Department of Botany, The University of British Columbia, Vancouver, BC, , CanadaV6T 1Z41
Pacific Agri-Food Research Centre, Summerland, BC, , CanadaV0H 1Z02
Author for correspondence: Hélène Sanfaçon. Fax +1 250 494 0755. e-mail sanfaconh{at}em.agr.ca
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Abstract |
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Introduction |
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The ToRSV protease is a serine-like protease, related to the 3C protease of picornaviruses and the 3C-like proteases of comoviruses and potyviruses (Hans & Sanfaçon, 1995 ). ToRSV cleavage sites characterized so far consist of Q/G or Q/S dipeptides (Hans & Sanfaçon, 1995
; Wang et al., 1999
; Carrier et al., 1999
). Site-directed mutagenesis of two ToRSV cleavage sites has confirmed that efficient processing by the protease requires the presence of a Q at position -1 and a S or a G at position +1 of the cleavage sites (Carrier et al., 1999
).
The N-terminal domain of the ToRSV P1 polyprotein, with a total coding capacity for a protein or a precursor of 67 kDa, has not been characterized. This domain includes a region that is identical to the amino acid sequence of P2 and a region that is unique to P1 (Rott et al., 1991b ). In this study, we present evidence for the presence of a novel cleavage site in the N terminus of the polyprotein that is recognized in vitro by the ToRSV protease. This would result in the release of two mature proteins from the N-terminal region of the ToRSV P1 (i.e. upstream of NTB). Sequence analysis revealed that the presence of two distinct protein domains in the N terminus of the P1 polyprotein may be a common feature of nepoviruses. In contrast, only one mature protein is released from the N-terminal region of the P1 polyprotein from comoviruses (Goldbach & Wellink, 1996
).
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Methods |
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In vitro transcription and translation, immunoprecipitations of translation products and trans-processing assays using purified recombinant active protease.
In vitro transcription and translation of cDNA clones were carried out using a TNT coupled transcription/translation system (Promega) in the presence of [35S]methionine at 30 °C for 2 h as described (Wang et al., 1999 ). To allow optimal proteolytic processing and to arrest the transcription/translation reaction, the samples were diluted 1:3 in processing buffer (100 mM TrisHCl, pH 8·0; 10 µg/ml RNase A; 1 mM DTT; 10%, v/v, glycerol) and incubated at 16 °C overnight. Translation products were separated by SDSPAGE (Laemmli, 1970
) and visualized by autoradiography. Immunoprecipitations of in vitro translation products were conducted as previously described (Hans & Sanfaçon, 1995
).
Recombinant active protease was purified from the expression products of plasmid pET15bPro-N-Pol as described (Wang et al., 1999 ). Upon expression, the protease was insoluble. Solubilization of the protease from the purified inclusion bodies with urea and renaturation of the protease by gradual dialysis was as described (Wang et al., 1999
). Aliquots of purified protease were stored in 50 mM TrisHCl, pH 8·0, 1 mM DTT and 10% (v/v) glycerol at -70 °C. Trans-processing assays were conducted by adding the purified protease diluted in the processing buffer (described above) to the translation products and incubating the samples at 16 °C overnight.
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Results |
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Processing at the cleavage site immediately upstream of the NTB domain in vitro
To characterize the N-terminal region of P1, in vitro translations were conducted using polyprotein precursors (X1-Pro and X2-Pro) that included various regions of P1. The coding region for these precursors was inserted into vector pCITE-4a(+), as described in Methods, and includes the T7 promoter and translational enhancing sequences to optimize the efficiency of in vitro translation. The X2-Pro precursor, encoded by plasmid pT7-X2-Pro, contained the domains for NTB, VPg and Pro and a region N-terminal of NTB with a coding capacity of 21 kDa, corresponding to the putative X2 protein (Fig. 2a). This precursor was predicted to contain one potential cleavage site located immediately upstream of the NTB domain (X2-NTB cleavage site) in addition to two previously identified cleavage sites (NTB-VPg and VPg-Pro; Wang et al., 1999
).
