Conservation of substrate specificities among coronavirus main proteases

Annette Hegyi1 and John Ziebuhr1

Institute of Virology and Immunology, University of Würzburg, Versbacher Straße 7, 97078 Würzburg, Germany1

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


   Abstract
Top
Abstract
Main text
References
 
The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CLpro. The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CLpro cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CLpro cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.


   Main text
Top
Abstract
Main text
References
 
Coronaviruses are positive-strand RNA viruses with exceptionally large genome sizes. Based on a similar polycistronic genome organization, common gene expression strategies and a conserved array of homologous domains in the viral polyprotein, the Coronaviridae have been united with the Arteriviridae in the order Nidovirales (den Boon et al., 1991 ; Cavanagh, 1997 ). Both coronaviruses and arteriviruses produce a nested set of subgenomic mRNAs using a unique discontinuous transcription mechanism (Spaan et al., 1983 ). Most probably, the noncontiguous 5' and 3' sequences are fused during negative-strand RNA synthesis (Sawicki & Sawicki, 1998 ; Sawicki et al., 2001 ) in a process that, based on arterivirus reverse-genetics data, has been suggested to resemble similarity assisted, copy-choice RNA recombination (van Marle et al., 1999 ).

It has been shown recently that all the protein functions required for coronavirus replication and transcription are encoded by the replicase gene (Thiel et al., 2001b ). This 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: ORF1a encodes a ~450 kDa protein, called pp1a, and ORFs 1a and 1b together encode the C-terminally extended frameshift protein, pp1ab, with a molecular mass of ~750 kDa. The two replicase polyproteins are processed extensively by viral proteases. The N-proximal region of pp1a/pp1ab is cleaved by virus-encoded papain-like proteases (Baker et al., 1989 ; Bonilla et al., 1997 ; Herold et al., 1998 ; Kanjanahaluethai & Baker, 2000 ; Lim et al., 2000 ; Ziebuhr et al., 2001 ), while the C-proximal region is processed by the coronavirus main protease, also called 3C-like protease (3CLpro). 3CLpro cleaves the replicase polyproteins at 11 conserved interdomain junctions (reviewed by Ziebuhr et al., 2000 ) and shares a remote similarity with the picornavirus 3C proteases (Gorbalenya et al., 1989b ; Liu et al., 1994 , 1997 , 1998 ; Liu & Brown; 1995 ; Lu et al., 1995 , 1998 ; Ziebuhr et al., 1995 , 1997 ; Grötzinger et al., 1996 ; Tibbles et al., 1996 ; Heusipp et al., 1997a , b ; Denison et al., 1999 ; Ziebuhr & Siddell, 1999 ). The substrate specificity of coronavirus main proteases is determined mainly by the P1, P2 and P1' positions (the amino acids flanking the protease cleavage sites are numbered from the N to the C terminus as follows: –P3–P2–P1{downarrow}P1'–P2'–P3'–; Schechter & Berger, 1967 ), which are occupied preferentially by LQ|S or LQ|A. Sequence comparisons and mutagenesis data revealed that coronavirus main proteases most probably employ a catalytic dyad of conserved His and Cys residues, rather than the catalytic His–Asp(Glu)–Cys triad present in other RNA virus 3C-(like) proteases (Bazan & Fletterick, 1988 ; Gorbalenya et al., 1989a ; Liu & Brown, 1995 ; Lu & Denison, 1997 ; Ziebuhr et al., 1997 , 2000 ). Furthermore, coronavirus main proteases have a unique C-terminal domain (Gorbalenya et al., 1989b ) that appears to be involved in proteolytic activity. Thus, truncations of this domain reduced significantly or abolished completely the proteolytic activities of the avain infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV) main proteases in both in vivo and in vitro experiments (Lu & Denison, 1997 ; Ziebuhr et al., 1997 ; Ng & Liu, 2000 ).

