Identification of a new cleavage site of the 3C-like protease of rabbit haemorrhagic disease virus

Pascale Joubert1, Carine Pautigny2, Marie-Françoise Madelaine2 and Denis Rasschaert1

Laboratoire de Virologie et Barrière d’Espèce, Unité de Pathologie Aviaire et de Parasitologie, INRA de Tours, 37380 Nouzilly, France1
Unité de Virologie et d’Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France2

Author for correspondence: Denis Rasschaert. Fax +33 2 47 42 77 74. e-mail rasschae{at}tours.inra.fr


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
The calicivirus rabbit haemorrhagic disease virus (RHDV) possesses a 3C-like protease which processes the RHDV polyprotein. In order to monitor the proteolytic activity of the RHDV 3C-like protease on its putative target sequences, i.e. the 10 EG dipeptide bonds distributed along the large polyprotein, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion protein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the coding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequences of the fusion proteins were cloned downstream of the T7 promoter and the proteolytic activity was evaluated by measuring the luciferase activity in both in vitro and ‘in vivo’ systems. The EG dipeptide bonds at positions 718–719, 1108–1109 and 1767–1768 were confirmed as cleavage sites and a new cleavage site EG (143–144) was identified.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Rabbit haemorrhagic disease virus (RHDV), the causative agent of a fulminating hepatitis in rabbits, is a member of the Caliciviridae (Ohlinger et al., 1990 ; Parra & Prieto, 1990 ). The genome of RHDV consists of a positive-stranded RNA of 7437 nt linked covalently to a VPg at the 5' end. In addition to the genomic RNA, a subgenomic RNA of 2·2 kb is found both in infected hepatocytes and in virions. Genomic and subgenomic RNAs are packaged in non-enveloped icosahedral capsids of about 35 nm in diameter (Meyers et al., 1991a , b ). The subgenomic RNA has the coding capacity for the major capsid protein VP60 and the small ORF present at the 3' end of the genome (ORF2). Most of the capsid protein might be translated from the 2·2 kb subgenomic RNA but it has been suggested that a fraction of the capsid protein could also result from polyprotein processing (Boniotti et al., 1994 ; Parra et al., 1993 ).

The genomic RNA of RHDV has been cloned as cDNA and completely sequenced (Meyers et al., 1991a ; Rasschaert et al., 1995 ). It contains two ORFs: ORF1, which encodes a large polyprotein of 257 kDa, and ORF2, which encodes a structural protein VP10. Sequence comparison has allowed the identification of non-structural and structural proteins on the large polyprotein. The non-structural proteins identified were RNA helicase, protease and RNA-dependent RNA polymerase, homologous to the 2C, 3C and 3D proteins of picornaviruses, respectively. The structural proteins identified on the polyprotein to date are the VPg and the capsid protein. Analysis of RHDV protein expression in infected hepatocytes (König et al., 1998 ) led to the identification of three proteins, pro1, pro2 and pro3, for which defined biological functions were not proposed. pro1 and pro2 are localized at the N-terminal extremity of the polyprotein and pro3 is localized between the helicase and VPg.

On the basis of amino acid sequence alignments and directed mutagenesis results, the catalytic triad of the 3C-like protease was found to consist of His-1135, Asp-1152 and Cys-1212, which is characteristic of trypsin-like cysteine proteases (Boniotti et al., 1994 ).

Previous studies have shown that the 3C-like protease of RHDV is involved in processing of the large polyprotein. Three cleavage site specificities of RHDV protease have already been characterized: EG (718–719), EG (1108–1109) and EG (1767–1768) (Boniotti et al., 1994 ; Wirblich et al., 1995 ; Alonso et al., 1996 ). An additional ET (1251–1252) dipeptide seemed to be cleaved by RHDV 3C-like protease, although with a lower efficiency, as studied in a bacterial expression system (Wirblich et al., 1995 ).

