INTRODUCTION

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The coronaviruses are a group of enveloped, positive-stranded RNA viruses that are associated predominantly with respiratory and gastrointestinal diseases in their natural hosts (28). The human coronaviruses (HCV), which are represented by the prototypes HCV 229E and HCV OC43, are responsible for 5 to 30% of all upper respiratory tract infections in humans, and their involvement in lower respiratory tract illness and gastroenteritis has also been documented (18, 25, 30).

The HCV 229E genome is comprised of approximately 27,000 nucleotides. Gene 1, which is located at the 5' end of the genome, encodes the viral RNA replicase and encompasses two large, overlapping open reading frames (ORFs), ORF1a and ORF1b (14). ORF1a encodes a polyprotein, pp1a, with a calculated molecular weight of 454,000. The downstream ORF, ORF1b, is expressed by ribosomal frameshifting as a fusion protein with pp1a (12), and the predicted gene product, pp1ab, has a calculated molecular weight of 754,000.

Proteolytic processing, and in particular the processing of replicase polyproteins, is a crucial step in the life cycle of many positive-stranded RNA viruses (7, 20). Generally, these processing events are carried out by virus-encoded proteinases. Coronaviruses are no exception, and sequence motifs characteristic of both papain-like cysteine proteinases and a chymotrypsin-like enzyme, the 3C-like proteinase, have been identified in the regions of pp1a and pp1ab encoded by ORF1a (8, 9, 14, 22). Recent studies have confirmed that these activities are indeed responsible for the proteolytic processing of replicase polyproteins and can be implicated in the generation of a functional replication complex (see, for example, references 4, 10, 15, 16, 23, and 31).

Sequence analysis of four different coronaviruses, HCV 229E (14), infectious bronchitis virus (5), murine hepatitis virus (MHV) (2, 22), and transmissible gastroenteritis virus (TGEV) (8), has suggested that either one infectious bronchitis virus or two (HCV 229E, MHV, and TGEV) papain-like proteinase activities are encoded in the amino-proximal region of pp1a and pp1ab. To date, no experimental evidence has demonstrated that the carboxyl-proximal domain, PCP2, is functional. In contrast, both in vivo and in vitro data have shown that the amino-proximal domain, PCP1, is active. Thus, the MHV PCP1 domain has been shown to be responsible for the release of two proteins from nascent replicase polyproteins in vitro, the amino-terminal protein p28 and the adjacent protein, p65 (1, 4). Deletion mutagenesis studies have identified the boundaries of the active MHV PCP1 proteinase, and codon mutagenesis has shown that the catalytic residues most likely are Cys-1137 and His-1288 (1). Finally, the MHV pp1a-pp1ab amino acids Gly-247 and Val-248 have been identified as the cleavage site for the release of p28 by MHV PCP1, and amino acids Gly-247 and Arg-246 have been identified as the major determinants for cleavage site recognition (6, 17).

In this paper, we report an analysis of the HCV 229E PCP1 activity. Our results show that the location and catalytic properties of the HCV 229E enzyme are similar to those described for MHV but that there are some peculiarities in the position and structure of the cleavage site used to release the amino-terminal protein, p9, of pp1a and pp1ab. These differences could not be predicted by previous sequence comparisons (8a, 17).

	MATERIALS AND METHODS

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Virus and cells. The methods for HCV 229E propagation in MRC-5 cells and for concentration of virus by use of polyethylene glycol have been described elsewhere (26, 32). HeLa cells (ATCC CCL2) were grown in monolayers in minimal essential medium with Earle's salts and containing 10% heat-inactivated fetal bovine serum, 25 mM HEPES, GLUTAMAX 1 (L-alanyl-L-glutamine), antibiotics, and nonessential amino acids. The recombinant vaccinia virus MVA-T7, which expresses the bacteriophage T7 RNA polymerase, was propagated in chicken embryo fibroblasts as described previously (29).

