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
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Abstract |
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Main text |
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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: P3P2P1
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 HisAsp(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 amyloseagarose 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
).
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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.
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Acknowledgments |
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References |
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Received 21 September 2001;
accepted 20 November 2001.