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As mentioned above, dipeptide Q620/G located immediately upstream of NTB (Rott et al., 1995 ) satisfied the criteria for ToRSV cleavage sites. Site-directed mutagenesis of this potential cleavage site was conducted. In plasmid pT7-X2-Pro
X2N, the codon for amino acid Q620 was precisely deleted. Maturation of this mutated precursor resulted in the production of the 88 kDa (X2-NTB) and 33 kDa (VPg-Pro) intermediates, whereas the 22 kDa (X2) and 66 kDa (NTB) proteins did not accumulate (Fig. 2c
, lanes 3 and 4). Based on these results, we tentatively propose dipeptide sequence Q620/G as the X2-NTB cleavage site. To confirm the potential nature of the cleavage site, we attempted direct N-terminal sequencing of the 66 kDa protein (NTB). However, the results were not conclusive as a result of cross-contamination of the NTB protein with another labelled protein that ran in close proximity in SDS gels (data not shown).
Processing at a novel cleavage site in the N-terminal domain of the P1 polyprotein
To test if additional cleavage site(s) upstream of the NTB domain could be detected in vitro, plasmid pT7-X1-Pro was constructed using the pCITE-4a(+) vector as previously described. The X1-Pro precursor contained the coding region for NTB, VPg and Pro and for a region upstream of NTB with a coding capacity of 45 kDa (Fig. 3a). Coupled transcription/translation reactions were conducted in vitro as described above and the translation products were separated by 11% or 7% SDSPAGE to allow visualization of small proteins and large precursors. As expected, a polyprotein with an apparent molecular mass of 146 kDa was observed following in vitro transcription/translation of this clone. After incubation of this polyprotein for 20 h at 16 °C, smaller proteins with apparent molecular masses of 26 kDa, 33 kDa, 42 kDa, 66 kDa and 105 kDa accumulated (Fig. 3b
and c
, lane 2). These proteins were not released following incubation of a polyprotein containing an inactive protease (plasmid pT7X1-ProH1283D; Fig. 3b
and 3c
, lane 1). To determine the nature of these proteins, two sets of experiments were conducted. First, three derivatives of the X1-Pro precursor were created: precursor X1-Pro
NV (containing a precise deletion of residue Q1212 at position -1 of the NTB-VPg cleavage site), precursor X1-Pro
X2N (containing a deletion of residue Q620 at position -1 of the putative X2-NTB cleavage site) and precursor X1-Pro
X2N
NV (containing deletions of residues Q1212 and Q620). Second, immunoprecipitation of the processing products was conducted using the anti-NTB, anti-VPg, anti-Pro and anti-X1 antibodies. The results of the mutagenesis experiments are shown in Fig. 3(b
and c
) and the results of the immunoprecipitation experiments are shown in Fig. 4
. These results are summarized in Table 2
. Using these approaches, the following processing products were identified: VPg-Pro (33 kDa protein, calculated molecular mass 32 kDa), X1-X2 (42 kDa protein, calculated molecular mass 45·5 kDa), NTB (66 kDa protein) and X1-X2-NTB (105 kDa protein, calculated molecular mass of 111 kDa).
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Taken together these results indicate that proteolytic processing occurred at the X2-NTB and NTB-VPg cleavage sites and at a new cleavage site in the N-terminal region of the polyprotein. Cleavage at the VPg-Pro site was not detected in the X1-Pro precursor. As mentioned above, examination of the deduced amino acid sequence of the RNA-1-encoded polyprotein at the putative location for the novel X1-X2 cleavage site revealed the presence of only one potential cleavage site that satisfied the criteria for ToRSV cleavage sites (dipeptide Q423/G). The calculated molecular mass of the putative X2-NTB-VPg-Pro precursor produced by the cleavage of this dipeptide is 119 kDa; this is slightly larger than the apparent molecular mass of the 110 kDa protein observed in the cleavage products of the X1-ProX2N
NV precursor. To resolve this discrepancy the cleavage products of the X1-Pro
X2N
NV precursor were separated by 7% SDSPAGE along with the X2-Pro precursor described in the previous section. The X2-Pro precursor includes all the viral sequence for the X2, NTB, VPg and Pro coding regions starting at the codon for amino acid G424. It has a calculated molecular mass of 122 kDa (the additional 3 kDa is due to an in-frame fusion of the viral sequence with sequences derived from the vector, Fig. 2
). The 110 kDa protein present in the cleavage products of the X1-Pro
X2N
NV precursor migrated slightly faster than the X2-Pro precursor (apparent molecular mass of 112 kDa, Fig. 3c
, lanes 6 and 8). The presence of X2, therefore, causes all proteins to run anomalously faster on SDSPAGE, as observed for X1-X2-NTB, X1-X2 and X2-NTB-VPg-Pro cleaved products and the X2-Pro precursor. These results support the hypothesis that the 110 kDa protein is the X2-NTB-VPg-Pro intermediate produced by cleavage at the putative Q423/G cleavage site.
