Institute of Virology and Immunology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany1
Department of Virology, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands2
Advanced Biomedical Computing Center, 430 Miller Dr., Rm 235, SAIC/NCI-FCRDC, Frederick, MD 21702-1201, USA3
Author for correspondence: John Ziebuhr. Fax +49 931 2013934. e-mail ziebuhr{at}vim.uni-wuerzburg.de
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Introduction |
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In contrast to the expression of the structural genes, nidovirus-encoded proteinases play a prominent role in the expression of the replicase gene. The replicase proteins are encoded by two large, 5'-proximal open reading frames (ORFs) that occupy approximately two-thirds to three-quarters of the genome (Fig. 1). The division of the replicase gene into ORFs 1a and 1b, which are connected by a ribosomal frameshift site (Brierley et al., 1987
), is one of the hallmarks of the nidoviruses. It results in the translation of an ORF1a protein and a carboxyl-extended ORF1ab frameshift protein; also known as replicase polyproteins 1a and 1ab (pp1a and pp1ab)
b. The size of the frameshift protein ranges from 3175 amino acids for the arterivirus EAV to about 7200 amino acids for the coronavirus MHV. The nidovirus ORF1a and ORF1ab translation products are polyprotein precursors which are cleaved by viral proteinases at a minimum of 10 (arteriviruses) or 13 (coronaviruses) sites.
Comparative sequence analyses (Boursnell et al., 1987 ; Gorbalenya et al., 1989b
; Bredenbeek et al., 1990a
; Snijder et al., 1990
; den Boon et al., 1991
; Lee et al., 1991
; Godeny et al., 1993
; Herold et al., 1993
; Meulenberg et al., 1993
; Eleouet et al., 1995
) and recent experimental data (van Dinten et al., 1997
, 1999
) suggest that several ORF1b-encoded replicase subunits are directly involved in viral RNA synthesis. The translational downregulation of one of these functions, the (putative) viral RNA-dependent RNA polymerase (RdRp), can also be found in a number of other virus systems of which the alphaviruses have been characterized in most detail (for a review, see Strauss & Strauss, 1994
).
With respect to the ORF1a-encoded subunits of the nidovirus replicase, two main functions have emerged so far. First, hydrophobic domains in the arterivirus ORF1a protein have been shown to mediate the membrane association of the replication complex and to be able to dramatically alter the architecture of host cell membranes (van der Meer et al., 1998 ; Pedersen et al., 1999
). A similar role can also be expected for the corresponding hydrophobic segments of the coronavirus replicase polyproteins (Shi et al., 1999
; van der Meer et al., 1999
). Second, the ORF1a-encoded regions of the nidovirus replicase polyproteins harbour a variety of proteolytic activities, which will be the topic of this review. Although our knowledge of the biochemical and structural properties of the nidovirus proteinases is still very limited, the available data underline the idea that, as for many other positive-stranded RNA viruses, these enzymes fulfil a crucial role in the regulation of the virus life-cycle.
This review article is organized into five main sections. In the first section, we introduce nidovirus proteinases and classify them into main and accessory proteinases. In the next two sections, we present a brief overview of the two classes of the nidovirus proteinases and, in this context, the coronavirus and arterivirus enzymes are compared to each other and to the prototypic proteinases. This is followed by a detailed description of the nidovirus proteinases themselves. The article is concluded by two sections that describe the regulatory role of proteinases during virus replication and then give an outline of future perspectives concerning nidovirus proteinases. The reader will note that the in vitro characterization of the proteinases of coronaviruses is more advanced than that of arteriviruses. In contrast, current knowledge on nidovirus proteolytic regulation in vivo is essentially derived from research on arteriviruses.
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Main and accessory proteinases of nidoviruses |
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Nidovirus main proteinases |
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The 3C-like proteinases of arteriviruses and coronaviruses occupy comparable positions in the replicase polyproteins (Fig. 2). They reside upstream of the ribosomal frameshift site and domains, including RdRp and HEL, which belong to the most conserved domains in this virus order. The 3C-like proteinases are autocatalytically processed at flanking sites§d (Fig. 3A
, B
) and direct the proteolytic processing of all downstream domains of the replicase polyproteins, in both cases at similarly positioned sites (Fig. 2
). This central role in the expression of the major replicative proteins justifies the designation of the 3C-like proteinases as the main proteinase of nidoviruses.
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The first experimental evidence for a coronavirus proteinase activity encoded in the 3'-proximal ORF1a sequence was reported for IBV (Liu et al., 1994 ). In this study, the expression of the putative RdRp domain was shown to involve a virus-encoded proteinase activity that mapped to a region previously predicted to contain a 3CLpro domain (Gorbalenya et al., 1989b
). It was concluded that the proteolytic activity observed was 3CLpro-mediated. Evidence supporting this hypothesis was obtained from three studies on the 3CLpro domains of IBV, MHV and HCoV 229E (Liu & Brown, 1995
; Lu et al., 1995
; Ziebuhr et al., 1995
). It is worth noting that, in these initial studies, the coronavirus 3CLpro proved to be active in quite different expression systems. The MHV 3CLpro was expressed in vitro in a rabbit reticulocyte lysate, the HCoV 229E 3CLpro was expressed in Escherichia coli, and the IBV 3CLpro was expressed in Vero cells using the recombinant vaccinia virus/T7 system. To further analyse the 3CLpro-mediated processing of the coronavirus replicase proteins, two of the systems mentioned above were exploited. In the case of IBV, the 3CLpro was co-expressed with substrate proteins derived from different portions of the replicase polyprotein(s) and 3CLpro-mediated proteolysis of substrates containing specific, mostly predicted, cleavage sites was observed (Liu et al., 1994
, 1997
, 1998
; Liu & Brown, 1995
; Ng & Liu, 1998
). The cleavage products were identified by their apparent molecular mass in SDSPAGE, and the cleavage sites were mapped by site-directed mutagenesis. For HCoV and MHV, the assay systems used were based upon bacterially expressed 3CLpro domains (Ziebuhr et al., 1995
; Herold et al., 1996
; Seybert et al., 1997
). This approach greatly facilitated the identification and N-terminal sequence analysis of 3CLpro cleavage sites by using both in vitro-translated and recombinant protein substrates (Ziebuhr et al., 1995
; Grötzinger et al., 1996
). Furthermore, it allowed for the determination of kinetic parameters using synthetic peptides combined with quantitative analysis of the substrate conversion (Ziebuhr & Siddell, 1999
). Finally, in the MHV system, it has even proved possible to perform amino-terminal microsequence analysis of metabolically labelled 3CLpro cleavage products isolated by immunoprecipitation from virus-infected cells (Lu et al., 1998
).
The information collected during studies with different coronaviruses can now be used to map the 3CLpro processing sites in the coronavirus replicase polyprotein (Fig. 2A). Taken together, at least 12 processing end-products (including the 3CLpro itself) are generated by 3CLpro-mediated cleavage. The processing products of HCoV, IBV and MHV that have been identified so far are summarized in Table 2
. Although the proteolytic processing of TGEV, another coronavirus with a known genome sequence (Eleouet et al., 1995
), has not yet been characterized experimentally, its close relationship with HCoV allows for reliable functional predictions.