In the present paper, the 10 potential EG sites and the ET site identified on the RHDV sequence were subcloned at the junction of the 3C-like protease and luciferase genes. The cleavage specificity of these potential sites was evaluated in two assays: (i) in vitro translation with the rabbit reticulocyte lysate (RRL) system; and (ii) the vaccinia-T7/RK13 cell (rabbit kidney cells) system (‘in vivo’). These experiments confirmed the three previously described EG dipeptide bonds and identified a new specific cleavage site EG (143–144) dipeptide bond.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Plasmid constructs.
Most DNA manipulations were performed using standard methods (Sambrook et al., 1989 ). PCR amplifications were carried out in two successive sets of cycles. PCR conditions were: 5 cycles of 1 min at 94 °C, 1 min at 37 °C, and 1 min at 72 °C, followed by 25 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C. All samples were held after the 30 cycles at 72 °C for 10 min. For all plasmids, regions of interest were verified by DNA sequence analysis.

The pBPL10 construct was the starting point for this work. This plasmid contains, under the T7 promoter, the 3C-like protease–luciferase genes fused in-frame with a multiple cloning site region at the junction of the two genes. In this construct, during PCR amplification, two mutations in the luciferase gene were introduced: (i) the EcoRI site was replaced by one BamHI site; and (ii) the initiation codon was removed and a SalI site was introduced at this location in order to minimize the potential background caused by internal initiation occurring during in vitro translation. The mutated luciferase gene was cloned at the EcoRI site of pBluescript SK(-) (Stratagene) after PCR amplification from pUG5-151 (Rasschaert et al., 1995 ) with primer 123 (5' gat atc gaa ttc gtc gac gcc aaa aac ata aag aaa ggc 3'; EcoRV, EcoRI and SalI sites are underlined, respectively) and primer 50 (5' aga tct gaa ttc tta caa ttt gga ctt tcc gcc 3'; EcoRI site and stop codon are underlined, respectively). The 980 bp fragment spanning codons 1108–1434 of ORF1 overlapping the putative RHDV protease was obtained by PCR amplification from pUG5-151 (Rasschaert et al., 1995 ) with primer 35b (5' tct aga ctc gag atg gag ggc ctg cct ggg ttc 3'; XhoI site and the initiation codon are underlined, respectively) and primer 108 (5' gag ctc aag ctt tct aga gct agc ttt cac ctt gtc aag agg cct gag 3'; HindIII, XbaI and NheI sites are underlined, respectively). The PCR product was cloned between XhoI and HindIII sites upstream of the mutated luciferase gene. At the junction of the protease and luciferase genes, the pBPL10 construct displays multiple cloning sites consisting, in succession, of the NheI, XbaI, HindIII, EcoRV, EcoRI and SalI sites (Fig. 1a).



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Fig. 1. Proteolytic cleavage of the EG10 dipeptide. Correlation between cleavage of the fusion proteins and recovery of luciferase activities. (a) Schematic diagram of pBPL10 and pBPLEG10 constructs. The hatched box, the open box and the solid box represent the plasmid pBluescript SK(-), the protease gene and the luciferase gene, respectively. The small grey-tinted box represents the cloning sites. Differences between pBPL10 and pBPLEG10 constructs (grey-tinted box) are shown. (b) Autoradiography after SDS–PAGE analysis of the labelled products obtained by coupled transcription/translation in RRL system with pBP3C, pBPLEG10KO, pBPLEG10, pBPL11, pBPL10 and pT7Luc plasmids. Molecular masses are indicated on the right. The coupled transcription/translation of pBP3C and pT7Luc allowed identification of the labelled protease and luciferase enzymes, respectively. (c) Relative luciferase activity of the pBPLEG10, pBPL11, pBPLEG10KO and pBPL10 transcription/translation products in RRL and vaccinia-T7/RK13 cells (VT7) systems. For pBPLEG10, the luciferase activity is artificially set at 100%. For pBPL10 and pBPL11, results represent the average of relative luciferase activities in eight assays. For pBPLEG10KO, results represent the average of relative luciferase activities in six assays.

