Laboratoire de Virologie et Barrière dEspèce, Unité de Pathologie Aviaire et de Parasitologie, INRA de Tours, 37380 Nouzilly, France1
Unité de Virologie et dImmunologie 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
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Abstract |
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Introduction |
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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 (718719), EG (11081109) and EG (17671768) (Boniotti et al., 1994 ; Wirblich et al., 1995
; Alonso et al., 1996
). An additional ET (12511252) 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 (143144) dipeptide bond.
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Methods |
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The pBPL10 construct was the starting point for this work. This plasmid contains, under the T7 promoter, the 3C-like proteaseluciferase 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 11081434 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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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 SDS12% 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 Trisphosphate, 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).
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 Eagles 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 manufacturers 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).
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Results and Discussion |
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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 SDS12% 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 SDSPAGE 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 SDSPAGE 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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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 (12511252) 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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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.
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Acknowledgments |
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References |
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Received 31 March 1999;
accepted 15 October 1999.