity. To test this hypothesis, we analysed the effect of a structurally conservative mutation by substituting Phe->Tyr (mutant Y160F). Interestingly, as Table 2 shows, proteolytic activity was, again, strongly reduced to a level similar to that of the Y160G mutant, suggesting a specific function of the polar hydroxyl group. We then constructed mutant Y160T in order to answer the question of whether the postulated function of the hydroxyl group is retained when attached to an aliphatic side chain. Obviously, the complete loss of activity in this mutant (Table 2) indicates both structural and functional constraints for the FIPV 3CLpro amino acid residue at position 160 (and probably also the equivalent positions in other coronavirus main proteases). The specificity of this function is supported further by the fact that the neighbouring (and also conserved) Met161 residue can be substituted (M161A) without significant effect on proteolytic activity. Our data thus lead us to propose that both the aromatic and hydroxyl properties of Tyr have been selected at this position. These properties, which cannot be delivered by the Gly residue conserved in other 3C-(like) proteases or another residue, may be involved in catalysis and/or substrate binding in a unique way.

One plausible role for the Tyr160 residue would be to take over the function of the third catalytic residue, which is predicted to be replaced by Gly83 (see above). On the basis of structural data, a similar model has been developed recently for another Tyr residue of a related 3C protease, the Tyr143 residue of HAV 3Cpro (Bergmann et al., 1997 ). To test our hypothesis, we have characterized the double mutant Y160G–G83D, in which two unique positions of the coronavirus main proteases were converted into the canonical form, and a negative control to this mutant, Y160G–G83A. Both mutants proved to be inactive (Table 2), indicating that either our hypothesis is not correct or other mutations are required to make this conversion functional.

We then addressed the function of two additional residues proposed previously to be involved in substrate recognition. First, we exchanged Leu and Ala for the conserved FIPV 3CLpro His162 residue. In both cases, the proteolytic activities dropped below the detection limit of the assay used in this study. The obvious indispensability of His162 for proteolytic activity supports the conclusions drawn from sequence comparisons (Gorbalenya et al., 1989b ; Lee et al., 1991 ; Herold et al., 1993 ; Eleouet et al., 1995 ) and is also consistent with mutagenesis data obtained previously for the equivalent His residue in HCoV 3CLpro (Ziebuhr et al., 1997 ). It is thus reasonable to believe that the His162 residue is the functional equivalent to the His191 residue shown to be present in the S1-specificity pocket of HAV 3Cpro (Bergmann et al., 1997 ). Second, we mutagenized the FIPV 3CLpro Ser138 residue. This residue has been predicted to be functionally equivalent to the Thr141 residue of human rhinovirus type 14 3Cpro (Gorbalenya & Snijder, 1996 ) and the Thr142 residue of poliovirus 3Cpro, both of which have been proposed to be involved in the formation of the S1 subsite of the substrate-binding pocket by hydrogen-bonding to the carboxamide side chain of the P1 Gln residue (together with the conserved His residue mentioned above) (Matthews et al., 1994 ; Mosimann et al., 1997 ). Both mutants, FIPV 3CLpro S138A and S138T, had reduced proteolytic activities. Importantly, the Ser->Thr mutant retaining a hydroxyl group was significantly more active than the Ser->Ala mutant retaining the side-chain size (Table 2). Similar results were obtained in a previous mutagenesis study in which the equivalent Thr1179 residue of equine arteritis virus 3C-like nsp4 protease was shown to tolerate substitutions without loss of proteolytic activity in most cases (Snijder et al., 1996 ). Some of these mutants, however, proved to be unable to cleave some but not other cleavage sites, which strongly suggested an involvement of the Thr1179 residue in substrate recognition (rather than catalysis). But, clearly, more experiments are needed to determine conclusively the functions of His162 and Ser138. Specifically, genetic data, including a wide range of substrates, and structural data need to be obtained.

Concluding remarks
In this study, we have presented further theoretical and experimental evidence that coronavirus main proteases have evolved unique structural and functional characteristics in the framework of a chymotrypsin-like fold. Using an in vitro assay, we have characterized more than two dozen FIPV and HCoV 3CLpro point mutations at eight very conserved positions thought to be part of the active centre. The observed effects on the proteolytic activities of the respective enzymes were rationalized within a model derived from a broad comparison of 3C-(like) proteases of coronaviruses and other viruses, especially potyviruses. The model, which is consistent with the obtained data, suggests that coronavirus main proteases use a catalytic Cys–His dyad that is not assisted by an acidic residue in the canonical sequence position. In addition to the Ser (Thr) and His residues present commonly in the substrate pocket of 3C-(like) proteases, coronavirus enzymes also employ the unique properties of a Tyr residue that replaces an otherwise conserved Gly residue. Finally, specific substitutions have been identified that confer significantly increased proteolytic activities on coronavirus main proteases. It will be interesting to test the in vivo effects of such 3CLpro mutants using the reverse-genetic systems established recently (Almazán et al., 2000 ; Yount et al., 2000 ; Thiel et al., 2001a ). Future studies, including structure analyses, are required to elucidate the peculiarities of the coronavirus 3CLpro active site in greater detail.


   Acknowledgments
 
We thank Andreas Kolb for providing FIPV strain 79-1146. The work was supported by grants from the Deutsche Forschungsgemeinschaft (GK Infektiologie, Zi 618/2-1, 2-2) and the Bayerische Forschungsstiftung (Neue Antiinfektiva). A.E.G. was supported with funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products or organization imply endorsement by the US Government.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 18 September 2001; accepted 9 November 2001.