In a previous study, we have shown that peptides representing different cleavage sites in the HCoV pp1a/pp1ab are not equally susceptible to proteolysis by recombinant 3CLpro (Ziebuhr & Siddell, 1999 ). To gain additional insights into the substrate preferences of coronavirus main proteases, we have now extended these studies and characterized recombinant main proteases of five coronaviruses in peptide-based cleavage assays. To this end, the 3CLpro-coding sequences of HCoV (strain 229E; Herold et al., 1993 ), porcine transmissible gastroenteritis virus (TGEV, strain Purdue-115; Eleouet et al., 1995 ), feline infectious peritonitis virus (FIPV, strain 79-1146; GenBank accession no. AF326575), IBV (strain Beaudette; Boursnell et al., 1987 ) and MHV (strain JHM; Lee et al., 1991 ) were cloned into the bacterial expression plasmid pMAL-c2 (New England Biolabs) and expressed as fusions with the Escherichia coli maltose-binding protein (MBP). This strategy has been proven previously to be suitable for the expression of the HCoV and MHV main proteases (Ziebuhr et al., 1995 ; Seybert et al., 1997 ). The fusion proteins were partially purified on amylose–agarose columns as described previously (Ziebuhr et al., 1995 ; Herold et al., 1996 ) and the 3CLpro domains with their authentic N- and C-termini were released by factor Xa cleavage (Fig. 1).



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 1. Bacterial expression, partial purification and factor Xa cleavage of recombinant coronavirus 3C-like proteases. Aliquots taken at each step of the purification protocol were analysed on 12·5% SDS–polyacrylamide gels and the proteins were stained with Coomassie brilliant blue. (A) Purification of the HCoV, FIPV and TGEV 3C-like proteases. Lanes M, protein molecular mass markers (kDa); 1, cleared lysate of IPTG-induced E. coli TB1(pMAL-3CL) cells (Ziebuhr et al., 1995 ); 2, pooled peak fractions from the amylose–agarose column; 3, factor Xa-cleaved MBP–HCoV 3CLpro fusion protein; 4, cleared lysate of IPTG-induced TB1(pMALc2-FIPV-3CL) cells (Hegyi et al., 2002 ); 5, pooled peak fractions from the amylose–agarose column; 6, factor Xa-cleaved MBP–FIPV 3CLpro fusion protein; 7, cleared lysate of IPTG-induced TB1(pMALc2-TGEV-3CL) cells; 8, pooled peak fractions from the amylose–agarose column; 9, factor Xa-cleaved MBP–TGEV 3CLpro fusion protein. (B) Purification of the MHV and IBV 3C-like proteases. Lanes M, protein molecular mass markers (kDa); 1, cleared lysate of IPTG-induced TB1(pMAL-MHVpro) cells (Seybert et al., 1997 ); 2, pooled peak fractions from the amylose–agarose column; 3, factor Xa-cleaved MBP–MHV 3CLpro fusion protein; 4, cleared lysate of IPTG-induced TB1(pMALc2-IBV-3CL) cells; 5, pooled peak fractions from the amylose–agarose column; 6, factor Xa-cleaved MBP–IBV 3CLpro fusion protein. The fusion proteins, MBP and the 3C-like main proteases are indicated by arrows.