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Special efforts have been made to identify the coronavirus 3CLpro counterpart of the third (acidic) residue present in the catalytic triad of CHL enzymes. In the original analysis of the IBV replicase sequence, Glu-2843 was aligned with the catalytic acidic residue of 3C and 3C-like proteinases (Gorbalenya et al., 1989b ). However, subsequent sequence analyses of other coronavirus replicases revealed that Glu-2841 is more conserved than Glu-2843. Also, Glu-2841 is the only residue in the region delimited by the catalytic His and Cys (Fig. 3A
; cf. Fig. 3B
) whose variability in coronaviruses (Asp in MHV, Glu in IBV, and Asn in HcoV and TGEV) could somehow be reconciled with the role of the third catalytic residue (Gorbalenya & Koonin, 1993
; Gorbalenya & Snijder, 1996
). Consequently, this position was probed by site-directed mutagenesis. However, variable results were obtained. For example, the substitution of Glu-2841 by Gln in IBV resulted in an active enzyme (Liu & Brown, 1995
). Likewise, the replacement of Asp-3398, the IBV Glu-2841 equivalent, by either Ala or Pro did not significantly alter the activity of the MHV 3CLpro (Lu & Denison, 1997
). In contrast, the replacement of the equivalently positioned Asn by Gly or Pro in the HCoV 3CLpro had different effects. The replacement of Asn with Gly did not change the rate of substrate conversion as compared to the wild-type enzyme in a peptide cleavage assay (Ziebuhr et al., 1997
). However, the replacement of Asn with Pro substantially reduced the rate of substrate conversion. It is important to note, however, that, in these experiments, the enzymatic activity was not rigorously tested (e.g. by comparison of purified wild-type and mutant 3CLpro domains in quantitative assays using a range of different substrates). Therefore, it would still be premature to draw definitive conclusions concerning the role of this (conserved) residue in the function of the coronavirus 3CLpro. However, keeping in mind that all other 3C/3C-like proteinases tested so far, including those of arteriviruses, only tolerated an exchange of Glu and Asp at this position, the data seem to indicate that the coronavirus 3CLpro lacks a corresponding acidic, catalytic residue in its sequence. It remains to be seen whether an alternative conserved acidic (or even non-acidic) residue outside the region delimited by the catalytic His and Cys assumes the catalytic role and occupies a position equivalent in space to that of the catalytic acidic residue of other 3C/3C-like proteinases.
The coronavirus 3CLpro displays additional features that clearly separate it from other virus-encoded 3C-like proteinases, including the arterivirus main proteinase. For example, it employs a novel version of the substrate-binding pocket core motif, which is characteristically Gly-X-His for most other 3C/3C-like proteinases. Thus, the Gly residue of this motif (Bazan & Fletterick, 1988 ; Gorbalenya et al., 1989a
, b
) is conserved in the vast majority of serine and cysteine proteinases with CHL folds and only very few proteinases tolerate substitutions with small amino acids (Ala or Cys) at this position. This conservation pattern indicates a strong selection pressure with regard to the space that this specific residue occupies. In contrast, in all coronavirus 3CLpro domains studied so far, Gly appears to be replaced by Tyr (Gorbalenya et al., 1989b
; Lee et al., 1991
; Herold et al., 1993
; Eleouet et al., 1995
) (Fig. 3A
). Given the unusual nature of this replacement and the very low level of overall similarity between the coronaviral and the other CHL proteinases (Fig. 5
), additional support for this theoretical assignment is needed. The Tyr residue of the Tyr-X-His motif has not yet been probed by mutagenesis. However, the replacement of the His residue (His-3127) by Ser completely abolished the proteolytic activity of the HCoV 3CLpro (Ziebuhr et al., 1997
). This inactivation was selective since a similar replacement of His-3136, another conserved His residue in this region, was not so detrimental (Ziebuhr et al., 1997
). Thus, the importance of the Tyr-X-His motif has been confirmed, implying that coronaviruses may indeed have accepted a Gly-to-Tyr replacement during evolution. It can be expected that this replacement is coupled to other substitution(s) in the active site to accommodate the bulky side chain of Tyr. The above data are also compatible with a model, originally developed and substantiated for other 3C/3C-like proteinases (Bazan & Fletterick, 1988
; Gorbalenya et al., 1989a
; Allaire et al., 1994
; Matthews et al., 1994
; Mosimann et al., 1997
), that implicates His-3127 (and its counterparts in other coronaviruses) in the formation of hydrogen bonds to the P1 glutamine side chain of 3CLpro substrates (Gorbalenya et al., 1989b
). The high degree of conservation of coronavirus cleavage sites upstream of the P1 position (Fig. 4
) suggests that the substrate-binding pocket of the coronavirus 3CLpro may have numerous additional contacts with its substrates. The determinants of these interactions remain to be elucidated.
The substrate specificity of 3CLpro resembles that of many other 3C/3C-like proteinases (Kräusslich & Wimmer, 1988 ; Dougherty & Semler, 1993
; Blom et al., 1996
) in so far as the P1 position of the substrate is exclusively occupied by Gln and small, aliphatic residues (Ser, Ala, Asn, Gly and Cys) are found at the P1' position (Fig. 4
). However, Asn and Cys are most uncommon as P1' residues outside of the coronaviruses, although a P1' Asn is found in rhinoviruses (Blom et al., 1996
) and, in a mutagenesis study, Cys proved to be a tolerable substitution in one of the encephalomyocarditis virus (EMCV) 3Cpro sites (Parks et al., 1989
). In four different coronaviruses, one 3CLpro cleavage site consistently contains Asn at the P1' position (Liu et al., 1997
; Lu et al., 1998
; Ziebuhr & Siddell, 1999
) and, for MHV, a P1' Cys residue was predicted for another site (Lee et al., 1991
). A peptide that mimicked an HCoV 3CLpro site with the P1' Asn residue was processed relatively poorly in vitro, which additionally points to the exceptional nature of Asn at this position (Ziebuhr & Siddell, 1999
).
Upon comparison of a large number of cleavage sites, most of which have experimentally been confirmed for at least one coronavirus (Fig. 2), it is evident that in addition to P1 and P1', the P2, P3, P4, P2' and P3' positions have a restricted variability (Fig. 4
). Among these, the P2 and P4 positions are most conserved with bulky hydrophobic residues (mainly Leu) at P2 and Val, Thr, Ser (and Pro) at P4 being clearly favoured (Fig. 4
). A similar complexity was previously described for the primary cleavage site determinants of the potyvirus 3C-like proteinase (NIa protein). The potyvirus sites could be transferred into an alien protein where they promoted selective cleavage of the protein by the cognate 3C-like proteinase in a reaction superficially resembling the cleavage of DNA by restriction endonucleases (Carrington & Dougherty, 1988
). The coronavirus 3CLpro cleavage sites can be predicted to possess similar properties. The efficiency of cleavage at specific sites is likely to be determined by the exact composition of the sites, since synthetic peptides mimicking different cleavage sites were processed in competition experiments at significantly different rates by the HCoV 3CLpro (Ziebuhr & Siddell, 1999
). In view of these data, it seems likely that together with the accessibility of potential cleavage sites in the context of the polyprotein the properties of the cleavage sites themselves contribute significantly to the coordinated, temporal release of specific polypeptides from the replicase polyproteins. This might lead to the (irreversible) activation or inactivation of specific functions in the course of the virus life-cycle, as has been demonstrated for a number of other positive-stranded RNA viruses.
Structural aspects of the coronavirus main proteinase
The coronavirus 3CLpro domains are the largest proteinases of their type. They consist of 302307 amino acids, whereas the prototypic poliovirus 3C proteinase contains only 182 residues. This size difference is due to the presence of a unique, carboxyl-terminal region of approximately 110 amino acids which appears to be required for proteolytic activity. Thus, a large number of different carboxyl-terminally truncated versions of the HCoV 3CLpro are inactive in assays using synthetic peptides (Ziebuhr et al., 1997 ; J. Ziebuhr, unpublished data). Also, the removal of 28 carboxyl-terminal amino acids from the MHV 3CLpro abolishes its activity in an in vitro translation system (Lu & Denison, 1997
). Recently, in apparent contrast to the HCoV and MHV data it was shown that a recombinant form of the IBV 3CLpro tolerated the introduction of six consecutive His residues near its carboxyl terminus without loss of activity (Tibbles et al., 1999
).
