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Short Communication |
‘The Calicilab’, Institut für Virologie, Medizinische Fakultät Carl Gustav Carus, Fiedlerstrasse 42, D-01307 Dresden, Germany
Correspondence
Jacques Rohayem
Jacques.Rohayem{at}tu-dresden.de
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ABSTRACT |
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MAIN TEXT |
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In noroviruses, the molecular mechanisms of peptide cleavage by the viral protease are poorly understood. It is also unclear whether active norovirus 3Cpro can be generated in vitro and purified after autocatalytic release from a larger precursor, i.e. 3CDpropol. Furthermore, the sequential release of the non-structural proteins from the norovirus polyprotein precursor has not been characterized.
In this study, it was postulated that (i) both 3Cpro and 3CDpropol are active forms of the viral protease, but display differential cleavage patterns of the norovirus polyprotein, and (ii) sequential processing of the cleavage sites in the polyprotein occurs. To address these hypotheses, the trans-cleavage activity of the purified, autocatalytically cleaved 3Cpro and 3CDpropol proteins was examined in a cell-free assay by using peptides bearing the scissile bonds from the norovirus polyprotein.
Expression and purification of autocatalytically released norovirus 3Cpro was performed as described previously (Rohayem et al., 2006a
, b) with slight modifications. Briefly, a cDNA fragment (729 bp) encompassing the norovirus 3C-like protease gene and bearing the VPg/3Cpro and 3Cpro/3Dpol cleavage sites at its 5' and 3' ends, respectively, was generated by PCR from norovirus clone pUSNorII (GenBank accession no. AY741811
[GenBank]
; Fig. 1). The cDNA was cloned into the pET-28b(+) vector (Novagen), resulting in expression vector pUSNorII-
VPg3Cpro3Dpol, which was used to transform Escherichia coli BL21(DE3)pLysS cells. Upon expression and after autocatalytic cleavage of 3Cpro from its precursor VPg3Cpro3Dpol, the protein was purified by precipitation with 75 % ammonium sulfate followed by cation-exchange chromatography. Fractions containing proteins were collected and used for the peptide-based assay. Protein concentration was determined with a BCA Protein Assay kit (Pierce) based on the Biuret reaction. Expression and purification of autocatalytically released norovirus 3CDpropol were performed as described above with slight modifications. Briefly, a cDNA fragment (2250 bp) encompassing the norovirus 3C-like and norovirus 3D-like genes and bearing the VPg/3Cpro cleavage site at its 5' end was generated by PCR from norovirus clone pUSNorII (GenBank accession no. AY741811
[GenBank]
; Fig. 1). The cDNA was cloned into the pET-28b(+) vector (Novagen). To express the complete 3CDpropol protein, the cleavage site between 3Cpro and 3Dpol was modified by using a QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions, resulting in the precursor protein VPg3Cpro(E1189A)3Dpol, bearing a mutation of Glu1189 to Ala1189 in the cleavage site between 3Cpro and 3Dpol. Upon expression and after autocatalytic cleavage of 3CDpropol from its precursor VPg3Cpro(E1189A)3Dpol, the protein was purified and its concentration was determined as described above.