 
Three sets of synthetic 15-mer peptides (Table 1), which represented corresponding 3CLpro cleavage sites in the replicase polyproteins of HCoV, TGEV and MHV, were synthesized by solid-phase chemistry (Merrifield, 1965 ) and used in competition experiments to determine relative cleavage efficiencies, expressed as (Vmax/Km)rel. The identity and purity of the peptides were confirmed by mass spectroscopy and HPLC (Jerini Bio-Tools). Two of the peptides, SP1 and SP4, represented the sites flanking the 3CLpro domain (P1|P2 and P2|P3; the mature proteins, which are released from pp1a/pp1ab by 3CLpro, are numbered continuously from P1 to P13, with P1 being the most N-terminal product; Ziebuhr et al., 2000 ) and two other peptides, SP5 and SP6, represented the sites flanking the processing product immediately upstream of the putative growth factor-like domain (P5|P6 and P6|P7; Gorbalenya et al., 1989b ; Lu et al., 1998 ; Ziebuhr & Siddell, 1999 ). It should be noted that the P5|P6 junction is a so-called noncanonical cleavage site because its P1' position is occupied by Asn, rather than Ala, Ser or Gly, which are usually found at this position. The competitive cleavage assays were done as described previously (Ziebuhr & Siddell, 1999 ). Briefly, two peptides (SP1 and another peptide, each at 300 µM) were incubated with recombinant 3CLpro (0·3 µM HCoV 3CLpro, 0·3 µM TGEV 3CLpro and 1·8 µM MHV 3CLpro) in 10 mM Bis–Tris–HCl buffer (pH 7·0) at 25 °C. Aliquots were removed, added to an equal volume of 2% trifluoroacetic acid and stored at -80 °C prior to analysis. The reaction products were separated by reverse-phase HPLC on a Delta Pak C18 column (3·9x150 mm; Waters) using a 5–90% linear gradient of acetonitrile in 0·1% trifluoroacetic acid, as previously described (Ziebuhr et al., 1997 ), and the elution was monitored at an absorbance wavelength of 215 nm. The 3CLpro cleavage efficiencies of specific sites in relation to a standard cleavage site (P1|P2, represented by SP1) were calculated using the methods described by Pallai et al. (1989) . The data of these experiments are summarized in Table 1. We obtained clear evidence for differential kinetics in the cleavage of the P1|P2, P2|P3, P5|P6 and P6|P7 sites. The sites flanking the 3CLpro domain were found to be cleaved most effectively in all three viruses analysed. In contrast, the noncanonical P5|P6 site was hydrolysed far less efficiently. Based on these results, a ranking of pp1a/pp1ab cleavage sites can be inferred for each of the viruses tested. The conservation of this ranking among prototypic viruses from the coronavirus groups I and II supports the biological significance of the data and leads us to predict that the order in which pp1a/pp1ab cleavage events occur may be very similar in all coronaviruses. It is also reasonable to suggest that the structural properties residing in the 15-mer peptides are critically involved in determining the order of cleavage events. Likewise, it has been possible in the poliovirus system to correlate peptide cleavage data with the half-life of specific processing intermediates (Pallai et al., 1989 ). Furthermore, secondary structure predictions (Gorbalenya et al., 1989b ) have indicated that most (if not all) of the coronavirus 3CLpro cleavage sites are located at (solvent-exposed) interdomain junctions, which should make these sites easily accessible to the trans-acting protease. However, we do not wish to exclude that, at least in some cases, higher order structures and folding of the replicase polyproteins may modulate the cleavage kinetics of specific sites.


View this table:
[in this window]
[in a new window]
 
Table 1. Relative 3CLpro cleavage efficiencies of synthetic peptides that represent corresponding cleavage sites in three coronavirus replicase polyproteins

 
The poor activities of the recombinant enzymes towards the SP5 peptides, which represent the noncanonical P5|P6 cleavage site, Gln|Asn, strongly suggests structural constraints for the coronavirus 3CLpro S1'-binding sites, as discussed previously (Ziebuhr & Siddell, 1999 ; Ziebuhr et al., 2000 ), and provides a plausible explanation for the conservation of small aliphatic residues (Ala or Ser) at the P1' position in coronavirus 3CLpro cleavage sites (Ziebuhr et al., 2000 ). The exceptional degree of sequence conservation among coronavirus P5|P6 sites leads us to speculate that (i) either the slow cleavage of the P5|P6 site has been preserved during evolution to extend the half-life of a precursor protein containing both P5 and P6 or (ii) the N terminus of P6, which is the conserved sequence NNE(L/I)MP, is required for the biological activity of the coronavirus P6 protein, which remains to be determined. In other words, functional constraints of the mature proteins may have dictated the conservation of this sequence and even accepted unfavourable cleavage kinetics in this case. Genetically engineered coronavirus mutants (Almazán et al., 2000 ; Thiel et al., 2001a ) carrying amino acid substitutions at the P5|P6 site should be extremely informative in identifying the selective forces that determined the conservation of this sequence.