In the absence of a structural model for the coronavirus 3CLpro, we can only speculate on the function of the carboxyl-terminal region. Obviously, several, not mutually exclusive, functions could be related to this domain, e.g. (i) maintenance of the overall folding of the enzyme, (ii) involvement in catalysis or (iii) substrate recognition, and (iv) a non-proteolytic function. It should be noted that two other groups of 3C-like proteinases, those of arteriviruses (Fig. 3B) and potyviruses (reviewed in Ryan & Flint, 1997
), also have carboxyl-terminal extensions, albeit of smaller sizes. Again no specific function(s) could be attributed to these domains.
The IBV 3CLpro appears to contain structural determinants that, in reticulocyte lysates, prime this proteinase for degradation by the concerted action of ubiquitin and the 26S ATP-dependent proteinase (Tibbles et al., 1995 ). The relevance of this observation to the turnover of the coronavirus proteinase in vivo has not yet been studied. For another distantly related proteinase that carries a protein destruction signal, the EMCV 3Cpro (Lawson et al., 1999
), a correlation between the kinetics of proteinase degradation in vitro and in vivo has been reported (Lawson et al., 1994
). Importantly, the 3C proteinase of another picornavirus, poliovirus, was shown to be stable (Lawson et al., 1999
). Thus, the proteinase degradation signal is a virus-specific structural feature in picornaviruses and, possibly, in coronaviruses.
Sequence comparisons have revealed that the coronavirus 3CLpro is flanked by two hydrophobic domains, HD1 and HD2 (Gorbalenya et al., 1989b ; Lee et al., 1991
; Herold et al., 1993
; Eleouet et al., 1995
), that are also conserved in arteriviruses (Fig. 2
). Recent data from in vitro translation experiments have shown that microsomal membranes are required for the efficient autoproteolytic processing of the 3CLpro from HD1 and HD2, most likely by assisting in the proper folding of these proteins (Tibbles et al., 1996
; Piñón et al., 1997
; Schiller et al., 1998
). However, after being released from the polyprotein, the 3CLpro activity does not depend on membranes, or any other cofactor(s), at least for its proteolytic activity in vitro (Ziebuhr et al., 1995
). It has been suggested that HD1 and HD2 may contribute to the intracellular localization of the 3CLpro itself and, possibly, of the virus replication complex in general (Gorbalenya et al., 1989b
). Recent data, obtained by using immunofluorescence and electron microscopy, strongly support this hypothesis (Heusipp et al., 1997a
; Bi et al., 1998
; Schiller et al., 1998
; Ziebuhr et al., 1998
; Denison et al., 1999
; Shi et al., 1999
; van der Meer et al., 1999
; Ziebuhr & Siddell, 1999
). Specifically, it has been found that the coronavirus nucleocapsid protein, numerous replicase gene-derived proteins and newly synthesized RNA co-localize to intracellular (mainly late endosomal) membranes (van der Meer et al., 1999
). However, there are also reports that favour a Golgi localization for the MHV replication complexes, at least in specific cell types (Bi et al., 1998
; Shi et al., 1999
). From the combined data, it can be concluded that coronavirus replication takes place at intracellular membranes and that a large number of non-structural, replicase gene-encoded proteins contribute to the formation and function of the coronavirus replication complex.
As outlined above, the coronavirus 3CLpro has a number of unique properties that remain poorly understood due to the lack of structural information about any of these enzymes. The currently available structures of three picornavirus 3C proteinases (Allaire et al., 1994 ; Matthews et al., 1994
; Mosimann et al., 1997
) and the results of inhibitor analyses (Tibbles et al., 1996
; Ziebuhr et al., 1997
) support the classification of the coronavirus 3CLpro as a two-
-barrel-fold protein. However, they are of limited use in understanding the unique features of these very distant relatives. Recently developed expression and purification systems (Ziebuhr et al., 1995
, 1997
; Seybert et al., 1997
) could provide a suitable basis for the crystallization of coronavirus 3CLpro domains and the elucidation of their structure.
Arterivirus main proteinase
The arterivirus 3C-like serine proteinase (3CLSP) was first identified by comparative sequence analysis of the ORF1a protein of EAV, the arterivirus prototype (den Boon et al., 1991 ). Subsequently, the 3CLSP domain was shown to reside in a 21 kDa cleavage product (nsp4; 204 residues in the case of EAV) derived from the central region of the ORF1a protein (Fig. 2B
) (Snijder et al., 1994
). In addition to this fully cleaved product, a number of 3CLSP-containing processing intermediates were identified in EAV-infected cells, the most abundant ones being nsp312, nsp38 and nsp34 (Snijder et al., 1994
; van Dinten et al., 1996
). The proteolytic activity of the 3CLSP was demonstrated in the recombinant vaccinia virus/T7 expression system (Snijder et al., 1996
), in which studies to characterize this proteinase (e.g. site-directed mutagenesis) were also carried out. The 3CLSP was shown to mediate (at least) eight cleavages in the EAV replicase; five in the carboxyl-terminal half of the ORF1a protein and three in the ORF1b-encoded polypeptide (Snijder et al., 1996
; Wassenaar et al., 1997
; van Dinten et al., 1999
). These cleavage sites were identified by comparison with other arterivirus sequences (Godeny et al., 1993
; Meulenberg et al., 1993
; Snijder et al., 1996
; van Dinten et al., 1996
) and, subsequently, confirmed by site-directed mutagenesis and expression studies (Snijder et al., 1996
; Wassenaar et al., 1997
; van Dinten et al., 1999
). Two sites (nsp4|5 and nsp6|7) were also confirmed by direct amino-terminal sequence analysis of cleavage products derived in an alphavirus expression system (Wassenaar et al., 1997
). Four of the known EAV 3CLSP cleavages (Fig. 2B
) occur at Glu|Gly sites, three at Glu|Ser dipeptides, and one (nsp9|10) between Gln and Ser. All of these sites are conserved in the known arterivirus sequences (Wassenaar et al., 1997
; van Dinten et al., 1999
) although, for a number of them, Ala (and in one case Lys) is predicted to be the P1' residue (Fig. 4
). In addition to the P1|P1' positions, some degree of conservation (although, less than in coronaviruses) is evident for the P2 and P4 positions (Fig. 4
). This appears to suggest that the residues at these positions contribute to substrate recognition but experiments to verify this hypothesis have not yet been reported. The sizes of the processing end-products generated by the 3CLSP (nsp312) are constant among different arteriviruses (Table 3
), which is in contrast to the virus-specific size heterogeneity of proteins carrying accessory proteolytic activities (nsp12 region; see below).
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The ability of the EAV 3CLSP to cleave specific sites in cis or in trans has not been studied in detail. It is likely that the two sites flanking the 3CLSP domain in the replicase polyproteins (nsp3|nsp4 and nsp4|nsp5) are cleaved in cis. However, the EAV 3CLSP has also been shown to be active in trans. For example, it has been found that, in the recombinant vaccinia virus/T7 expression system, an nsp4 expression product was able to cleave the nsp9|10 and nsp10|11 sites in a separately expressed, ORF1b-encoded polyprotein (van Dinten et al., 1999 ).