Site-directed mutagenesis of the possible Q423/G cleavage site between X1 and X2 was conducted. In construct pT7-X1-ProX1X2, amino acid Q423 was precisely deleted. Following proteolytic processing of this precursor, the 26 kDa (X1) protein was not produced (Fig. 3b
, lane 3). The 26 kDa and 110 kDa (X2-NTB-VPg-Pro) proteins were also not produced in a triple mutant containing mutations of the X1-X2, X2-NTB and NTB-VPg cleavage sites (compare construct pT7-X1-Pro
X1X2
X2N
NV with construct pT7-X1-Pro
X2N
NV; Fig. 3b
and c
, lanes 6 and 7). Based on these results, we tentatively propose dipeptide Q423/G as the X1-X2 cleavage site. Confirmation of this cleavage site by direct sequencing of the cleaved products was not possible as the 110 kDa (X2-NTB-VPg-Pro) protein was produced in low amounts. Also, the putative mature X2 protein (predicted molecular mass of 21 kDa) was not detected in our assays despite attempts to visualize this protein by 15% SDSPAGE (data not shown). Our inability to detect the mature X2 protein may have been due to the intrinsic instability of the protein and/or to the small number of methionine residues present in the protein. The X2 protein released from this precursor contains fewer methionine residues than the X2 protein released from the X2-Pro precursor, in which the N terminus of X2 was fused to vector sequences containing four methionine residues.
Intramolecular processing of P1 cleavage sites in vitro
To investigate if P1 cleavage sites could be processed in trans by the protease in vitro, precursors containing various regions of P1 were incubated in the presence of purified recombinant ToRSV protease (Fig. 5). Plasmid pET-MPCAT was used as a positive control for protease activity. This plasmid allowed the expression of a precursor containing the cleavage site between the RNA-2-encoded movement protein (MP) and coat protein (CP) that is cleaved in trans by the protease (Hans & Sanfaçon, 1995
).The MP-CAT precursor with an apparent molecular mass of 65 kDa (calculated molecular mass of 59 kDa) was processed into two proteins with apparent molecular masses of 36 kDa and 27 kDa. These corresponded approximately to the predicted sizes for the C-terminal half of MP (32 kDa) and a fusion protein (CP-CAT) containing the N-terminal region of CP and CAT (27 kDa). The nature of these proteins was confirmed by immunoprecipitation experiments using antibodies against MP and CAT (Hans & Sanfaçon, 1995
; data not shown). Cleavage by exogenously added protease was not detected in two large P1-derived polyprotein precursors containing an inactive protease. Precursor X1-ProH1283D included all the potential cleavage sites upstream of Pro and precursor NTB-PolH1283D contained the domains for NTB, VPg, Pro and Pol and therefore all the potential cleavage sites downstream of NTB. Trans-cleavage was also not detected upon addition of the exogenous protease to three smaller precursors that did not include the protease domain: a precursor containing the C-terminal half of NTB followed by VPg (plasmid pT7-C-NTB-VPg; Fig. 5
), a precursor containing the entire NTB and VPg domains (data not shown) and a precursor containing the C-terminal region of X1 followed by X2. These results suggest that processing at P1 polyprotein cleavage sites is predominantly an intramolecular event.
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Discussion |
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The results presented in this study and earlier (Carrier et al., 1999 ) suggest that P1 processing occurs predominantly in cis. Indeed, precursors in which the protease domain was mutated or absent were not cleaved in vitro by purified recombinant ToRSV protease supplied exogenously. The accumulation of several intermediates in vitro (X1-X2-NTB, X1-X2) supports the notion that they are not further cleaved by the protease. Interestingly, although trans-cleavage was not detected in the P1 polyprotein of tomato black ring virus (TBRV, a nepovirus of subgroup B; Hemmer et al., 1995
), trans-cleavage was observed at the X-NTB cleavage site of grapevine fanleaf virus (GFLV, a nepovirus of subgroup A; Margis et al., 1994
). Our results do not exclude the possibility that some trans-cleavage of the ToRSV P1 cleavage sites may occur on the full-length P1 polyprotein.