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). Peptides were synthesized by Invitrogen and purified by HPLC on a reversed-phase column (RP-HPLC). Lyophilized peptides were dissolved in Tris buffer (40 mM Tris, 150 mM NaCl, pH 7.2) at a final concentration of 1 mg ml–1. Proteolytic cleavage was examined in a reaction mix containing 30 µM peptide and 4 µM 3Cpro in a final volume of 100 µl. Digestion experiments were performed at 30 °C. The reaction was stopped after 17 h by addition of 0.5 % trifluoroacetic acid (TFA). Proteolytic cleavage was monitored by RP-HPLC analysis using the following conditions: YMC-Pack ODS-AQ column (YMC Europe), 4.6 mmx12 cm, 15 % B/85 % A to 65 % B (with 0.1 % TFA as solvent A and 65 % acetonitrile/35 % water/0.1 % TFA as solvent B), linear gradient in 90 min, 1 ml min–1 flow rate and 23 °C. These experimental conditions are further referred to as ‘standard conditions’. A215 was determined and peak areas were calculated by integration. The cleavage efficiencies of the peptides by the viral protease were reported as F (%), the fraction of substrate that is converted to product based on the integrated peak area (O'Leary & Baughn, 1972; Pallai et al., 1989). Comparative analysis of cleavage efficiencies was performed by using a non-parametric Mann–Whitney U-test using GraphPad InStat version 3.0a for Macintosh (GraphPad Software). Statistical significance was set at P<0.05. Overnight incubation of the peptides with 3Cpro followed by analysis of the reaction by RP-HPLC provided evidence of proteolytic cleavage of peptides p37/2C and 2C/p20 by 3Cpro (Fig. 2a). These results are in accordance with previous reports on the activity of norovirus 3Cpro in cell-free (Belliot et al., 2003; Liu et al., 1999; Seah et al., 2003) and cell-based (Chang et al., 2006; Seah et al., 1999, 2003; Sosnovtsev et al., 2006) systems. However, no cleavage products were observed when peptides VPg/3C, 3C/3D or p20/VPg were incubated with 3Cpro, suggesting resistance of these peptides to proteolytic processing by 3Cpro in trans. Interestingly, peptide VPg/3C was cleaved by 3CDpropol in trans (Fig. 2a). This processing of the VPg/3C scissile bond was not observed previously in a cell-free system using the polyprotein precursor of Norwalk virus (Blakeney et al., 2003; Hardy et al., 2002), suggesting a possible difference in processing of the polyprotein precursor scissile bonds that depends on the experimental system used. In our study, and for the first time to our knowledge, a highly purified, autocatalytically cleaved norovirus protease, its precursor and highly purified synthetic peptides reflecting the authentic cleavage sites of the norovirus polyprotein precursor were used. Our observations suggest differential cleavage patterns of the scissile bonds in the norovirus polyprotein by 3Cpro and 3CDpropol.
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-cyano-4-hydroxy-trans-cinnamic acid as the matrix, as described by Thomas et al. (2004). Spectra were calibrated externally by using masses of abundant autolysis products of trypsin. NanoESI (electrospray ionization)-MS and MS/MS analyses were performed on a hybrid quadrupole time-of-flight mass spectrometer QSTAR Pulsar i (MDS Sciex) equipped with an automated chip-based nanoflow ion source (Advion). Spectra were processed with BioAnalyst QS v.1 software (MDS Sciex). First, the fractions containing the products of proteolysis of the p37/2C peptide were examined. For the first product of proteolysis, MALDI MS could not determine the exact mass of the cleaved product, displaying a spectrum at about 700 Da. Therefore, ESI-MS and MS/MS (fragmentation) analysis was applied, and the expected sequence GPEDLAV of the singly charged peptide was confirmed by fragmentation of this precursor. The second product of proteolysis displayed a mass corresponding to the expected peptide fragment LLGDYELQ (calculated M+H, 950.341 Da). Next, the fractions containing the products of proteolysis of the 2C/p20 peptide were examined. The first product displayed a mass corresponding to expected peptide fragment GPALTTF (calculated M+H, 706.270 Da). The second product peak had a mass corresponding to the expected fragment RLDEFELQ (calculated M+H, 1049.561 Da). Finally, the fractions containing the products of limited proteolysis of the VPg/3C peptide were examined. The first product displayed a mass corresponding to the expected peptide fragment YNEKLSFE (calculated M+H, 1029.489 Da). The second product had a peak with mass corresponding to the expected fragment APPSIWS (calculated M+H, 757.388 Da). These observations confirm the specificity of norovirus 3Cpro and 3CDpropol for the scissile bond Gln–Gly, as well as for Glu–Ala in the case of 3CDpropol. To examine whether the P2 and P3 positions in the peptide sequence modulate cleavage of the p20/VPg and 3C/3D peptides, two additional peptides bearing mutations at positions P2 [p20/VPg(TP2L)] and P3 [3C/3D(TP3E)] were designed. Neither peptide was cleaved by 3CDpropol or 3Cpro, suggesting that the specificity of 3Cpro activity is modulated by the sequence of the scissile bond.