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Nidovirus accessory proteinases |
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The arteriviruses EAV, lactate dehydrogenase-elevating virus (LDV) and porcine reproductive and respiratory syndrome virus (PRRSV) encode a cassette of three adjacent proteolytic domains that are known (from amino terminus to carboxyl terminus) as papain-like cysteine proteinase 1 (PCP1
), papain-like cysteine proteinase 1
(PCP1
) and cysteine proteinase of nsp2 (CP2). The digits in the names stand for the non-structural proteins (nsp; numbered from amino to carboxyl terminus) in which the proteolytic domains reside. PCP1
and PCP1
may reside in one protein (nsp1; EAV) or in two separate proteins (nsp1
and nsp1
; LDV and PRRSV) that precede nsp2, which itself contains CP2. The EAV PCP1
domain is an inactivated enzyme and, therefore, the cleavage between the PCP1
and PCP1
domains does not occur, resulting in an amino-terminal cleavage product (nsp1) that contains both PCP1 domains. In contrast, due to the activity of PCP1
and PCP1
, the equivalent LDV and PRRSV proteins are cleaved into nsp1
and nsp1
. In both PCP1
and PCP1
, but not in CP2, the catalytic Cys (Fig. 6B
, C
) is immediately followed by a conserved aromatic residue. This sequence signature is a hallmark of cellular papain-like proteinases and was initially used to characterize viral proteinases as being papain-like. Therefore, in the paper describing the identification of CP2 (Snijder et al., 1995
), the enzyme was distinguished from PCP1
and PCP1
. Subsequently, however, a distant variant of the papain-like fold was identified in a human ubiquitin carboxyl-terminal hydrolase (Johnston et al., 1997
) in which, as in the arterivirus CP2 (Fig. 6C
), the conserved aromatic residue is replaced by Gly. Hence, the original reasons for discriminating between CP2 and PCP1
/PCP1
no longer exist. CP2 may adopt a papain-like fold and, if this is confirmed, its name should be modified accordingly.
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The coronaviruses MHV, HCoV and TGEV encode two accessory proteinases (Fig. 2). They are called coronavirus papain-like proteinases 1 and 2, PL1pro and PL2pro, respectively. IBV encodes only one accessory proteinase. It is called coronavirus papain-like proteinase, PLpro (Fig. 2
). Despite the relationship implied in the names, only a marginal similarity between the coronaviral and prototypic cellular proteinases is evident in an alignment based on the comparison of (predicted) secondary structures (Herold et al., 1999
; Fig. 6A
). However, a statistically reliable, albeit local, primary structure similarity was detected between coronavirus PLpro domains and the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV), a picornavirus (Gorbalenya et al., 1991
; A. E. Gorbalenya, unpublished data). This similarity was used to identify a papain-like catalytic centre in the FMDV Lpro, which was subsequently confirmed by mutagenesis experiments (reviewed in Ryan & Flint, 1997
) and X-ray crystallography (Guarné et al., 1998
). Obviously, this relationship suggests that FMDV Lpro and coronavirus PLpro domains have evolved under a similar selective pressure that separates them from other RNA virus papain-like proteinases including those of arteriviruses (for a list of these proteinases see Gorbalenya & Snijder, 1996
). It is conceivable that common phenotypic features exist that are conserved in Lpro and PLpro domains. It is also worth noting that the functional separation of coronavirus PLpro domains and 3CLpro into accessory and main proteinases also applies to the FMDV Lpro and 3Cpro, respectively. Thus, it is justified to treat the FMDV Lpro as the prototypic viral proteinase for coronavirus PLpro domains (Table 1
).
Comparative sequence analyses of the coronavirus accessory proteinases do not provide definitive support for the clustering of the PL1pro and PL2pro domains into two groups (as their names appear to imply) or for the association of the IBV PLpro with one of these groups (A. E. Gorbalenya, unpublished observations). Indeed, the alignment of PL1pro and PL2pro (Fig. 6A) shows that only very few conserved residues are present exclusively in one of the two groups. Instead, and despite the low overall level of similarity in pairwise comparisons (1332% identical residues), coronavirus accessory proteinases, including the IBV PLpro, have eight absolutely conserved residues (Herold et al., 1999
). Among these are the catalytic Cys-His dyad as well as three Cys residues that are involved in the formation of a zinc-binding finger. The activity or activities of this zinc finger must be both essential and compatible with the different functions commonly found in related, but paralogous proteinases (i.e. enzymes that evolved by duplication rather than speciation). The conservation of this structural element embedded in the central region of these enzymes clearly discriminates the accessory proteinases of coronaviruses from their arterivirus counterparts.
The presence of only one PLpro domain in IBV is most intriguing and can be interpreted in different ways. For example, in the course of coronavirus evolution, a duplication of the PLpro domain might have occurred after the divergence of IBV from the rest of the coronavirus family. This scenario would imply that the PLpro duplications have occurred independently in the arterivirus and coronavirus lineages. Alternatively, a (single) duplication could have taken place in a common nidovirus progenitor. In this case, the second PLpro domain in IBV must have been deleted or diverged beyond recognition. Thus, the IBV PLpro would be orthologous with either the PL1pro or the PL2pro group (i.e. these enzymes would have diverged from a common ancestor by speciation rather than by duplication). It has been noted before (Gorbalenya et al., 1991 ) that the IBV PLpro and the PL2pro domains of the other coronaviruses are collinear in the replicase polyproteins (Fig. 2A
), which favours the hypothesis that they might be orthologous proteins.
Based on a limited sequence similarity with a streptococcal cysteine proteinase, which belongs to a prokaryotic subset of papain-like proteinases, the existence of another accessory proteinase in IBV was initially postulated (named Streptococcus pneumoniae-like proteinase, SPL) (Gorbalenya et al., 1989b ). SPL was predicted in a region of pp1a/pp1ab that partly overlaps with the PLpro domain identified 2 years later (Gorbalenya et al., 1991
; Lee et al., 1991
). The predicted catalytic Cys and His residues of SPL are not conserved in other coronavirus replicase polyproteins and, furthermore, the relevant Cys residue immediately follows the catalytic His residue of PLpro. To our knowledge, such an overlapping organization of the active sites of two different enzymes has not been observed elsewhere and, clearly, this raises doubts about the correct identification of SPL and PLpro. Given the conservation of PLpro in all coronaviruses and the solid experimental support for its existence, it is safe to assume that the SPL domain is not functional.
Processing of the amino-proximal region of the coronavirus replicase polyproteins by accessory papain-like cysteine proteinases
The first data on the processing of coronavirus replicase polyproteins were obtained by translation of genomic MHV RNA in rabbit reticulocyte lysates. In pulsechase experiments, a 28 kDa protein, p28, which is initially synthesized as part of a larger precursor protein, was identified (Denison & Perlman, 1986 ). This protein was also detected in virus-infected cells (Denison & Perlman, 1987
). The genesis of p28 has been analysed in detail. First, in vitro translation experiments, combined with peptide mapping, were used to show that p28 represents the amino-terminal polypeptide (Soe et al., 1987
). Second, it was shown that the proteolytic activity involved is virus-encoded and maps to the predicted PL1pro domain (Baker et al., 1989
, 1993
; Gorbalenya et al., 1991
). Third, the scissile bond that is cleaved to release the carboxyl terminus of p28 was found to be located between Gly-247 and Val-248 (Dong & Baker, 1994
; Hughes et al., 1995
).