 
The pBPLEG1, pBPLEG2, pBPLEG3, pBPLEG4, pBPLEG5, pBPLEG6, pBPLEG7, pBPLEG8, pBPLEG9, pBPLEG10, pBPLET, pBPLA and pBPL11 constructs were obtained by direct cloning of the corresponding annealed phosphorylated oligonucleotides (Table 1) at NheI–SalI sites of the pBPL10. Annealing was performed by heating for 10 min at 100 °C and cooling to 35 °C, in a volume of 200 µl containing 5 nmol of each oligonucleotide, 50 mM Tris–HCl buffer, pH 8 and 100 mM NaCl. The annealed oligonucleotides were inserted into the NheI–SalI-cut dephosphorylated pBPL10 vector at a molar ratio of 5:1 (duplex:vector). The plasmid pBP3C was constructed by ligating the 980 bp fragment overlapping the RHDV protease (codons 1108–1434 of ORF1) between the XhoI and HindIII sites of vector pBluescript SK(-).


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Table 1. Sequences of the annealed oligonucleotides

 
The pBPLEG10KO construct was obtained by inserting a mutated protease gene into the NcoI–SalI-cut dephosphorylated pBPL10 vector. The mutation was targeted to the catalytic site of the protease gene. Cys-1212 and its two neighbouring amino acids were replaced by the PCR method. The primers used were: 372 (5' tct aga cca tgg agg gcc tgc ctg ggt tc 3'; XbaI and BglII sites are underlined, respectively), 626 (5' cag cca gat ctc acc gtg ggt ggt ctg 3'; BglII site is underlined), 627 (5' ggt gag atc tgg ctg ccg ttg tat gac tcc a 3'; BglII site is underlined) and 628 (5' tct aga gtc gac gcg ggc ttt gcc ctc cat aac g 3'; XbaI and SalI sites are underlined, respectively). Primers 626 and 627 harbour a BglII cloning site which allows Asp-1211 to be replaced by Glu, Cys-1212 by Ile and Gly-1213 by Trp. The mutated protease gene was obtained by two-step PCR amplification from pBPLEG10. Firstly, a 312 bp fragment spanning codons 1108–1214 of ORF1 was obtained by PCR amplification with primers 372/626 and was cloned into pGEM-T vector (Promega) generating the pG372/626 construct. Secondly, a 672 bp product spanning codons 1210–1434 of ORF1 and ending with the EG10 site described in Table 2 was obtained by PCR amplification with primers 627/628 and was also cloned into the pGEM-T vector (Promega) generating the pG627/628 construct. The NcoI–BglII fragment of pG372/626 was ligated into NcoI–BglII-cut dephosphorylated pG627/628 plasmid, generating the pG3CKO construct. Finally, the NcoI–SalI fragment of pG3CKO was ligated into the NcoI–SalI-cut dephosphorylated pBPL10 vector generating the pBPLEG10KO construct. Mutated clones were identified by incorporation of the BglII site and the mutated protease gene sequence was confirmed by sequence analysis.


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Table 2. The nucleotide sequences of the potential proteolytic processing sites

 
{blacksquare} In vitro transcription and translation assays.
Eukaryotic in vitro transcription/translation assays were performed with the TNT7 system as specified by the manufacturer (Promega).

To visualize the cleavage efficiency at the different sites, in vitro expression from the constructs was carried out with [35S]Met radiolabel as a tracer. The reaction mix, which contained 0·5 µg circular plasmid DNA, 0·5 µl T7 RNA polymerase, 28·6 µCi Pro-mix L-[35S] in vitro cell labelling mix (Amersham), 20 units RNasin, 0·5 µl amino acid mixture minus Met, 1 µl TNT reaction buffer and 12·5 µl RRL, was incubated for 2 h at 30 °C. Five microlitres of translation reaction product was separated by electrophoresis on SDS–12% polyacrylamide gel (Laemmli, 1970 ) and the distribution of radiolabel was determined by autoradiography.