Preparation of antigen and antiserum. A 632-bp SphI/KpnI cDNA fragment corresponding to nucleotides 412 to 1043 of the genomic RNA of HCV 229E was excised from plasmid pJ12E6 (14) and ligated with SphI/KpnI-digested pQE30 DNA (Diagen, Hilden, Germany). The ligated DNA was transformed into competent Escherichia coli JM109, and individual clones were analyzed by restriction enzyme digestion and sequencing. The correct construct was designated pI1a.1.

Construction of DNAs encoding carboxyl-terminally extended pp1a and pp1ab proteins. Polyadenylated RNA was isolated from HCV 229E-infected MRC-5 cells and reverse transcribed (oligonucleotide 1; Table 1) (13). Then, 2 µl of the reaction mixture was used as a template in a PCR (oligonucleotides 2 and 3; Table 1) to amplify a DNA that corresponds to nucleotides 387 to 12850 of the HCV 229E genomic RNA. Elongase polymerase mixture (Life Technologies, Eggenstein, Germany) was used for all PCR amplifications with the recommended buffer conditions. The cycle conditions were as follows: initial denaturation, 94°C for 30 s; 12 cycles at 94°C for 30 s, 50°C for 30 s, and 68°C for 12 min; 18 cycles at 94°C for 30 s, 50°C for 30 s, and 68°C for 12 min, with 15 s for extension per cycle; and final elongation, 72°C for 10 min.

Construction of pT7-IRES-Pap. The construction of plasmid pT7-IRES-Pap is complex and is illustrated in Fig. 1. The starting plasmids, pPap, pBluescript II KS+, pTM3, and pJ12E6, have all been described elsewhere (13, 14, 24) (Stratagene, Heidelberg, Germany). Briefly, a DNA fragment containing the T7 RNA polymerase promoter and the encephalomyocarditis virus internal ribosomal entry site (IRES) element, derived from pTM3, was cloned into pBluescript II KS+; subsequently, most of the multiple cloning site was removed, resulting in plasmid pT7-IRES(CX). An NcoI site was introduced into pJ12E6 by in vivo recombination PCR (oligonucleotides 16 and 17; Table 1) (12), and HCV 229E ORF1a nucleotides 1 to 1207 were cloned behind the T7-IRES element of pT7-IRES(CX) to produce plasmid pT7-IRES-1a/N. Finally, the small NotI/SpeI fragment of pT7-IRES-1a/N was used to replace the small ApaI/SpeI fragment of pPap to produce plasmid pT7-IRES-Pap. The nucleotide sequence of pT7-IRES-Pap was determined, and two PCR-derived nucleotide misincorporations that led to changes in the deduced amino acid sequence compared to the published sequence (14) (GAG [Glu-1023]  GGG [Gly]; AAA [Lys-1316]  TAA [*]) were identified. Thus, pT7-IRES-Pap encodes a protein corresponding to the amino-terminal 1,315 amino acids of pp1a and pp1ab.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Construction of pT7-IRES-Pap. The diagram illustrates the original plasmids and strategy used to construct plasmid pT7-IRES-Pap. Relevant restriction sites, the major steps in the cloning procedure, important functional elements in the RNA, and proteins encoded by the various plasmids are indicated. UTR, untranslated region; nts, nucleotides.

Codon and deletion mutagenesis of pT7-IRES-Pap. Codon mutations were introduced into pT7-IRES-Pap by in vivo recombination PCR (oligonucleotides 18 to 25; Table 1) (12). Deletions were made in the HCV 229E ORF1a coding region of pT7-IRES-Pap by PCR. Oligonucleotide 26 was used as a downstream primer, and oligonucleotide 27, 28, 29, or 30 was used as an upstream primer (Table 1). The resulting PCR products were digested with NcoI, religated with T4 DNA ligase, and transformed into E. coli Top 10F' bacteria (Invitrogen, Leck, The Netherlands). The nucleotide sequences of the resulting plasmids were determined to exclude PCR-derived nucleotide misincorporations. The proteins encoded by this plasmid series correspond to the initiating methionine of HCV 229E ORF1a followed by pp1a-pp1ab amino acids 578 to 1315 (pT7-IRES-Papdel2-577), 861 to 1315 (pT7-IRES-Papdel2-860), 976 to 1315 (pT7-IRES-Papdel2-975), and 1037 to 1315 (pT7-IRES-Papdel2-1036).