In subsequent studies, immunoprecipitation experiments with region-specific antisera revealed that additional proteolytic cleavages within the amino-proximal region of the MHV-A59 replicase polyproteins generate polypeptides of 65, 50, 240 and 290 kDa (p65, p50, p240 and p290) (Denison et al., 1992 , 1995
). Pulsechase experiments indicated that the 290 kDa protein is the precursor of the 50 kDa and 240 kDa proteins (Denison et al., 1992
). The experimental data also suggested that p65 is adjacent to the carboxyl terminus of p28 (Denison et al., 1995
). Subsequently, a second PL1pro cleavage site was identified (Bonilla et al., 1995
) and characterized by amino acid sequence analysis (Bonilla et al., 1997
). This cleavage occurs at the Ala-832|Gly-833 peptide bond and it was concluded that p65 encompasses the MHV-A59 ORF1a-encoded amino acids 248832.
A slightly different processing pattern has been reported for the JHM strain of MHV (Baker et al., 1989 ; Gao et al., 1996
; Schiller et al., 1998
). In this case, cleavage products of 28, 72 and 250 kDa were identified. Pulsechase experiments revealed that the 72 kDa protein, p72, is further processed to a 65 kDa protein, p65 (Gao et al., 1996
), whereas the 250 kDa protein, p250, is processed to a 210 kDa protein, p210 (Schiller et al., 1998
). In the same study, another p250-derived, 40 kDa processing product, p40, was described, but a clear precursorproduct relationship between p250 and p40 has not yet been established. Furthermore, the JHM-specific polypeptides, p250, p210 and p65, were found to comigrate with the A59-specific polypeptides, p290, p240 and p65, and, therefore, it is reasonable to assume that they represent identical proteins. Schiller et al. (1998)
also concluded from these data that the precursor of p65 is p72 and it is unique to MHV-JHM. They also propose that the carboxyl-terminal cleavage site of p72 is the peptide bond between Gly-904 and Val-905.
Information on the proteolytic processing of the amino-proximal region of the replicase polyproteins of HCoV and IBV is quite limited. For both viruses, only the amino-terminal cleavage products have been characterized so far. By using a monospecific antiserum directed against the HCoV ORF1a-encoded amino acids 41250, a polypeptide with an apparent molecular mass of 9 kDa, p9, was identified in HCoV-infected cells (Herold et al., 1998 ). This protein, p9, was shown to be generated by PL1pro-mediated cleavage between Gly-111 and Asn-112, which is equivalent to the p28 cleavage site recognized by the MHV PL1pro. In the same study, polypeptides with apparent molecular masses of 93 kDa (p93), 170 kDa (p170) and 230 kDa (p230) were specifically immunoprecipitated from lysates of metabolically labelled, HCoV-infected cells. The identity of these proteins and potential precursorproduct relationships remain to be determined.
For IBV, the amino-terminal processing product of the replicase polyproteins has been suggested to be a protein with an apparent molecular mass of 87 kDa (Lim & Liu, 1998 ). Conflicting data have been published with respect to the proteinase responsible for the processing of the 87 kDa protein. Thus, in an initial report, the involvement of a cellular proteinase in the production of p87 in vitro was proposed (Liu et al., 1995
). Recently, however, it was shown that mutagenesis of the putative active-site residues of the previously predicted PLpro domain (Gorbalenya et al., 1991
; Lee et al., 1991
) completely abolished the production of the 87 kDa protein in a Vero cell transient expression system (Lim & Liu, 1998
). This result and additional data presented in this paper suggest that the production of p87 is mediated by the virus-encoded PLpro, rather than a cellular proteinase. In the same study, site-directed mutagenesis of a candidate PLpro cleavage site suggested that p87 is released by cleavage of the Gly-673|Gly-674 peptide bond. Interestingly, no evidence has been obtained for the existence of an additional, upstream cleavage site that would be equivalent to the sites involved in the release of p28 and p9 from the MHV and HCoV replicase polyproteins, respectively.
Enzymatic and structural properties of coronavirus accessory proteinases
On the basis of previous predictions (Gorbalenya et al., 1991 ; Lee et al., 1991
; Herold et al., 1993
), mutagenesis studies of putative Cys-His catalytic dyads of three different coronavirus PLpro domains were done. The available data suggest that Cys-1137/His-1288, Cys-1054/His-1205 and Cys-1274/His-1437 are the putative catalytic residues of the MHV-JHM PL1pro, the HCoV PL1pro and the IBV PLpro, respectively (Baker et al., 1993
; Herold et al., 1998
; Lim & Liu, 1998
). The Cys-1137/His-1288 dyad of MHV-JHM corresponds to Cys-1121/His-1272 in the MHV-A59 replicase polyproteins (Bonilla et al., 1994
) and the involvement of His-1272 in proteolysis has been experimentally confirmed (Bonilla et al., 1995
). Recently, the papain-like fold of the coronavirus accessory proteinases was modelled in some detail (Herold et al., 1999
). In this analysis, a number of additional residues were postulated to be involved in the formation of the active site of coronavirus PLpro domains. These residues were probed by point mutations in the HCoV PL1pro and shown to be essential for proteolytic activity. However, their precise roles remain to be elucidated.
A theoretical analysis of the primary and (predicted) secondary structures of coronavirus papain-like proteinase domains has also recently delineated a conserved zinc finger structure. This structure is organized as a separate, (possibly) -sheet domain, connecting the left- and right-hand domains of a papain-like fold in an unprecedented way (Figs 6A
and 7
) (Herold et al., 1999
). Several striking implications of this analysis have been tested experimentally using the HCoV PL1pro, expressed and purified as an E. coli maltose-binding fusion protein (MBPPL1pro). First, using a spectrometric method, equimolar binding of Zn2+ by MBPPL1pro was documented. Second, denaturation/renaturation experiments using MBPPL1pro revealed a strong Zn2+-dependence for proteolytic activity and a tight association of Zn2+ with PL1pro. And third, the replacement of the four cysteine residues predicted to coordinate Zn2+ resulted in a selective inactivation of the enzyme as judged by in vitro cis- and trans-cleavage assays. Thus, it was concluded that the Zn2+ ion is tetrahedrally coordinated by Cys-1126, Cys-1128, Cys-1154 and Cys-1157 of the HCoV PL1pro. The zinc finger domain replaces a poorly conserved structure of variable size which connects the two domains of the papain fold in numerous papain-like proteinases (Fig. 7
). For example, the analogous structure in the FMDV Lpro is composed of only two parallel
-strands (Guarné et al., 1998
). This accounts, at least partially, for the smaller size of this picornavirus proteinase compared with the coronavirus homologues.
|
Sequence analyses of three cleavage sites have provided information on the substrate specificity of PL1pro. For both of the MHV PL1pro cleavage sites identified so far, Gly-247|Val-248 and Ala-832|Gly-833, extensive mutation studies have been done (Dong & Baker, 1994 ; Hughes et al., 1995
; Bonilla et al., 1997
). A number of conclusions can be drawn. First, the most important structural element of MHV PL1pro cleavage sites appears to be the P1 residue. This position is invariably occupied by Gly or Ala, with Gly being the clearly preferred residue. Second, only an extremely limited number of amino acid substitutions are tolerated at the P2 position. For example, in the case of the Gly-247|Val-248 cleavage site, nine different substitutions of P2 obviated the function of the cleavage site (Dong & Baker, 1994
; Hughes et al., 1995
). Third, although the P1' position tolerated a quite large number of substitutions, a clear preference for small, uncharged amino acids was found. Again, Gly and Ala were the most active amino acids in different assays. Fourth, a basic residue is, most likely, part of the substrate signature because substrates with either Arg or Lys at the P5 position and Arg at the P2 position gave high cleavage rates. In this respect, it is also noteworthy that the presence of Arg at the P2 position was sufficient to compensate for an inactivating Arg-by-Met substitution at the P5 position (Bonilla et al., 1997
). Finally, the data indicate that the positions P3, P4, P2' and P3' tolerate a large number of amino acid substitutions. Therefore, it is thought that these residues do not contribute significantly to substrate recognition.