To measure luciferase activities, in vitro transcription/translation assays were performed without the addition of radiolabel. The reaction mix contained: 0·1 µg circular plasmid DNA, 0·3 µl T7 RNA polymerase, 12 units RNasin, 0·3 µl amino acid mixture minus Met, 0·3 µl amino acid mixture minus Leu, 0·6 µl TNT reaction buffer and 7·5 µl RRL, and was incubated for 2 h at 30 °C. Five microlitres of translation reaction mixture was diluted with 100 µl luciferase buffer (25 mM Tris–phosphate, pH 7·4, 8 mM magnesium chloride, 1 mM DTT, 1 mM EDTA, 1% Triton X-100, 1% BSA and 15% glycerol) prior to measuring the luciferase activity as described by Nguyen et al. (1988) with a luminometer (Autolumat LB 953 Berthold).

{blacksquare}In vivo’ expression assays.
Transient expression in RK13 cells (ATCC CCL 37) was performed as described by Fuerst et al. (1986) . Briefly, RK13 cells were seeded onto 24-well cell culture plates 48 h prior to transfection. Cells were infected with recombinant vaccinia vTF7-3 in Eagle’s minimum essential medium at an m.o.i. of 20 p.f.u. per cell. After incubation at 37 °C for 1 h, 1 µg plasmid DNA was transfected into cells with lipofectin according to the manufacturer’s instructions (Gibco BRL). After incubation for 5 h at 37 °C, the cells were harvested with 200 µl luciferase buffer (Nguyen et al., 1988 ). After clarification (14000 g for 2 min), the luciferase activity was measured from two aliquots of 100 µl of the supernatant as described by Nguyen et al. (1988) with a luminometer (Autolumat LB 953 Berthold).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
In previous studies, three EG dipeptide bonds were identified as cleavage sites for RHDV 3C-like protease (Boniotti et al., 1994 ; Wirblich et al., 1995 ; Alonso et al., 1996 ). Analysis of the RHDV polyprotein sequence revealed 10 EG dipeptide bonds distributed along the polyprotein sequence: EG1 (54–55), EG2 (143–144), EG3 (338–339), EG4 (718–719), EG5 (776–777), EG6 (823–824), EG7 (1108–1109), EG8 (1169–1170), EG9 (1435–1436), EG10 (1767–1768) (Rasschaert et al., 1995 ). In the absence of a simple efficient cell culture system for studying the proteolytic activity of RHDV 3C-like protease on these different sites, we tried to define a strategy that would allow the identification of the cleavage sites by using a sensitive reporter gene (luciferase) in an expression system that could be tested both in vitro and in rabbit epithelial cells (T7 polymerase experiments). It has been shown (D. Rasschaert, personal communication) that when the luciferase was expressed in the C-terminal position of a fusion protein, if the protein in the N-terminal position was larger than 30 kDa, the resulting fusion protein displayed a dramatic loss of luciferase activity. We took advantage of this to test the proteolytic activity of the 3C-like protease of RHDV on several potential sites. In order to test the efficiency of this approach, four constructs were made: pBPL10, pBPLEG10, pBPL11 and pBPLEG10KO. pBPL10 contains, under the T7 promoter, the 3C-like protease–luciferase genes fused in-frame with a multiple cloning site region at the junction of the two genes. pBPLEG10 is a modified pBPL10 plasmid which contains, at the junction of the protease and luciferase genes, the coding sequence for the EG10 dipeptide bond flanked by its three amino acids on both sides of the dipeptide (Fig. 1a). This sequence, at the beginning of the capsid gene, was the first cleavage site defined for the RHDV protease (Boniotti et al., 1994 ; Wirblich et al., 1995 ; Alonso et al., 1996 ). pBPL11 is a modified pBPLEG10 lacking the coding sequence for the EG dipeptide but keeping the coding sequence for the three amino acids on both sides of the dipeptide (Table 2). Thus, as with pBPLEG10, this construct has an AUG codon situated just upstream of the luciferase gene. pBPLEG10KO is a modified pBPLEG10 encoding an inactivated RHDV protease by modification of the Cys-1212, which is recognized as the nucleophilic residue of the catalytic triad (Boniotti et al., 1994 ).