Metabolic labeling, cell lysis, and immunoprecipitation. Metabolic labeling of virus-specific polypeptides was done essentially as described previously (32). Briefly, 2 × 10⁶ HeLa cells in 56-cm² dishes were mock infected or infected with HCV 229E at a multiplicity of 10 PFU per cell. After 1 h, the supernatant was replaced with 10 ml of fresh medium. Radioactive labeling of newly synthesized proteins was done for 3 h at 33°C, between either 4 to 7 h postinfection or 7 to 10 h postinfection. Before labeling, the cells were washed twice with methionine- and cysteine-free Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 2% dialyzed fetal bovine serum. Pro-Mix L-³⁵S in vitro cell-labeling mixture (SJQ 0079; Amersham, Braunschweig, Germany) was added to the cells to yield concentrations of 100 µCi of L-[³⁵S]methionine and 42 µCi of L-[³⁵S]cysteine per ml of medium. After labeling, the cells were lysed, and immunoprecipitation was done with IS1720 or preimmune serum essentially as described by Ziebuhr et al. (32). Proteins were analyzed by electrophoresis in sodium dodecyl sulfate (SDS)-10 to 17.5% polyacrylamide gradient gels.

T7 RNA polymerase-mediated transient expression. Proteins encoded by circular DNA (plasmids) or linear DNA (PCR products) were expressed in HeLa cells with recombinant vaccinia virus MVA-T7 as a source of bacteriophage T7 RNA polymerase (29). To do this, 2 × 10⁵ HeLa cells in 10-cm² dishes were washed twice with OptiMEM (Life Technologies) and transfected with 5 µg of DNA by use of 12.5 µl of Lipofectin (Life Technologies) according to the manufacturer's protocol. After 2 h, the transfection mixture was removed, and the cells were washed twice with medium and infected with MVA-T7 at a multiplicity of 5 PFU per cell. When linear DNA was transfected, the intracellular proteins were metabolically labeled for 6 h starting at 2 h postinfection. When circular DNA was used for transfection, the intracellular proteins were labeled for 2 h starting at 4 h postinfection. Cell labeling, lysis, and immunoprecipitation were done as described above for HCV 229E-infected cells.

In vitro trans-cleavage assay. RNA was synthesized in vitro by use of a MEGAscript T7 kit (Ambion, Austin, Tex.). For the preparation of a labeled substrate, RNA transcribed from the PCR product encoding pp1a-pp1ab amino acids 1 to 956 was translated (100 ng/µl of reaction mixture) in a reticulocyte lysate (Promega, Heidelberg, Germany) in the presence of [³⁵S]methionine as described previously (13). Proteins with putative proteolytic activity were translated in a separate reaction in which [³⁵S]methionine was replaced with [³²S]methionine at a concentration of 50 µM. Both the substrate and the enzyme translation reactions were stopped after 1 h of incubation at 30°C by the addition of 0.1 volume of TL-stop mix (10 µg of cycloheximide per µl, 100 ng of RNase A per µl, 5 mM [³⁵S]methionine). Then, 1 volume of substrate and 2.5 volumes of enzyme reaction mixtures were mixed and incubated for 3 h at 30°C. Immunoprecipitation with IS1720 and protein analysis in SDS-polyacrylamide gels was done as described above.