To characterize the differential cleavage patterns of 3Cpro and 3CDpropol, cleavage efficiencies of the scissile bonds were compared. Therefore, the activity of the norovirus protease 3Cpro in comparison to its putative precursor 3CDpropol was measured in the peptidolytic assay run under standard conditions. As shown in Fig. 2(a)
, norovirus 3Cpro cleaved peptides p37/2C and 2C/p20 more efficiently than did 3CDpropol (P=0.0143 and P<0.001, respectively, by non-parametric Mann–Whitney U-test). However, peptide VPg/3C was cleaved only by 3CDpropol. These data suggest differential cleavage patterns of the norovirus polyprotein by 3Cpro and 3CDpropol. It also suggests that the Gln–Gly scissile bond between the P1 and P1' positions of the peptide is an important modulator of cleavage specificity by the 3C-like protease. Indeed, the only peptides that were cleaved by 3Cpro in trans displayed a Gln–Gly scissile bond. The VPg/3C scissile bond, consisting of Glu–Ala, was not cleaved by 3Cpro, but was cleaved by 3CDpropol. Hence, the P1 and P1' positions seem to play important roles in modulating the differential cleavage of the scissile bonds. Interestingly, peptides 3C/3D and p20/VPg, which were not cleaved in trans, displayed a Glu–Gly scissile bond, thus differing in the P1 and P1' positions from the above-mentioned cleavage sites. However, the Glu–Gly scissile bond of the 3C/3D junction was cleaved in cis, indicating that this dyad may play a role in regulating cis versus trans cleavage. Cleavage in cis by the 3C-like protease has already been discussed by Someya et al. (2002), based on the 3B/3C model in poliovirus proposed by Khan et al. (1999). Someya et al. (2002) postulated that, in noroviruses, an intramolecular autocatalytic cleavage in cis at the VPg/3C junction can occur through an extended conformation of the VPg3Cpro domain, allowing the cleavage site to contact the active site of the protease.
Finally, we postulated that resistance of the predicted cleavage sites to proteolysis by norovirus 3Cpro and/or 3CDpropol may sustain a possible cleavage ranking of the polyprotein precursor by 3Cpro and/or 3CDpropol. To examine this hypothesis, competition experiments involving a peptide of interest (PI), a competitor (PC) and the viral protease were performed as described by others (Pallai et al., 1989). Peptidolytic assays were run under standard conditions. The relative efficiency was given by the ratio (Vmax/Kmax)PI/(Vmax/Kmax)PC=log(1–FPI)/log(1–FPC), where FPI and FPC are the fraction of substrate peptide of interest converted to product and the fraction of competitor peptide converted to product, respectively (O'Leary & Baughn, 1972; Pallai et al., 1989). Comparative analysis of the relative cleavage efficiencies was performed by using a non-parametric Mann–Whitney U-test as described above. The VPg/3C peptide was cleaved more efficiently by 3CDpropol than was p37/2C (P=0.0143, Fig. 2b) when co-incubated with 2C/p20, suggesting a preferential cleavage of the VPg/3C scissile bond followed by the p37/2C scissile bond in the norovirus polyprotein. p37/2C co-incubated with 2C/p20 was cleaved more efficiently by 3Cpro than by 3CDpropol (P=0.0143, Fig. 2b), suggesting that the p37/2C scissile bond may be cleaved preferentially by 3Cpro. According to the observed relative cleavage efficiencies of the peptides by 3Cpro or 3CDpropol, a ranking for processing of the norovirus polyprotein is proposed (Fig. 2c). This ranking is in accordance with observations on processing of the polyprotein precursor in cell-free expression systems (Belliot et al., 2003), as well as in murine norovirus cultivated in the RAW264.7 cell line (Sosnovtsev et al., 2006). This ranking may be based on the half-life of the precursor proteins, as observed during the processing of poliovirus polyprotein (Pallai et al., 1989), where a similar mechanism regulating availability of the non-structural proteins of the replication complex was postulated. Therefore, it is conceivable that polyprotein processing by norovirus 3Cpro regulates the formation of the replication complex through a ranking order of stability of the non-structural proteins.