The substrate specificity of the HCoV PL1pro was determined by sequence analysis of the carboxyl-terminal cleavage site of p9 (Herold et al., 1998 ). By and large, the cleavage site corresponds well to the general pattern outlined above for the MHV PL1pro. Again, a Gly residue was found at the P1 position and the P1' and P5 positions were occupied by small uncharged (Asn-112) and basic (Lys-107) residues, respectively. Furthermore, sequence alignment revealed that, within the replicase polyproteins of three different coronaviruses, the relevant PL1pro cleavage site is an integral part of the only significantly conserved sequence block upstream of PL1pro. An analysis of this block suggested that the PL1pro cleavage site might have migrated by two residues in either the MHV or HCoV/TGEV evolutionary lineage from its initial position in the common coronavirus ancestor. Such an event would have taken place under a complex and evolving selective pressure upon this locus. For the recently described IBV PLpro cleavage site (Lim & Liu, 1998
), site-directed mutagenesis suggests an involvement of the pp1a/pp1ab amino acids Gly-673 and Gly-674 in the release of the p87 carboxyl terminus. However, in the absence of protein sequence data it is still premature to draw conclusions about the IBV PLpro substrate specificity.
The first detailed reports of PL1pro activity detected no trans activity for the MHV-JHM enzyme using an in vitro translation assay (Baker et al., 1989 ). Similarly, the IBV PLpro was not active in trans upon transient expression in Vero cells (Lim & Liu, 1998
). Recently, however, evidence has been presented that two other related PL1pro domains, those of MHV-A59 and HCoV, are active in trans, at least in vitro (Bonilla et al., 1997
; Herold et al., 1998
). These and other results (Baker et al., 1993
; Bonilla et al., 1995
; Teng et al., 1999
) suggest that a number of factors can profoundly modulate proteolytic processing by the PL1pro, both in cis and in trans.
In MHV-A59, a domain||e of 233 amino acids (pp1a/pp1ab residues 10841316) was shown to be required for the cis cleavage that generates p28 (Bonilla et al., 1995 ). Subsequently, however, it was found that this form of the enzyme had a lower activity compared to a form containing amino acids 10621364. In addition, cleavage at both MHV-A59 PL1pro processing sites seems to be modulated by sequences downstream of PL1pro. Thus, for example, it was found that a polypeptide encompassing amino acids Lys-869 to Pro-2028 showed a more than fivefold enhanced cleavage activity compared to a polypeptide encompassing Lys-869 to Gln-1314 (Teng et al., 1999
). The region absent in the smaller protein contained the X domain, which is highly conserved among Togaviridae and Coronaviridae (Gorbalenya et al., 1991
), and the putative PL2pro domain (Lee et al., 1991
; Bonilla et al., 1994
) (Fig. 2
). Thus, it is tempting to speculate that sequences downstream of PL1pro might be involved in the regulation of its activity, as has been previously suggested (Gorbalenya et al., 1991
). The question of whether these domains provide specific activities or simply affect the overall conformation of the protein has yet to be answered. Intriguingly, no evidence for a proteolytic activity of the PL2pro domain has been obtained so far (Gao et al., 1996
; Teng et al., 1999
).
With respect to PL1pro substrate(s), it has been shown that the production of p28 requires the presence of downstream sequences (Baker et al., 1993 ; Bonilla et al., 1995
). This observation was confirmed and extended by a study in which the purified PL1pro domain (amino acids 10621364) was assayed in trans using a set of in vitro-translated, ORF1a-derived substrates (Teng et al., 1999
). The data showed that a minimum of 622 amino acids from the amino-proximal region of the replicase polyproteins are required to obtain high rates of cleavage by PL1pro. Consistently, recombinant forms of PL1pro failed to cleave synthetic peptides containing cognate cleavage sites (Teng et al., 1999
). It can be concluded that the region delimited by amino acids 302622 (or even amino acids further downstream) specifically contributes to proteolysis at the p28 site, probably by enhancing presentation of the substrate or activating the enzyme.
Only very limited information is currently available about the requirements of the accessory proteinase-mediated cleavages in other coronaviruses. In HCoV, as in MHV, the sequence between the p9 cleavage site (which is equivalent to the MHV p28 cleavage site) and the catalytic domain was shown to contain determinant(s) required for PL1pro-mediated processing in trans in reticulocyte lysates (Herold et al., 1998 ). In IBV, the situation appears to be different. It was shown, in a recombinant vaccinia virus/T7 expression system, that cleavage at the p87 site in cis tolerates a large deletion of approximately half the region between the cleavage site and the catalytic domain of PLpro. Also, again in contrast to the MHV data, the proteolytic reaction of PLpro appeared to be insensitive to the presence of approximately 300 amino acids flanking the catalytic domain on its carboxyl terminus (Lim & Liu, 1998
). The above data suggest that the dependence of PLpro-mediated cleavages upon specific sequences, which flank the cleavage site(s) and the catalytic domain, may be conserved in some, but not all, coronaviruses.
In common with the coronavirus main proteinases, the coronavirus accessory proteinases have also been shown to deviate significantly from the prototypic enzymes. The availability of purified, recombinant PL1pro domains from two coronaviruses (Herold et al., 1999 ; Teng et al., 1999
) should now make these enzymes amenable to detailed structural analyses. This information should help in understanding the complex organization of viral proteins carrying PLpro domains and provide insights to their structurefunction relationships.
Arterivirus nsp1 papain-like proteinases
The activity of in vitro-translated EAV nsp1 papain-like cysteine proteinase (PCP1, the origin of the suffix,
, is clarified below) facilitated its early experimental characterization. Comparative sequence analysis had predicted the presence of a cysteine proteinase, with limited sequence similarity to cellular and other viral papain-like cysteine proteinases, in the amino-proximal region of the EAV replicase polyproteins (den Boon et al., 1991
). Upon translation of RNA transcripts encoding this region, a 29 kDa, amino-terminal cleavage product was rapidly generated (Snijder et al., 1992
). Uncleaved precursor proteins could not be detected and pulsechase studies suggested the almost immediate (co-translational) release of an amino-terminal product, which was named nsp1 (Fig. 2B
). The same protein was also detected in virus-infected cells, where it appeared to be produced with similar kinetics (Snijder et al., 1994
). Furthermore, the EAV nsp1 proteinase was found to be active in E. coli when expressed as part of a bacterial fusion protein (Snijder et al., 1992
). Amino-terminal sequence analysis of an E. coli-derived cleavage product showed that the nsp1|2 scissile bond was located between Gly-260 and Gly-261 of the EAV ORF1a and ORF1ab polyproteins.
The subsequent sequence analyses of the genomes of two other arteriviruses, LDV (Godeny et al., 1993 ; Palmer et al., 1995
) and PRRSV (Lelystad strain; Meulenberg et al., 1993
) revealed that their nsp1 region was substantially larger than that of EAV (about 380 versus 260 residues). Furthermore, this region was found to contain two, instead of one, PCP domains. The sequence analysis also revealed an overall collinearity between the amino-terminal regions of EAV and PRRSV/LDV (den Boon et al., 1995
). Specifically, the carboxyl-terminal proteinase domain of PRRSV/LDV could be aligned with the EAV nsp1 PCP1
, whereas the amino-terminal domain of PRRSV/LDV matched with a PCP remnant that was identified upstream of the EAV PCP1
in nsp1. This new domain was named PCP1
. The functionality of both of these domains was experimentally confirmed for PRRSV/LDV by den Boon et al. (1995)
. First, PCP1
was shown to mediate the rapid liberation of an amino-terminal, 2022 kDa cleavage product (nsp1
) (Fig. 2B
). Second, the PRRSV/LDV PCP1
was shown to act like its EAV orthologue in that it cleaves the nsp1|2 junction. Thus, both PCP1
and PCP1
contribute to the production of the nsp1
protein (Fig. 2B
) (den Boon et al., 1995
).