The rapid proteolysis of the SP1 and SP4 substrates suggests that the autocatalytic release of 3CLpro from the viral polyproteins is an early processing event. If this conclusion is correct, most 3CLpro cleavages within pp1a/pp1ab should occur in trans. A similar conclusion has been reached in another study using alternative approaches (Lu et al., 1996 ). The conserved ranking of pp1a/pp1ab cleavage sites, with the N-terminal 3CLpro autoprocessing sites being, in most cases, the most efficiently cleaved substrates, prompted us to address the question of whether this site would be a suitable substrate for main proteases from all coronavirus groups. To answer this question, we incubated the partially purified and factor Xa-activated HCoV, IBV, FIPV, MHV and TGEV main proteases with the HCoV SP1 peptide, which represents the N-terminal HCoV 3CLpro cleavage site, and analysed the reaction products by reverse-phase chromatography. As shown in Fig. 2, all recombinant coronavirus main proteases tested in this experiment cleaved this peptide, albeit with slightly different kinetics. Surprisingly, FIPV 3CLpro proved to be even more active towards the HCoV-derived substrate than the cognate enzyme (Fig. 2, B and D). We conclude from this experiment that, despite the considerable sequence diversity among coronavirus main proteases (Ziebuhr et al., 2000 ), the substrate specificities are highly conserved. The results lead us to believe that both the development of universally applicable 3CLpro assays and the design of broad-spectrum inhibitors blocking all coronavirus main proteases should be feasible.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Conservation of substrate specificities among the 3C-like proteases from prototypic viruses of the coronavirus groups I (HCoV, TGEV and FIPV), II (MHV) and III (IBV). The synthetic peptide used in this experiment was H2N–VSYGSTLQAGLRKMA–COOH (bold-face letters indicate the scissile dipeptide bond). The peptide sequence represents the N-terminal autoprocessing site of HCoV 3CLpro and corresponds to the HCoV pp1a/pp1ab amino acids 2958–2972. The different coronavirus 3C-like proteases were incubated with 0·5 mM of the peptide indicated above for 30 min at 25 °C in buffer consisting of 20 mM Tris–HCl (pH 7·5), 200 mM NaCl, 1 mM EDTA and 1 mM DTT. Shown is the extent of substrate proteolysis as determined by reverse-phase HPLC. A, elution profile of the uncleaved peptide substrate (incubated with buffer); B, peptide incubated with HCoV 3CLpro (0·3 µM); C, peptide incubated with TGEV 3CLpro (0·3 µM); D, peptide incubated with FIPV 3CLpro (0·3 µM); E, peptide incubated with MHV 3CLpro (0·9 µM); F, peptide incubated with IBV 3CLpro (0·9 µM).

 

   Acknowledgments
 
We thank Paul Britton, Rosa Casais and David Brown for the cDNA clone pFRAG-2 (Casais et al., 2001 ) containing the IBV 3CLpro sequence. The work was supported by grants from the Deutsche Forschungsgemeinschaft (GK Infektiologie, SI 357/2-2, ZI 618/2-1) and the Bayerische Forschungsstiftung (Neue Antiinfektiva).


   References
Top
Abstract
Main text
References
 
Almazán, F., González, J. M., Pénzes, Z., Izeta, A., Calvo, E., Plana-Durán, J. & Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences, USA 97, 5516-5521.[Abstract/Free Full Text]

Baker, S. C., Shieh, C. K., Soe, L. H., Chang, M. F., Vannier, D. M. & Lai, M. M. (1989). Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. Journal of Virology 63, 3693-3699.[Medline]

Bazan, J. F. & Fletterick, R. J. (1988). Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proceedings of the National Academy of Sciences, USA 85, 7872-7876.[Abstract]

Bonilla, P. J., Hughes, S. A. & Weiss, S. R. (1997). Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59. Journal of Virology 71, 900-909.[Abstract]