Amino-terminal protein sequence analysis. trans-Cleavage assays with 100 µl of substrate and 250 µl of enzyme reaction mixtures were done as described above, except that the in vitro-synthesized substrates were radiolabeled separately with either [³⁵S]methionine or [³⁵S]cysteine. The products of the cleavage reactions were immunoprecipitated with IS1720, separated by electrophoresis in SDS-10% polyacrylamide gels, and transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Munich, Germany) (32). The areas of the membranes containing cleavage products were identified by autoradiography and isolated. The bound proteins were then subjected to 20 cycles of Edman degradation by use of a pulsed-liquid protein sequencer (ABI 467A; Applied Biosystems, Weiterstadt, Germany). The eluate from each cycle was mixed with scintillation cocktail, and the radioactivity was measured.

	RESULTS

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Identification of four proteins derived from the amino-terminal region of the HCV 229E pp1a and pp1ab polyproteins in vivo. To facilitate the analysis of proteolytic processing events at the amino terminus of the HCV 229E replicase polyproteins, we first generated a polyclonal rabbit antiserum containing antibodies specific for a region of pp1a/pp1ab corresponding to amino acids 41 to 250 (Fig. 2A). This serum, IS1720, reacted strongly with the bacterial fusion protein used for immunization but not with other bacterial proteins (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Detection of p9 in HCV 229E-infected cells. (A) Schematic representation of HCV 229E ORF1a; the relative position of the bacterially expressed fusion protein is shown. The putative catalytic amino acids of PCP1, PCP2, and the 3C-like proteinase are indicated, as are the boundaries of the domain of the 3C-like proteinase. (B) Metabolically labeled lysates from mock- or HCV 229E-infected HeLa cells were analyzed by electrophoresis with an SDS-containing 10 to 17.5% polyacrylamide gradient gel before or after immunoprecipitation with preimmune serum or IS1720 immune serum. The cells were labeled from 4 to 7 h or 7 to 10 h postinfection. Either 1-µl lysates from mock- or HCV 229E-infected cells were analyzed directly (lanes 1 to 3) or 140-µl lysates were analyzed after immunoprecipitation with preimmune serum (lanes 4 to 6) or IS1720 immune serum (lanes 7 to 9). Protein molecular weight markers (in thousands; lanes M) (CFA 626; Amersham), p9, and infection-specific higher-molecular-weight polypeptides are indicated.

HCV 229E PCP1 is likely to be responsible for the generation of the p9 protein. It has been shown that MHV PCP1 is a papain-like proteinase responsible for the cleavage of amino-terminal p28 from pp1a and pp1ab (1, 17). Therefore, it seemed likely that the cleavage of p9 would also be mediated by a homologous enzyme, HCV 229E PCP1. To test this hypothesis, we produced a series of PCR products that contained a T7 RNA polymerase promoter, an EMCV IRES element at the 5' end, and different 3' extensions representing HCV 229E ORF1a from codons 1 to 111 (DNA 4/5) to codons 1 to 2058 (DNA 4/15) (Fig. 3A). These PCR DNA templates could be synthesized in quantitative amounts and were sufficiently homogeneous to be used in transfection experiments without further purification (data not shown). Seven of these DNAs were transfected into HeLa cells. Subsequently, the cells were infected with recombinant vaccinia virus MVA-T7. Newly synthesized proteins were metabolically labeled from 2 to 8 h postinfection, and cell protein lysates were then immunoprecipitated with IS1720 serum. The precipitated proteins were analyzed by gel electrophoresis and autoradiography.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Demonstration of HCV 229E PCP1 activity in transfected cells. (A) Schematic representation of HCV 229E ORF1a, the in vitro ligation product, and the positions of the PCR primers used to generate PCR products. The putative catalytic amino acids of PCP1 and PCP2 are indicated, as are the boundaries of the domain of the 3C-like proteinase. (B) Metabolically labeled lysates from mock-infected (lane 1) or DNA-transfected (lanes 2 to 9) HeLa cells that had been coinfected with MVA-T7 were immunoprecipitated with IS1720 antiserum. The immunoprecipitated proteins were analyzed by electrophoresis in an SDS-containing 10 to 17.5% polyacrylamide gradient gel. Protein molecular weight markers (in thousands; lanes M), p9, p93, and p102 are indicated.