In summary, our results suggest a differential processing of the norovirus polyprotein precursor depending upon the presence of 3Cpro or its precursor 3CDpropol. However, it remains unclear whether a synergetic processing of the non-structural proteins by norovirus 3Cpro and/or 3CDpropol occurs. Furthermore, structural features of the peptide affecting its interaction with the protease cleavage site may contribute to this regulation. Understanding factors that regulate the interaction between the viral protease, its substrates and possible cellular factors may shed light on regulation of polyprotein processing in noroviruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Blakeney, S. J., Cahill, A. & Reilly, P. A. (2003). Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase. Virology 308, 216–224.[CrossRef][Medline]
Chang, K. O., Sosnovtsev, S. V., Belliot, G., King, A. D. & Green, K. Y. (2006). Stable expression of a Norwalk virus RNA replicon in a human hepatoma cell line. Virology 353, 463–473.[CrossRef][Medline]
Hardy, M. E., Crone, T. J., Brower, J. E. & Ettayebi, K. (2002). Substrate specificity of the Norwalk virus 3C-like proteinase. Virus Res 89, 29–39.[CrossRef][Medline]
Khan, A. R., Khazanovich-Bernstein, N., Bergmann, E. M. & James, M. N. (1999). Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors. Proc Natl Acad Sci U S A 96, 10968–10975.
Liu, B. L., Viljoen, G. J., Clarke, I. N. & Lambden, P. R. (1999). Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli. J Gen Virol 80, 291–296.[Abstract]
O'Leary, M. H. & Baughn, R. L. (1972). Acetoacetate decarboxylase. Identification of the rate-determining step in the primary amine catalyzed reaction and in the enzymic reaction. J Am Chem Soc 94, 626–630.[CrossRef][Medline]
Pallai, P. V., Burkhardt, F., Skoog, M., Schreiner, K., Bax, P., Cohen, K. A., Hansen, G., Palladino, D. E., Harris, K. S. & other authors (1989). Cleavage of synthetic peptides by purified poliovirus 3C proteinase. J Biol Chem 264, 9738–9741.
Rohayem, J., Jager, K., Robel, I., Scheffler, U., Temme, A. & Rudolph, W. (2006a). Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis. J Gen Virol 87, 2621–2630.
Rohayem, J., Robel, I., Jager, K., Scheffler, U. & Rudolph, W. (2006b). Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. J Virol 80, 7060–7069.
Seah, E. L., Marshall, J. A. & Wright, P. J. (1999). Open reading frame 1 of the Norwalk-like virus Camberwell: completion of sequence and expression in mammalian cells. J Virol 73, 10531–10535.
Seah, E. L., Marshall, J. A. & Wright, P. J. (2003). Trans activity of the norovirus Camberwell proteinase and cleavage of the N-terminal protein encoded by ORF1. J Virol 77, 7150–7155.
Someya, Y., Takeda, N. & Miyamura, T. (2002). Identification of active-site amino acid residues in the Chiba virus 3C-like protease. J Virol 76, 5949–5958.
Sosnovtsev, S. V., Belliot, G., Chang, K. O., Prikhodko, V. G., Thackray, L. B., Wobus, C. E., Karst, S. M., Virgin, H. W. & Green, K. Y. (2006). Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. J Virol 80, 7816–7831.
Thomas, H., Havlis, J., Peychl, J. & Shevchenko, A. (2004). Dried-droplet probe preparation on AnchorChip targets for navigating the acquisition of matrix-assisted laser desorption/ionization time-of-flight spectra by fluorescence of matrix/analyte crystals. Rapid Commun Mass Spectrom 18, 923–930.[CrossRef][Medline]
Received 15 December 2006;
accepted 10 March 2007.
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