Based on the results of site-directed mutagenesis, dipeptides Q423/G and Q620/G are proposed to be the putative X1-X2 and X2-NTB cleavage sites, respectively. In a previous study (Carrier et al., 1999 ), we showed that the identity of the amino acid at position -2 of the cleavage site played a role in the efficiency of cleavage at two ToRSV cleavage sites. While previously characterized ToRSV cleavage sites contain a C or a V at position -2, introduction of an A at position -2 of the Pro-Pol and X-MP cleavage sites resulted in efficient cleavage at these sites. In contrast, introduction of other amino acids (R, G, F) resulted in inefficient cleavage. The presence of an A at position -2 of the putative X1-X2 cleavage site is consistent with these results. The presence of a T at position -2 of the presumed X2-NTB cleavage site suggests that this is also an acceptable amino acid at this position. Efficiency of processing at cleavage sites including a T at position -2 was not directly tested in our previous mutagenesis study (Carrier et al., 1999
).
The results presented here suggest that in ToRSV, the region upstream from NTB contains two distinct protein domains delineated by the putative X1-X2 and X2-NTB cleavage sites. The predicted molecular mass of these proteins is 46 kDa for the X1 protein (assuming that translation is initiated at the first AUG codon) and 21 kDa for the X2 protein. Comoviruses and nepoviruses have very similar genomic organizations in that the NTB, VPg, Pro and Pol domains are located in the C-terminal region of the P1 polyprotein. However, the size of the region N-terminal of NTB varies with different viruses (Fig. 6). In cowpea mosaic virus (CPMV, a comovirus), this region contains a single protein domain with a molecular mass of 32 kDa (Co-Pro protein; Goldbach & Wellink, 1996
). In nepoviruses, this region has a coding capacity for a protein (or a precursor) with a predicted molecular mass of 67 kDa for ToRSV, 71 kDa for peach rosette mosaic virus (PRMV, a nepovirus of subgroup C; Lammers et al., 1999
), 60 kDa for grapevine chrome mosaic virus and TBRV (two nepoviruses of subgroup B; Greif et al., 1988
; Le Gall et al., 1989
) and 45 kDa for GFLV (a nepovirus of subgroup A; Ritzenthaler et al., 1991
). Indirect evidence based on comparisons of the amino acid sequence of the N-terminal region of the P1 polyprotein of nepoviruses and comoviruses suggests that the presence of two distinct protein domains may be a common feature of nepoviruses. Indeed, two regions of sequence similarity were identified (Fig. 6
). The first region of similarity is an alanine-rich sequence which has been identified in the X1 domain of the ToRSV P1 polyprotein and in the equivalent regions of the P1 polyproteins of all other nepoviruses for which sequence information is available (Rott et al., 1995
; Mayo & Robinson, 1996
; Lammers et al., 1999
). This sequence is not present in the comovirus genome. The second region of similarity is present in the X2 domain of the ToRSV P1 polyprotein, in the Co-Pro domain of the comovirus polyproteins and in the corresponding regions of all other nepovirus polyproteins (Ritzenthaler et al., 1991
; Rott et al., 1995
; Mayo & Robinson, 1996
; Lammers et al., 1999
). Taken together these results raise the possibility that P1 polyproteins from other nepoviruses may also contain an additional cleavage site at their N terminus. PRMV, a nepovirus of subgroup C, is closely related to ToRSV. The cleavage site specificity of the PRMV protease was suggested to be similar to that of the ToRSV protease (Lammers et al., 1999
). Using the criteria established for the specificity of the ToRSV protease, the deduced amino acid sequence of the PRMV RNA-1- encoded polyprotein was examined. A single potential Q/S cleavage site was identified in the N-terminal region of P1 that would allow the release of two proteins: one equivalent to the ToRSV X1, with a predicted molecular mass of 44·5 kDa, and one equivalent to the ToRSV X2, with a predicted molecular mass of 26 kDa (Fig. 6
). Prediction of cleavage sites in nepoviruses of subgroups A and B is more difficult since the cleavage sites recognized by these proteases differ considerably from those of nepoviruses of subgroup C (Sanfaçon, 1995
; Mayo & Robinson, 1996
and references therein) and since a consensus sequence for those cleavage sites has not been established.
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Acknowledgments |
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Footnotes |
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References |
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Received 2 June 2000;
accepted 21 July 2000.