In order to verify the coding capacity of our constructs, the pBPL10, pBPLEG10, pBPL11, pBPLEG10KO plasmids and two control plasmids, pBP3C and pT7Luc (provided by the manufacturer), were used for eukaryotic in vitro transcription/translation with the TNT7 system (Promega) and with [35S]Met as a tracer. Labelled products were separated by electrophoresis on SDS–12% polyacrylamide gels and the distribution of radiolabel was determined by autoradiography (Fig. 1b). The lanes corresponding to pBPLEG10, pBPL10, pBPL11 and pBPLEG10KO all coded for a band of varying intensity with an apparent molecular mass of 98 kDa which corresponds to the molecular mass of the fusion protein. Two additional proteins of 62 and 36 kDa were also identified after transcription/translation of pBPLEG10. These two proteins apparently resulted from the proteolytic cleavage of the protein fusion (98 kDa) yielding the luciferase (62 kDa) and the protease (36 kDa). The blurred appearance of the 36 kDa band stems from the migration of 36 and 34 kDa products which correspond to the expected translation product and to the product initiated at the first internal AUG codon, respectively. A weak band of 62 kDa was also observed in pBPL11 and pBPLEG10KO transcription/translation. This protein probably stemmed from an internal initiation translation at the AUG located just upstream of the luciferase gene. This kind of internal initiation is considered to be a common phenomenon during translation in the RRL system. As this AUG is absent in pBPL10, the 62 kDa band was not observed, but faster-migrating, labelled proteins were present, also resulting from internal initiations at in-frame AUG. A band with an apparent molecular mass of 36 kDa was not visualized in any of the transcription/translation products of pBPL10, pBPL11 or pBPLEG10KO.

Subsequently, we wanted to check whether the presence of the 62 kDa band, and possibly its amount, correlated well with the luciferase activity resulting from the transcription/translation experiments. In this way, the luciferase activities resulting from the transcription/translation of pBPL10, pBPL11 and pBPLEG10 were measured in the RRL system. To standardize our results, the luciferase activity of pBPLEG10 was artificially set at 100% in all experiments. The relative luciferase activities of pBPL10 and pBPL11 were 20 and 30%, respectively (Fig. 1c). The luciferase activity stemming from in vitro transcription/translation of pBPLEG10 is related to the visualization of the intense 62 kDa band and the presence of the 36 kDa band as shown by autoradiography (Fig. 1b, lane pBPLEG10). The residual luciferase activity (20%), resulting from in vitro transcription/translation of pBPL10, defined the background which came from translation initiation at an internal AUG codon located near the 3' end of the protease gene, yielding a product migrating slightly slower than the luciferase (Fig. 1b). Moreover, the transcription/translation of pBPL11 yielded a luciferase activity of 30%, which is 10% greater than that of pBPL10. This increase in background may be related to the presence of the AUG codon upstream of the luciferase gene which allows internal initiation yielding a small amount of 62 kDa protein (Fig. 1b, lane pBPL11). To confirm these results, the luciferase activities resulting from the transcription/translation of pBPL10, pBPL11, pBPLEG10 and pBPLEG10KO were measured in the vaccinia-T7/RK13 cell system (Fig. 1c). This eukaryotic system allows the transcription/translation experiments to take place in a cellular environment and seems to prevent the phenomenon of internal initiation observed during translation in the RRL system. As for the RRL system, the luciferase activity of pBPLEG10 was artificially set at 100%. The luciferase activities of pBPL10, pBPL11 and pBPLEG10KO were below 8%. These results confirmed that the residual luciferase activity observed in the RRL system came from internal initiation during the translation. Finally, we obtained the cleavage inhibition either by mutation of the RHDV protease catalytic site or by mutation of the cleavage site. The correlation between light emission and luciferase amount reflected a direct dependency of relative luciferase activity on the cleavage efficiency.