Boursnell, M. E. G., Brown, T. D. K., Foulds, I. J., Green, P. F., Tomley, F. M. & Binns, M. M. (1987). Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. Journal of General Virology 68, 57-77.[Abstract]

Brierley, I., Boursnell, M. E., Binns, M. M., Bilimoria, B., Blok, V. C., Brown, T. D. & Inglis, S. C. (1987). An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV. EMBO Journal 6, 3779-3785.[Abstract]

Casais, R., Thiel, V., Siddell, S. G., Cavanagh, D. & Britton, P. (2001). A reverse genetics system for the avian coronavirus infectious bronchitis virus. Journal of Virology 75, 12359-12369.[Abstract/Free Full Text]

Cavanagh, D. (1997). Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Archives of Virology 142, 629-633.[Medline]

den Boon, J. A., Snijder, E. J., Chirnside, E. D., de Vries, A. A., Horzinek, M. C. & Spaan, W. J. (1991). Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. Journal of Virology 65, 2910-2920.[Medline]

Denison, M. R., Spaan, W. J., van der Meer, Y., Gibson, C. A., Sims, A. C., Prentice, E. & Lu, X. T. (1999). The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis. Journal of Virology 73, 6862-6871.[Abstract/Free Full Text]

Eleouet, J. F., Rasschaert, D., Lambert, P., Levy, L., Vende, P. & Laude, H. (1995). Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology 206, 817-822.[Medline]

Gorbalenya, A. E., Donchenko, A. P., Blinov, V. M. & Koonin, E. V. (1989a). Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Letters 243, 103-114.[Medline]

Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, V. M. (1989b). Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Research 17, 4847-4861.[Abstract]

Grötzinger, C., Heusipp, G., Ziebuhr, J., Harms, U., Süss, J. & Siddell, S. G. (1996). Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E. Virology 222, 227-235.[Medline]

Hegyi, A., Friebe, A., Gorbalenya, A. E. & Ziebuhr, J. (2002). Mutational analysis of the active centre of coronavirus 3C-like proteases. Journal of General Virology 83, 581-593.[Abstract/Free Full Text]

Herold, J., Raabe, T., Schelle-Prinz, B. & Siddell, S. G. (1993). Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology 195, 680-691.[Medline]

Herold, J., Siddell, S. & Ziebuhr, J. (1996). Characterization of coronavirus RNA polymerase gene products. Methods in Enzymology 275, 68-89.[Medline]

Herold, J., Gorbalenya, A. E., Thiel, V., Schelle, B. & Siddell, S. G. (1998). Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate. Journal of Virology 72, 910-918.[Abstract/Free Full Text]

Heusipp, G., Grötzinger, C., Herold, J., Siddell, S. G. & Ziebuhr, J. (1997a). Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells. Journal of General Virology 78, 2789-2794.[Abstract]

Heusipp, G., Harms, U., Siddell, S. G. & Ziebuhr, J. (1997b). Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E. Journal of Virology 71, 5631-5634.[Abstract]

Kanjanahaluethai, A. & Baker, S. C. (2000). Identification of mouse hepatitis virus papain-like proteinase 2 activity. Journal of Virology 74, 7911-7921.[Abstract/Free Full Text]

Lee, H. J., Shieh, C. K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J., Bagdzhadzhyan, A. & Lai, M. M. (1991). The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180, 567-582.[Medline]

Lim, K. P., Ng, L. F. & Liu, D. X. (2000). Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products. Journal of Virology 74, 1674-1685.[Abstract/Free Full Text]

Liu, D. X. & Brown, T. D. (1995). Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein. Virology 209, 420-427.[Medline]

Liu, D. X., Brierley, I., Tibbles, K. W. & Brown, T. D. (1994). A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. Journal of Virology 68, 5772-5780.[Abstract]

Liu, D. X., Xu, H. Y. & Brown, T. D. (1997). Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites. Journal of Virology 71, 1814-1820.[Abstract]