Codon mutagenesis of HCV 229E PCP1. The results shown above suggest that HCV 229E PCP1 is responsible for the cleavage of p9 from the replicase polyproteins. To strengthen this conclusion and to provide experimental data to support the prediction of Cys-1054 and His-1205 as the catalytic residues of this proteolytic activity (14), we carried out codon mutagenesis of the HCV 229E PCP1 domain.

View larger version (79K): [in this window] [in a new window]   FIG. 4.   Codon mutagenesis of HCV 229E PCP1. HeLa cells were mock transfected (lane 1) or DNA transfected (lanes 2 to 11) and then infected with MVA-T7. The cells were metabolically labeled, and cell protein lysates were immunoprecipitated with IS1720 antiserum. The immunoprecipitated proteins were analyzed by electrophoresis with an SDS-containing 10 to 17.5% polyacrylamide gradient gel. Protein molecular weight markers (in thousands; lanes M) and p9, p93, p102, p137, and p146 are indicated.

Mapping of the PCP1 domain. In order to identify the amino-terminal and carboxyl-terminal borders of the active HCV 229E PCP1 proteinase, we used an in vitro trans-cleavage assay in combination with deletion mutagenesis. This approach allows for the modification of the enzyme without introducing changes in the substrate molecule and significantly simplifies the interpretation of the results.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Mapping the borders of HCV 229E PCP1. (A) Schematic representation of HCV 229E ORF1a and the polypeptides tested for proteolytic activity. The putative catalytic amino acids of PCP1 are also indicated. (B) trans-Cleavage assay with amino-terminally and carboxyl-terminally truncated pp1a and pp1ab proteins. To produce a substrate for the trans-cleavage assay, the amino-terminal 956 amino acids of pp1a and pp1ab were translated in vitro in a reticulocyte lysate in the presence of [35S]methionine. The polypeptides to be tested for proteolytic activity were translated in vitro in a reticulocyte lysate in the presence of [32S]methionine. After the termination of translation, 1 volume of substrate reaction mixture was incubated without (lanes 1 and 10) or with (lanes 2 to 9 and 11 to 14) 2.5 volumes of enzyme reaction mixture for 3 h at 30°C. The cleavage reaction products were then immunoprecipitated with IS1720 antiserum, and the proteins were analyzed by electrophoresis in an SDS-containing 10 to 17.5% polyacrylamide gradient gel. Protein molecular weight markers (in thousands; lanes M), the uncleaved substrate, and the two cleavage products, p9 and a larger protein, are indicated. The lower panel shows a longer exposure of the low-molecular-weight region of the gel.

PCP1 cleaves the Gly-111-Asn-112 peptide bond. The in vitro trans-cleavage assay described above also allowed us to determine the HCV 229E PCP1 cleavage site used for the generation of p9. Thus, the amino-terminal 956 amino acids of pp1a and pp1ab were translated in vitro with either [³⁵S]cysteine or [³⁵S]methionine as the radiolabel. These substrates were incubated together with in vitro-synthesized enzyme and, after transfer to PVDF membranes, the position of the carboxyl-terminal proteolytic product was determined by autoradiography. This area of the membrane was then isolated, and the bound protein was subjected to 20 cycles of Edman degradation. The results are shown in Fig. 6. Peaks of radioactivity were found at position 10 when the substrate was labeled with [³⁵S]cysteine and at position 18 when the substrate was labeled with [³⁵S]methionine.

View larger version (30K): [in this window] [in a new window]   FIG. 6.   Identification of the PCP1 cleavage site. Preparative-scale trans-cleavage reactions were carried out with [35S]methionine- or [35S]cysteine-labeled substrate. After immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and electrophoretic transfer to PVDF membranes, the position of the carboxyl-terminal cleavage product was determined by autoradiography, the isolated proteins were subjected to 20 cycles of Edman degradation, and the distribution of radiolabeled amino acids was determined. The amino acid sequence of pp1a and pp1ab from positions 112 to 131 is shown. The amino acids Cys-121 and Met-129 are underlined.