Subsequently, we cloned the encoding sequences of the dipeptide bonds together with their amino acid environment: EG1, EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9 and ET (Table 2). All constructs were tested for fusion protein production and cleavage by SDS–PAGE analysis. Autoradiography (Fig. 2) showed that in vitro expression of pBPLEG2, pBPLEG4, pBPLEG7 and pBPLEG10 yielded one major band which was identified as luciferase. Two other constructs, pBPLEG3 and pBPLEG5, directed the synthesis of a fusion protein which was cleaved to a 62 kDa band, although at a reduced amount (Fig. 2). Expression of pBPLEG1, pBPLEG6, pBPLEG8, pBPLEG9 and pBPLET constructs led to the synthesis of an intact fusion protein, indicating that those sites were not recognized by the 3C-like protease. The relative luciferase activity was also tested after in vitro and ‘in vivo’ transcription/translation. In vitro expression showed that relative luciferase activity of pBPLEG2, pBPLEG4, pBPLEG7 and pBPLEG10 ranked between 95 and 100%, whereas pBPLEG5 and pBPLEG3 scored between 50 and 60%, and pBPLEG1, pBPLEG6, pBPLEG8, pBPLEG9 and pBPLET was between 20 and 40%. pBPLEG2, pBPLEG4, pBPLEG7 and pBPLEG10 ‘in vivo’ expression induced a luciferase activity between 100 and 140%. Transfection of pBPLEG5 and pBPLEG3 resulted in a luciferase activity of 20%. Moreover, pBPLEG1, pBPLEG6, pBPLEG8, pBPLEG9 and pBPLET induced a luciferase activity equivalent to the background. Thus, luciferase activities in the RRL system and vaccinia-T7/RK13 cell system for each construct are comparable (Table 3). Regarding the efficiency of cleavage of RHDV protease at each EG dipeptide bond, all results of luciferase activity (Table 2) are in good correlation with the results from SDS–PAGE analysis (Fig. 2). We conclude that four sites, EG2, EG4, EG7 and EG10, lead to very efficient proteolysis activity. The insertion of five sites, EG1, EG6, EG8, EG9 and ET between the protease and the luciferase did not result in cleavage of the fusion protein. Two sites, EG3 and EG5 induced a weak but significant proteolytic activity. Our results confirmed the three sites, EG4, EG7 and EG10, defined in previous studies (Alonso et al., 1996 ; Wirblich et al., 1995 ; Boniotti et al., 1994 ) and allowed us to define a fourth site, EG2.



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Fig. 2. Proteolytic activity of the RHDV protease. Autoradiography after SDS–PAGE analysis of the in vitro translation products obtained from constructs (pBPLEG1 to 10 and pBPLET) with a tracer. Each lane is identified by construct name. The position of the luciferase is indicated on the right.

 

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Table 3. Luciferase activity scores obtained by in vitro and in vivo translation of each pBPL construct