Liu, D. X., Shen, S., Xu, H. Y. & Wang, S. F. (1998). Proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3C-like proteinase and identification of two novel cleavage products. Virology 246, 288-297.[Medline]

Lu, Y. & Denison, M. R. (1997). Determinants of mouse hepatitis virus 3C-like proteinase activity. Virology 230, 335-342.[Medline]

Lu, Y., Lu, X. & Denison, M. R. (1995). Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59. Journal of Virology 69, 3554-3559.[Abstract]

Lu, X., Lu, Y. & Denison, M. R. (1996). Intracellular and in vitro-translated 27-kDa proteins contain the 3C-like proteinase activity of the coronavirus MHV-A59. Virology 222, 375-382.[Medline]

Lu, X. T., Sims, A. C. & Denison, M. R. (1998). Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro. Journal of Virology 72, 2265-2271.[Abstract/Free Full Text]

Merrifield, R. B. (1965). Automated synthesis of peptides. Science 150, 178-185.[Medline]

Ng, L. F. & Liu, D. X. (2000). Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site. Virology 272, 27-39.[Medline]

Pallai, P. V., Burkhardt, F., Skoog, M., Schreiner, K., Bax, P., Cohen, K. A., Hansen, G., Palladino, D. E., Harris, K. S., Nicklin, M. J. & Wimmer, E. (1989). Cleavage of synthetic peptides by purified poliovirus 3C proteinase. Journal of Biological Chemistry 264, 9738-9741.[Abstract/Free Full Text]

Sawicki, S. G. & Sawicki, D. L. (1998). A new model for coronavirus transcription. Advances in Experimental Medicine and Biology 440, 215-219.[Medline]

Sawicki, D. L., Wang, T. & Sawicki, S. G. (2001). The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus. Journal of General Virology 82, 385-396.[Abstract/Free Full Text]

Schechter, I. & Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27, 157-162.[Medline]

Seybert, A., Ziebuhr, J. & Siddell, S. G. (1997). Expression and characterization of a recombinant murine coronavirus 3C-like proteinase. Journal of General Virology 78, 71-75.[Abstract]

Spaan, W., Delius, H., Skinner, M., Armstrong, J., Rottier, P., Smeekens, S., van der Zeijst, B. A. & Siddell, S. G. (1983). Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO Journal 2, 1839-1844.[Medline]

Thiel, V., Herold, J., Schelle, B. & Siddell, S. G. (2001a). Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. Journal of General Virology 82, 1273-1281.[Abstract/Free Full Text]

Thiel, V., Herold, J., Schelle, B. & Siddell, S. G. (2001b). Viral replicase gene products suffice for coronavirus discontinuous transcription. Journal of Virology 75, 6676-6681.[Abstract/Free Full Text]

Tibbles, K. W., Brierley, I., Cavanagh, D. & Brown, T. D. (1996). Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus. Journal of Virology 70, 1923-1930.[Abstract]

van Marle, G., Dobbe, J. C., Gultyaev, A. P., Luytjes, W., Spaan, W. J. & Snijder, E. J. (1999). Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proceedings of the National Academy of Sciences, USA 96, 12056-12061.[Abstract/Free Full Text]

Ziebuhr, J. & Siddell, S. G. (1999). Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab. Journal of Virology 73, 177-185.[Abstract/Free Full Text]

Ziebuhr, J., Herold, J. & Siddell, S. G. (1995). Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. Journal of Virology 69, 4331-4338.[Abstract]

Ziebuhr, J., Heusipp, G. & Siddell, S. G. (1997). Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase. Journal of Virology 71, 3992-3997.[Abstract]

Ziebuhr, J., Snijder, E. J. & Gorbalenya, A. E. (2000). Virus-encoded proteinases and proteolytic processing in the Nidovirales. Journal of General Virology 81, 853-879.[Free Full Text]

Ziebuhr, J., Thiel, V. & Gorbalenya, A. E. (2001). The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond. Journal of Biological Chemistry 276, 33220-33232.[Abstract/Free Full Text]

Received 21 September 2001; accepted 20 November 2001.