United States Department of Agriculture Agricultural Research Service and Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583, USA1
Author for correspondence: Roy French. Fax +1 402 472 4020. e-mail rfrench{at}unlnotes.unl.edu
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Abstract |
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Introduction |
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TEV encodes a serine proteinase (P1) that acts in cis to cleave itself from the amino-terminal domain of the polyprotein (Verchot et al., 1991 , 1992
). TEV P1 proteinase cleavage occurs between polyprotein residues Y304 and S305 (Verchot et al., 1991
). Because the WSMV polyprotein contains YS at positions 283 and 284, Stenger et al. (1998)
speculated that WSMV P1 proteinase cleavage may occur between these two residues. Insertion of 12 non-viral nucleotides immediately downstream of this putative cleavage site into an infectious clone of WSMV had no effect on viability but insertion of a bacterial glucuronidase (GUS) gene at the same site abolished infectivity (Choi et al., 2000b
). One explanation for the aforesaid observations is that WSMV P1 proteinase cleavage occurs at a site other than Y283S284, making it of interest to define the cleavage site experimentally. In this report, we describe coupled in vitro transcriptiontranslation experiments designed to map the WSMV polyprotein domain involved in P1 proteolysis. We further demonstrate that the WSMV P1 proteinase cleavage site is processed in vivo such that insertion of GUS immediately downstream of the P1 proteinase cleavage site in an infectious clone of WSMV results in the accumulation of excised GUS protein in tissues of infected plants.
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Methods |
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A DNA fragment containing the NPT II gene was synthesized by PCR with P.f.u. polymerase using pWSMV-JNPT (Choi et al., 2000b ) as template and primers X-NPT5 and NPT-3 (Table 1
). The NPT II PCR product was digested with XhoI and SalI and then inserted into the XhoI site of pACT2 to create pACT-XNPT. A nested set of DNA fragments (X21, X22, X23, X24 and X25) from the WSMV P1 cistron was generated by PCR using pACYC-WSMV (Choi et al., 1999
) as template with a forward primer (P1-5) and one of the following reverse primers: P1-X21 (for X21), P1-X22 (for X22), P1-X23 (for X23), P1-X24 (for X24) or P1-X25 (for X25); sequences of these and other primers used in this study are given in Table 1
. The DNA fragments X21 to X25 were digested with XhoI and inserted into the XhoI site located between the GAL4 activation domain and NPT II in pACT-XNPT. The resultant plasmids, pCP1-NPT21, -NPT22, -NPT23, -NPT24 and -NPT25, have WSMV P1 sequences fused to NPT II.
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Insertion of GUS at the P1/HC-Pro junction in an infectious clone of WSMV.
A unique SalI site was engineered in the infectious clone of WSMV (pACYC-WSMV) (Choi et al., 1999 ) immediately downstream of the codons for the P1 proteinase cleavage site identified in this study (Fig. 2
). Three nucleotides (GTC) were inserted between nt positions 1189 and 1190 of pACYC-WSMV by site-directed mutagenesis to produce pWSMV-S1RN. The GUS gene linked to the nucleotide sequence encoding a WSMV NIa proteinase cleavage site was isolated from pWSMV-GUSJ1 (Choi et al., 2000b
) and inserted into the SalI site of pWSMV-S1RN to generate pWSMV-GUS-S1RN. In vitro transcripts of pWSMV-S1RN and pWSMV-GUS-S1RN were transcribed with SP6 RNA polymerase and inoculated to wheat (Triticum aestivum L.) seedlings as described (Choi et al., 1999
). Inoculated plants were evaluated for GUS activity by histochemical staining in situ (Choi et al., 2000b
). Native mobility of GUS produced by pWSMV-GUS-S1RN was assessed following electrophoresis of infected plant protein extracts under non-denaturing conditions by activity gel (zymograph) detection using ELF-97
-D-glucuronide as described (Steinberg et al., 2000
). Purified wild-type GUS protein (Sigma) was used as a positive control in the electrophoretic assay, whereas protein extracted from mock-inoculated or WSMV-S1RN infected plants served as negative controls.