The putative PCP1 active-site residues were shown to be Cys-76/His-146 and Cys-76/His-147 in PRRSV and LDV, respectively, but a conserved putative nsp1
|1
cleavage site could not be identified (Godeny et al., 1993
; Meulenberg et al., 1993
; den Boon et al., 1995
). Although a Lys residue was found in place of the presumed catalytic Cys residue, which explains the proteolytic deficiency of the EAV PCP1
domain, the sequences around EAV His-122 display convincing sequence similarity with the region surrounding the PCP1
active-site His-146/147 of LDV/PRRSV (Fig. 6B
). The consequences of the inactivation of the EAV PCP1
domain are unclear but the partial conservation of the inactivated PCP1
suggests that this part of the replicase probably contains an additional, non-proteolytic function that is conserved in all arteriviruses (den Boon et al., 1995
; Gorbalenya & Snijder, 1996
).
The identification of Cys-164 and His-230 as the probable EAV PCP1 catalytic dyad (den Boon et al., 1991
) was supported by data from site-directed mutagenesis (Snijder et al., 1992
) which showed that the replacement of the putative active-site residues Cys-164 (by Ser or Gly) or His-230 (by Val, Ala or Gly) completely inactivated proteinase function. Deletion mutagenesis was used to delimit the minimal domain required for activity to residues 123263. The putative PCP1
active-site residues in PRRSV and LDV are Cys-276/His-345 and Cys-269/His-340, respectively (den Boon et al., 1995
). Sequence comparison suggests that the PCP1
domains of both PRRSV and LDV cleave between Tyr and Gly (Tyr-384|Gly-385 and Tyr-380|Gly-381, respectively; Fig. 6B
).
Attempts to achieve cleavage in trans using arterivirus PCPs were unsuccessful (den Boon et al., 1995 ; Snijder et al., 1992
). A similar situation was also observed for another virus proteinase, the Sindbis virus capsid protein, which was shown to act exclusively in cis (Choi et al., 1991
). In the case of Sindbis virus, the carboxyl terminus of the proteinase was found to remain in the P1 substrate site subsequent to the autocatalytic cis cleavage of the capsid protein. The structure analysis of this protein revealed that the carboxyl-terminal Trp residue seals the active site of the proteinase, rendering the enzyme inactive after the release of the downstream protein. An analogous organization may be adopted by the arterivirus PCPs, which would explain their readily detectable cis activity and the absence of trans activity. A limited mutagenesis study of the nsp1|2 cleavage site revealed that its P1 position is more sensitive to replacements than its P1' position and that sequences downstream of P2' are not required for processing (Snijder et al., 1992
).
Arterivirus nsp2 cysteine proteinase
The arterivirus CP2 is the most carboxyl-terminally located member of the array of three cysteine proteinase domains present in the amino-terminal 500 residues of the replicase polyproteins (Fig. 2B). CP2 is located in the amino-terminal region of nsp2 and is highly conserved among arteriviruses, although it has only been studied experimentally for EAV (Snijder et al., 1995
). The cleavage of the nsp2|3 junction appears to be the single processing step mediated by CP2. The size of the resulting nsp2 cleavage product is quite variable, ranging from 571 residues in EAV to 1195 residues in the VR2332 strain of PRRSV (Nelsen et al., 1999
). For EAV, it has been shown that cleaved nsp2 is an essential co-factor for cleavage of the nsp4|5 site by the nsp4 3CLSP (Wassenaar et al., 1997
).
The activity of the EAV nsp2 CP2 has been analysed in vivo using infected cells and eukaryotic expression systems (Snijder et al., 1994 , 1995
), where the proteinase cleaves rapidly and probably in cis. Trans-cleavage activity could also be demonstrated in a eukaryotic expression system, albeit with relatively low efficiency (Snijder et al., 1995
). Because its activity could not be demonstrated in vitro there is only indirect evidence to support the idea that the EAV CP2 cleaves between two Gly residues (Gly-831|Gly-832). Specifically, it has been shown that mutagenesis of the putative P1 residue (Gly-831 to Pro) abolished processing of the nsp2|3 site (Snijder et al., 1996
). Also, the proposed site is conserved among all arteriviruses (Fig. 6C
).
Comparative sequence analysis and site-directed mutagenesis have characterized the CP2 as a cysteine endopeptidase whose conserved domain encompasses about 100 residues. Like the PCP domains, this viral enzyme most clearly resembles viral papain-like cysteine proteinases. Residues Cys-270 and His-332 are assumed to form the CP2 catalytic dyad because their replacement completely inactivated proteolytic activity (Snijder et al., 1995 ). Although the distance between these active-site residues resembles that found in arterivirus papain-like proteinases (Fig. 6B
, C
), there is one important difference. The putative catalytic Cys-270 is flanked by Gly-271, and not by a bulky, hydrophobic residue which is almost always found at this position in viral proteinases and is a hallmark of this group of proteinases (Gorbalenya & Snijder, 1996
; although see above and Johnston et al., 1997
). An attempt to convert CP2 into an active canonical papain-like proteinase through the replacement of Gly-271 by Trp failed (Snijder et al., 1995
), indicating that the fixation of Gly in CP2 is likely to be tightly coupled with additional changes. Unlike the two other arterivirus papain-like proteinases and the FMDV Lpro, CP2 does not cleave immediately downstream of the active-site His. Indeed, about 500 residues separate CP2 and its cleavage site in EAV and this distance can even extend to more than 1100 residues in PRRSV-VR2332 (Nelsen et al., 1999
) (Fig. 6C
). The entire CP2 domain is highly conserved among arteriviruses, which is a remarkable difference to the PCP1
and PCP1
domains. Among the conserved residues are a number of cysteines and one aspartate residue (Fig. 6C
). While the replacement of the conserved or neighbouring acidic residues did not influence CP2 activity, the substitution of the conserved Cys residues in EAV abolished (Cys-319, Cys-349 and Cys-354) or reduced (Cys-344 and Cys-356) processing at the nsp2|3 site (Snijder et al., 1995
). It is conceivable that the three conserved and essential Cys residues may be part of a zinc finger, which would resemble the situation found in coronavirus PLpro domains where the structural importance of the zinc finger for the proteinase activity has recently been shown (Herold et al., 1999
; see also above).
The arterivirus nsp2 appears to be a multi-domain protein. It contains highly conserved regions, e.g. a domain with a number of conserved Cys residues in its carboxyl-terminal half, but there are also sequences that cannot be aligned among arteriviruses. A detailed description of the functions associated with these domains remains to be obtained, but it has recently become clear that nsp2 is involved in the generation of a membrane-associated replication complex (van der Meer et al., 1998 ; Pedersen et al., 1999
). This may also have consequences for the proteolytic activity of CP2. Both large and small deletions in the EAV nsp2 region that separates the CP domain and its cleavage site were found to interfere with proteolytic processing at the nsp2|3 junction (Snijder et al., 1995
).