	DISCUSSION

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The data presented in this paper represent the first characterization of the HCV 229E PCP1 proteinase. In many respects, the results that we obtained parallel those of earlier studies on the MHV PCP1 proteinase (1, 6, 17); in other respects, they reveal some intriguing and significant differences.

First, in keeping with previous predictions (14), mapping of the HCV 229E PCP1 domain indicates that the HCV 229E proteinase shares a common location with its counterpart from MHV, approximately 900 to 1300 amino acids from the amino terminus of the replicase polyproteins pp1a and pp1ab. However, despite this overall congruity, a closer analysis of the data reveals some specific differences. For example, our results located the amino-terminal border of the active HCV 229E PCP1 domain between pp1a-pp1ab amino acids 861 and 975. Bonilla et al. (3) reported that the active MHV PCP1 domain lies between pp1a-pp1ab amino acids 1084 and 1316. When the HCV 229E and MHV pp1a and pp1ab sequences are optimally aligned in this region (8a), the MHV pp1a-pp1ab amino acid Ala-1084 is found to correspond to the HCV 229E pp1a-pp1ab amino acid Thr-1025. Thus, the HCV 229E PCP1 proteinase domain seems to be extended at the amino terminus relative to the MHV PCP1 proteinase domain. Further detailed experiments will be required to assess the significance of this observation.

Second, as indicated by a sequence alignment analysis (14), our results, combined with those of Baker et al. (1), suggest that homologous residues (Cys-1054 and His-1205 for HCV 229E and Cys-1121 and His-1272 for MHV) probably act as the catalytic amino acids for both enzymes. Also, on the basis of our results, it is most likely that coronavirus PCP1 enzymes are able to function in trans, not only in vitro (4) but also in transfected cells.

Third, it is striking that the cleavage sites used by the HCV 229E and MHV PCP1 proteinases at the amino terminus of the replicase polyproteins are different in both position and sequence. Thus, the MHV activity cleaves a protein, p28, from the amino terminus of pp1a and pp1ab, and the recognition site is NH₂-243ArgGlyTyrArgGlyValLysProIleLeu252-COOH, with cleavage between Gly-247 and Val-248 (6, 17). The HCV 229E PCP1 proteinase cleaves a protein, p9, from the amino terminus of pp1a and pp1ab, and the recognition site is NH₂-107LysArgGlyGlyGlyAsnValThrTyrThr116-COOH, with cleavage between Gly-111 and Asn-112. Thus, not only the position and recognition sequence but also the scissile bond of PCP1-mediated processing are clearly different for these two viruses.

The coronavirus PCP1 substrates described in this paper and by others (4, 6, 17) obey a general pattern which includes cleavage between small uncharged residues: a basic amino acid at the P5 position and relative flexibility at the P2, P3, and P4 positions. Within this framework, the viruses differ by using, for instance, different small residues in the P1 and P1' positions. The MHV p28-p65 cleavage site, the HCV 229E PCP1 cleavage site, a homologous sequence from TGEV, and their neighboring sequences can be aligned as two ungapped blocks, AI and AII (Fig. 7A). Block AI comprises the aligned cleavage sites but is not statistically significant and cannot be selected without prior knowledge of the locations of (putative) functionally equivalent residues which are conserved in the essential P1 and P5 positions of the HCV 229E and MHV PCP1 cleavage sites. According to the alignment of Fig. 7A, a region around the PCP1 cleavage site might have evolved by accepting replacements as well as insertions or deletions immediately downstream of this site in the three coronavirus lineages. This alignment also predicts that Arg-106 and Gly-110 will occupy, respectively, the P5 and P1 positions of a putative PCP1 cleavage site in the TGEV pp1a and pp1ab polyproteins.