 
The sharp difference of efficiency in the cleavage according to EG dipeptide bonds suggests that the amino acids at P4, P3, P2, P2', P3' and P4' positions are important for RHDV protease activity. Notably, EG9 (KVKEGKKR) and EG10 (NVMEGKAR) are very similar in terms of their environment. Nevertheless, the EG9 dipeptide bond is not cleaved by RHDV protease, unlike the EG10 site. Essential differences between these two sites result from the nature of the amino acids at the P4, P2 and P3' positions. At the P2 position, there is a Met (hydrophobic amino acid) for EG10 and a Lys (polar amino acid) for EG9. At the P4 position, there is an Asn for EG10 and another Lys for EG9. Therefore, a basic environment at the N-terminal extremity seems to hamper cleavage, whereas the nature of the amino acid at the P2 position seems to be important for cleavage. To confirm the importance of the nature of the amino acid at the P2 position, we constructed pBPLA. This plasmid contains at the junction of the 3C-like protease and the luciferase genes the coding sequence for a mutated EG10 site (Table 2). The mutation consists of replacement of the Met at P2 position by a Ser. Luciferase activities resulting from the in vitro and ‘in vivo’ transcription/translation of pBPLA were 35 and <8%, respectively (Table 3). This shows that replacement of Met by a Ser at position P2 of the EG10 site resulted in a dramatic decrease in cleavage activity. Furthermore, Wirblich et al. (1995) examined the cleavage specificity of the RHDV protease which was determined by the amino acid situated at the P2 position of the EG7 site and concluded that, at this position, there is a preference for large, hydrophobic amino acids. Nevertheless, the comparison between cleaved and uncleaved sites did not lead to a clear consensus regarding the environment of EG dipeptide bonds.

According to the protein analysis realized by König et al. (1998) , EG2, EG4, EG7 and EG10 sites would define the primary processing of the polyprotein leading to the release of the maturation products, p16, p60, p43, p72 and VP60 (Fig. 3). EG3 and EG5 dipeptide bonds could explain the existence of p37 and p23/2 products. Previous studies seemed to show that ET (1251–1252) dipeptide bond could be cleaved by RHDV protease (Wirblich et al., 1995 ) to process the p72 precursor in p15 and p58 products (Fig. 3). In our system, the ET dipeptide bond is not cleaved by RHDV protease. A more efficient cleavage at EG3, EG5 and ET dipeptide bonds could depend on the contribution of viral and/or cellular co-factors which might not be present in our experiments. In several other animal positive-stranded RNA virus systems, it has been demonstrated that viral co-factors strongly influence polyprotein processing. Notably, in hepatitis C virus, NS4A is an important NS3 protease co-factor required for cleavage at the NS3/4A, NS4A/4B and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B (Failla et al., 1994 ; Tanji et al., 1995 ; Bartenschlager et al., 1995 ). Alternatively, another form of 3C-like protease might be required to process these sites. For instance, in poliovirus, the cleavage of P1 to capsid proteins is more efficiently performed by the 3CD intermediate than by the 3C-like protease (Ypma-Wong et al., 1988 ).



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Fig. 3. (a) Schematic representation of the RHDV polyprotein. Boxes represent the identified proteins. The biological functions of pro1, pro2 and pro3 are not yet identified. Positions of the dipeptide bonds are indicated. (b) The grey-shaded boxes indicate the polypeptides generated by the hypothetical processing of the polyprotein. The enclosed box represents the assumed processing of p29 which has not been demonstrated (König et al., 1998 ).

 
The mechanism leading to the cleavage between p29 and p14 products remains unknown (Fig. 3). No EG dipeptide bond in this part of the RHDV polyprotein might explain this cleavage. Nevertheless, although RHDV protease seems to tolerate only a limited number of amino acids at the P1 position, the QG dipeptide bond could be a possible target for this protease as reported for picornaviruses (Cohen et al., 1996 ). Indeed, the previous study of the substrate specificity of the RHDV protease showed that even if the optimal substrate at the P1 position is a glutamic acid, cleavage is also possible with glutamine and aspartic acid at this position (Wirblich et al., 1995 ). Moreover, there are two QG dipeptide bonds within the putative sequence of the junction of p29 and p14 products.

Further studies will focus on the identification of cleavage sites differing from the EG dipeptide, on the need for viral co-factors to completely process the RHDV polyprotein and on the relative cleavage efficiencies and specificities of the 3C and 3CD forms.


   Acknowledgments
 
We thank Jean-François Vautherot for the critical reading of the manuscript.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 31 March 1999; accepted 15 October 1999.