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Proteinases as regulators of nidovirus replication |
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As mentioned above, two alternative pathways can be followed for the processing of the carboxyl-terminal part of the EAV ORF1a protein (Fig. 8). Either the nsp4|5 (major pathway) or the nsp5|6 and nsp6|7 (minor pathway) sites are processed (Wassenaar et al., 1997
). Cleavage at either of these sites is believed to render the alternative site(s) non-accessible. It remains to be shown which factors determine the selection of the site that is initially cleaved by the 3CLSP. It is conceivable that the association of cleaved nsp2 with the nsp38 precursor triggers the 3CLSP to cleave the nsp4|5 site. In this complex, nsp2 is likely to have a strong interaction with nsp3 (Snijder et al., 1994
). If this model is correct, a decisive role in the major pathway could be assigned to CP2 (since it generates nsp2). Furthermore, it should be remembered that three processing end-products of the ORF1a polyprotein (nsp2, nsp3 and nsp5) contain hydrophobic domains that presumably mediate the anchoring of the EAV replication complex to intracellular membranes (van Dinten et al., 1996
; van der Meer et al., 1998
) and result in modification of these membranes into characteristic double-membrane vesicles (Pedersen et al., 1999
). Thus, it is reasonable to speculate that a specific, membrane-associated folding or post-translational organization of the nsp25 complex may determine the selection of a particular processing pathway.
|
The question of whether the two processing pathways of the carboxyl-terminal part of the ORF1a polyprotein are also used to process the corresponding part of the ORF1ab (frameshift) polyprotein remains to be addressed. The experiments that uncovered the two processing pathways were based on expression of only the EAV ORF1a polyprotein in the recombinant vaccinia virus/T7 expression system. This indicates that ORF1b-encoded sequences are not required for the utilization of alternative pathways. However, it cannot be excluded that the extension of the ORF1a protein with the ORF1b-encoded polypeptide might affect or modulate processing of this region.
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Concluding remarks |
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The diversity of nidovirus proteinases is so pronounced that even the most sensitive computer-aided methods do not reveal any specific relationship between arterivirus and coronavirus enzymes that would justify their grouping and separation from other proteolytic enzymes. However, this dissimilarity at the sequence level is in sharp contrast to the collinearity of the arrays of conserved replicative domains (including the proteinases) and to the similar distribution of proteinase cleavage sites in the nidovirus replicase polyproteins. Thus, the grouping of the nidovirus proteinases is essentially based on the organization and expression of the replicase polyproteins and not on sequence similarity of the proteolytic enzymes. Despite the lack of evidence from structural studies, the hypothesis of divergent evolution from a common nidovirus ancestor, which contained a replicase (gene) with an organization resembling that of the contemporary nidoviruses, provides the most parsimonious explanation for the observed diversity of the proteolytic enzymes. It remains a goal for future studies to identify the selection pressures that, in the main nidovirus lineages, have driven the profound divergence of the proteinases and their targets, as well as the selective forces that determined the conservation of these specific patterns during evolution. As a step toward this goal, a comprehensive characterization of the proteolytic enzymes of the toroviruses, the third genus within the Nidovirales, should be conducted (for a review on toroviruses, see Snijder & Horzinek, 1993 ). With respect to genome size, toroviruses are intermediate between arteriviruses and coronaviruses, and, possibly, the torovirus proteinases bridge the huge gap that separates the proteolytic enzymes of the other two nidovirus genera. The sequence data currently available for toroviruses does not include proteinases, and, furthermore, no experimental data on the processing of the replicase polyproteins have been reported. However, based on the substrate specificity of nidovirus main proteinases and the alignment of the nidovirus replicase polyproteins (A. E. Gorbalenya, unpublished data), a number of potential cleavage sites can be discerned in the published sequence (Snijder et al., 1990
) of the ORF1b-encoded region of the replicase polyprotein of Berne virus.
As outlined in this review, the study of the processing pathways of the nidovirus replicase polyproteins led to the identification of a large number of intermediate and end-products that are believed to represent components of the viral replication complex. These results provide a solid basis for the elucidation of individual protein functions involved in various stages of the nidovirus life-cycle. Obviously, apart from the proteinases themselves, the initial studies were focussed on proteins harbouring putative replicative functions (Heusipp et al., 1997b ; van Dinten et al., 1997
, 1999
) or functions related to the localization of the replication complex. Given the organization and extraordinary size of nidovirus replicase polyproteins, it can be expected that the assembly and function of the viral replication machinery is considerably more complex than that of most other RNA viruses. For example, it has been speculated that Nidovirales and plant closteroviruses (the largest plant positive-stranded RNA viruses with genome sizes up to 20 kb) have evolved specific strategies to build and maintain large RNA genomes (Dolja et al., 1994
; Agranovsky, 1996
). For nidoviruses, only a limited number of protein domains encoded by the replicase gene have been correlated with specific activities or functions. The larger part of the replicase gene encodes proteins for which no counterparts have been identified in other cellular or viral systems. For example, the portion of the nidovirus replicase polyproteins between the main proteinase and the carboxyl terminus of pp1a is extensively processed to produce up to six proteins (Wassenaar et al., 1997
; Liu et al., 1997
; Lu et al., 1998
; Ng & Liu, 1998
; Ziebuhr & Siddell, 1999
). For most of them, however, no functional assignments have been made. Without doubt, reverse-genetic systems, at the moment restricted to arteriviruses, will prove to be valuable tools in the investigation of specific functions involved in different aspects of virus replication and transcription.
Nidovirus-encoded proteinases, like many other viral proteinases, are highly effective regulators of virus replication and, indirectly, possibly even of virion biogenesis (van Dinten et al., 1999 ). Consequently, they represent ideal targets for therapeutic intervention. This would be highly desirable for a number of economically important nidovirus infections. The elaborated substrate specificity of the nidovirus main proteinases should facilitate the identification of selective low molecular weight compounds that can be targeted to the active site of virus proteinases without affecting cellular functions. Over the past years, both rational drug design based on X-ray crystallography and high-throughput screening of compound libraries have aided in the development of such inhibitors. The success of this approach is impressively illustrated by the recent advances in the treatment of human immunodeficiency virus (HIV) infections using HIV proteinase inhibitors (for reviews see Flexner, 1998
; Patick & Potts, 1998
; Wlodawer & Vondrasek, 1998
). In the case of the nidovirus proteinases, it can be expected that additional potential target structures will be identified as the elucidation of interactions between proteinases and RNA or protein components of the replication complex proceeds.
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Acknowledgments |
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![]() |
Footnotes |
---|
c The name 3C indicates that the associated proteinase occupies a position between two other domains, 3B and 3D, in the P3 region of the picornavirus polyprotein (Rueckert & Wimmer, 1984
).
d § Autocatalytic cleavage can occur in cis or in trans. Technically, the nidovirus proteinases were characterized in two types of assays, with the proteinase and its substrate residing in the same molecule, or, alternatively, in different molecules. The first assay type can be called a monomolecular reaction since it permits, although not exclusively, cis cleavage. The second assay type is a bimolecular reaction that is commonly called trans cleavage. If a proteinase proved to be active in both types of assay, it has been shown to cleave in trans but is considered likely to also cleave in cis. In contrast, if the proteinase is only active in the first assay type, it is thought to cleave only in cis. A nidovirus proteinase that is active only in the bimolecular reaction assay has not yet been described. We will use the terms cis and trans cleavage as defined above, although the reader should be aware that cis-cleavage activity has not rigorously been proven for any of the nidovirus proteinases.
e || This domain and the corresponding domain of HoCV (Herold et al., 1998 ) could be called minimal. We avoid using this word as it may give the impression that the domain in question is necessary and sufficient for proteolytic activity. However, as discussed, the presence of additional domains in the proteinase assay needs to be considered in the interpretation of the results.
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