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Multiple amino acid alignments of three coronavirus pp1a and pp1ab sequences around the PCP1 cleavage site. The gap-free alignments (blocks) shown in uppercase letters were generated by use of the MACAW program (27) with a Gibbs sampler (21) and the Blossum62 scoring table (11). The statistical significance (19) of the alignments was assessed with a searching space comprised of the MHV A59, HCV 229E, and TGEV pp1a sequences. Fragments of the sequences not aligned by the program are shown in lowercase type. Roman bold type indicates residues conserved in any two sequences; italic bold type indicates a positive residue occupying the P5 position relative to the (putative) PCP1 cleavage site, whose position is indicated in the MHV A59 and HCV 229E sequences with ><. The positions of the fragments aligned within the pp1a and pp1ab sequences and sequence database accession numbers are indicated on the left and right sides of the alignments, respectively (GB, GenBank; SP, Swissprot). (A) Alignment constrained by the need to have the P5 and P1 residues of the PCP1 cleavage sites of MHV A59 and HCV 229E in equivalent positions. The statistical significances of the similarities in block AI and block AII were 1.0e+0 (statistically nonsignificant) and 1.2e3, respectively. (B) Alignment including the statistically most favorable block encompassing the PCP1 cleavage sites in MHV A59 and HCV 229E. The statistical significances of the similarities in block BI and block BII were 1.0e+0 (statistically nonsignificant) and 4.4e5, respectively.

It is important to note, however, that an alternative alignment of the region encompassing the PCP1 cleavage site can be deduced by a comparative sequence analysis (Fig. 7B). This alignment includes a block-ungapped BII, the only statistically significant block identified within the amino-terminal region of the pp1a and pp1ab polyproteins upstream of the PCP1 domain. Most notably, in the analysis shown in Fig. 7B, the MHV and HCV 229E cleavage sites are shifted by two residues relative to one another. This alignment suggests that no insertions or deletions have been accepted in a region delimited between the cleavage site and the downstream conserved region (tripeptide Asp-Gln-Tyr) in the three coronavirus lineages. If this suggestion is correct, then the position of the PCP1 cleavage site has migrated in these polyproteins over the course of evolution. Also, for TGEV, cleavage at either Gly-110-Ala-111 or Thr-107-Gly-108, both of which conform to PCP1 site rules (see above), would be compatible with this model.

Irrespective of which analysis correctly reflects the ancestral relationships among the proteins of the three coronaviruses, both alternatives can be reconciled if it is assumed that the PCP1 cleavage site region is multifunctional and under complex selective pressure driven by both divergent and convergent evolution.

It is also worth noting that, in contrast to the situation with MHV (4), our data provide no indication of further HCV 229E PCP1-mediated cleavages in the replicase polyproteins, at least within the first 1,500 amino acids.

Very little is known about the function of HCV 229E protein p9 or, indeed, of any of the proteins derived from coronavirus pp1a and pp1ab proteins by PCP1 activity. This lack of knowledge is partly due to the fact that there are no obvious sequences from which a putative function can be deduced. Immunofluorescence assays of HCV 229E-infected cells with IS1720 serum showed a punctate pattern of staining in the perinuclear region, like that found with antisera specific for the HCV 229E 3C-like proteinase, antisera specific for the putative metal-binding and helicase protein (p71) (16), and a monoclonal antibody specific for p41, a 3C-like proteinase-mediated processing product encoded by ORF1b (15). This result suggests that at least one of the proteins reacting with IS1720 serum remains associated with the viral replication complex and therefore may have a role in RNA replication and transcription.

Now that we have identified the trans-active domain of HCV 229E PCP1 and a corresponding substrate recognition sequence, we will try to (over)express a biologically active form of the PCP1 protein in bacteria or eucaryotic cells. This approach has been very successful for HCV 229E 3C-like proteinase (31, 32) and would allow for detailed biochemical and structural studies on the papain-like proteinases of coronaviruses. Structural studies on a purified form of HCV 229E PCP1 would be very desirable because this enzyme is an obvious target for the design of synthetic inhibitors to control coronavirus infections.