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Results and Discussion |
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A second series of templates (B1B6) based on pCSP1-8 bore nested 5'-deletions in the P1 domain (Fig. 1B). For each template, the upstream primer used to amplify the PCR products included an initiation codon to replace the native WSMV polyprotein initiation codon that was removed as part of the 5'-deletion. In this experiment, the uncleaved full-length translation product became progressively smaller in size, corresponding to the size of the deletion. For templates B1B3, two prominent cleaved polypeptide products were observed. The larger cleaved product remained constant in size, and was interpreted as being the carboxy-terminal HC-Pro polypeptide. The smaller cleavage product varied in size according to the extent of the 5'-deletion and was interpreted as the cleaved P1 polypeptide. The translation product of template B4 had impaired proteolytic activity, with only minor amounts of the large cleavage product detected. No evidence of proteolysis was detected for the translation products of templates B5 and B6. These results indicated that the proteolytic domain and cleavage site of P1 reside downstream of nt 755 (amino acid residue 208). Thus, as is the case for TEV, the proteolytic domain is located in the carboxy-terminal region of P1.
To fine map the P1 proteinase cleavage site, a third series of in vitro transcriptiontranslation templates (C2C6) bearing WSMV sequences from nt 638 to 11621210 was constructed in which downstream HC-Pro sequences were replaced with NPT II sequences (Fig. 1C). Translation of template C1 (retaining WSMV sequences from nt 638 through the 3'-proximal end of HC-Pro) produced two cleavage products, the larger of which corresponded to full-length HC-Pro and a smaller product represented the carboxy-terminal portion of P1. Apparently, templates C2 and C3 did not encode the intact proteinase cleavage site and produced only uncleaved translation products. In contrast, templates C4C6 encoded the functional proteinase cleavage site, thereby producing cleaved polypeptides corresponding to the carboxy-terminal portion of P1. Unexpectedly, the second predicted polypeptide cleavage product corresponding to NPT II sequences was not detected. Furthermore, sizes of the uncleaved translation products were less than expected for the P1NPT II fusions. Sequence determined for the NPTII insert of pACT-XNPT revealed an unintended frame-shift mutation in the NPT II sequence, such that the P1NPT II translation products would be prematurely terminated to yield a full-length uncleaved polypeptide of
29 kDa. The small size (9 kDa) of the predicted carboxy-terminal polypeptide released by P1 proteolysis explains why the second proteolytic cleavage product was not detected by autoradiography. Nonetheless, these results indicate that HC-Pro sequences are not required for P1 proteolysis and that the P1 cleavage site of WSMV occurs downstream of residue G348 and upstream of residue G353.
Hallmark serine proteinase motifs are present in WSMV P1
Collectively, the in vitro transcriptiontranslation experiments indicated that the WSMV P1 proteinase cleavage site resides considerably downstream from the YS283284 dipeptide. CLUSTAL X failed to align the known P1 proteinase cleavage site of TEV with the region of the WSMV polyprotein implicated by the in vitro transcriptiontranslation experiments. This is not surprising considering that the P1 domain is the least conserved and most variable in length among monopartite viruses of the family Potyviridae (Ward et al., 1995 ) However, when the WSMV Y352 residue was manually aligned with the Y304 residue of TEV, upstream amino acid residues required for TEV proteolysis came into alignment with similar residues present in WSMV (Fig. 3
). Specifically, the WSMV polyprotein contained H257, D267 and S303 that were conserved at similar positions in the modified alignment, relative to all other taxa examined. These three residues are required for proteinase activity of TEV P1 (Verchot et al., 1991
, 1992
) and likely constitute the catalytic H-D-S triad associated with serine proteinases (Bazan & Fletterick, 1990
). The serine residue was flanked by glycine residues (GXSG), another hallmark of the serine proteinase H-D-S catalytic triad (Ryan & Flint, 1997
). However, the spacing between the H, D and S residues is somewhat more compact for potyviruses and tritimoviruses than that of the serine proteinase encoded by ORF 1 of the polerovirus Potato leafroll virus (Sadowy et al., 2001
) or other serine proteinases of the picornavirus super-group (Ryan & Flint, 1997
). Additionally, the WSMV motif FIVMG325329 aligned with a similar motif in TEV, TVMV and PPV (FIVRG), and BrSMV (FVVQG), that is also required for TEV P1 proteolysis (Verchot et al., 1991
). Other than these motifs, there was little conservation of intervening sequences among different species of the family Potyviridae (Fig. 3
).
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The WSMV P1 domain includes all known essential motifs of a serine proteinase with the probable site of peptide bond hydrolysis between Y352 and G353, rather than between Y and S residues as in TEV. Thus, mature WSMV P1 protein would be 48 amino acid residues longer than P1 of TEV. The P1 proteinase cleavage site predicted for a second tritimovirus, BrSMV (Götz & Maiss, 1995 ; Fig. 3
), would yield a longer P1 polypeptide (403 amino acids) with hydrolysis between Y and S residues. However, differences between species of the same genus may not be unusual, as the potyvirus TVMV has S275 as the amino-terminal residue of HC-Pro, indicating that P1 cleavage occurs between F and S residues (Mavankal & Rhoads, 1991
). Thus, although monopartite species of the family Potyviridae retain serine proteinase activity to cleave the N-terminal region of the polyprotein, the length of the mature P1 polypeptide varies, as does the residues between which the peptide bond is hydrolysed.
WSMV P1 proteinase functions in vivo
Dolja et al. (1992) inserted GUS between the P1 and HC-Pro domains of TEV without destroying infectivity so long as the polyprotein was proteolytically processed to remove P1. Although insertion of a 12 base sequence containing a SalI site between WSMV nt 991 and 992 did not affect infectivity, subsequent insertion of GUS at the engineered SalI site in WSMV abolished infectivity (Choi et al., 2000b
). In light of the present study, these results are not surprising, as elements of the catalytic domain were separated by over 600 amino acid residues due to insertion of GUS. However, since the initial 12 base insertion retained infectivity, evidently some variation in the spacing of critical motifs required for P1 proteinase is allowed.
To provide additional evidence that the P1 proteinase cleavage site of WSMV occurs between amino acid residues Y352 and G353, the GUS reporter gene was repositioned adjacent to and downstream of this dipeptide in the infectious clone of WSMV. Excision of GUS from the viral polyprotein would occur by P1 proteolysis at the amino end and by NIa-catalysed cleavage at the carboxy end at an engineered NIa proteinase cleavage site (Fig. 2). In vitro transcripts derived from the GUS-containing construct pWSMV-GUS-S1RN produced systemic infection of wheat and GUS activity was demonstrated in systemically infected wheat leaves by a histochemical assay (data not shown). Active GUS protein also was detected in a total protein sample extracted from WSMV-GUS-S1RN-infected plants and subjected to PAGE under non-denaturing conditions (Fig. 4
). A substantial fraction of active GUS protein extracted from pWSMV-GUS-S1RN-infected plants exhibited electrophoretic mobility similar to that observed for purified wild-type GUS protein obtained from a commercial source. This result confirms that GUS protein was excised from the polyprotein and provides additional in vivo evidence that the WSMV P1 proteinase cleavage site was correctly identified. The presence of some active GUS protein in a second, slower migrating band unique to pWSMV-GUS-S1RN indicated that proteolysis was incomplete. Incomplete proteolysis may have resulted from the co-translational requirement for P1 autoproteolysis (Verchot et al., 1992
), and/or alteration of local topology of the cleavage site in which native HC-Pro sequences were replaced with GUS sequences. Nonetheless, identification of the P1 proteinase cleavage site permitted insertion of a foreign gene sequence at a second genomic location in WSMV without compromising infectivity and may expand the utility of WSMV as a gene expression vector in monocotyledonous hosts.
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Acknowledgments |
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References |
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Received 23 August 2001;
accepted 26 October 2001.