Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis.

The NIa proteinase encoded by tobacco etch potyvirus catalyzes six processing events, three of which occur by an autoproteolytic mechanism. Autoproteolysis is necessary to cleave the boundaries of both NIa and the 6-kDa protein, which is located adjacent to the N terminus of NIa in the viral polyprotein. As a consequence, NIa may exist in a free form or in a transient polyprotein form containing the 6-kDa protein. While the majority of NIa molecules localize to the nuclei of infected cells, a fraction of the NIa pool is attached covalently to the 5' terminus of genomic RNA in the cytoplasm. To determine whether the presence of the 6-kDa protein affects the nuclear transport properties of NIa, we have generated transgenic plants that express genes encoding a reporter enzyme, beta-glucuronidase (GUS), fused to NIa or NIa-containing polyproteins. The NIa/GUS fusion protein was detected by histochemical analysis in the nucleus. Similarly, an NIa/GUS fusion protein that arose by autoproteolysis of a 6-kDa/NIa/GUS polyprotein was found in the nucleus. In contrast, fusion protein consisting of 6-kDa/NIa/GUS, which failed to undergo proteolysis because of the presence of a Cys-to-Ala substitution in the proteolytic domain of NIa, was detected in the cytoplasm. The inhibition of NIa-mediated nuclear transport was not due to the Cys-to-Ala substitution, since this alteration had no effect on translocation in the absence of the 6-kDa protein. These results indicate that the 6-kDa protein impedes nuclear localization of NIa and suggest that subcellular transport of NIa may be regulated by autoproteolysis.     

Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector

We previously described an expression cassette that relies on the tobacco etch virus (TEV) nuclear inclusion a (NIa) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. However, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22].Studies were conducted to evaluate whether the disruption of NIa nuclear localization would affect the levels of proteins produced via the cassette. Modifications comprised either removal of its nuclear localization signals (NLSs), removal of the VPg domain (which includes the NLSs), and fusion to the 6 kDa protein, previously demonstrated to be a viral cytoplasmic anchor [28]. In in vitro translation reactions and in vivo protoplast experiments the modified NIa retained sequence-specific proteolysis. Moreover, the removal of the NLSs correlated with an increase in GUS reporter accumulation. The modified cassette, pPRO10, led to the synthesis of up to three viral coat protein (CPs) in addition to NIa. However, the accumulation of proteins in protoplasts depended upon the position of the CP coding sequence within the cassette as well as on the stability of the protein.     

Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo.

The NIa protein of plant potyviruses is a bifunctional protein containing an N-terminal VPg domain and a C-terminal proteinase region. The majority of tobacco etch potyvirus (TEV) NIa molecules are localized to the nucleus of infected cells, although a proportion of NIa is attached covalently as VPg to viral RNA in the cytoplasm. A suboptimal cleavage site that is recognized by the NIa proteinase is located between the two domains. This site was found to be utilized in the VPg-associated, but not the nuclear, pool of NIa. A mutation converting Glu-189 to Leu at the P1 position of the processing site inhibited internal cleavage. Introduction of this mutation into TEV-GUS, an engineered variant of TEV that expresses a reporter protein (beta-glucuronidase [GUS]) fused to the N terminus of the helper component-proteinase (HC-Pro), rendered the virus replication defective in tobacco protoplasts. Site-specific reversion of the mutant internal processing site to the wild-type sequence restored virus viability. In addition, the trans-processing activity of NIa proteinase was tested in vivo after introduction of an artificial cleavage site between the GUS and HC-Pro sequences in the cytoplasmic GUS/HC-Pro polyprotein encoded by TEV-GUS. The novel site was recognized and processed in plants infected by the engineered virus, indicating the presence of excess NIa processing capacity in the cytoplasm. The potential roles of internal NIa processing in TEV-infected cells are discussed.     

Molecular Cloning, Expression, and Purification of Nuclear Inclusion A Protease from Tobacco Vein Mottling Virus

The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The K_m and k_cat values were about 0.2 mM and 0.071 s^–1, respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.     

Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.     

Expression of a self-processing,pathogen resistance-enhancing gene construct in <Emphasis Type="Italic">Arabidopsis</Emphasis>

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.Revisions requested 17 January 2005; Revisions received 17 January 2005     

Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase.

The genome of tobacco etch virus contains a single open reading frame with the potential to encode a 346-kilodalton (kDa) polyprotein. The large polyprotein is cleaved at several positions by a tobacco etch virus genome-encoded, 49-kDa proteinase. The locations of the 49-kDa proteinase-mediated cleavage sites flanking the 71-kDa cytoplasmic pinwheel inclusion protein, 6-kDa protein, 49-kDa proteinase, and 58-kDa putative polymerase have been determined by using cell-free expression, proteolytic processing, and site-directed mutagenesis systems. Each of these sites is characterized by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly (in which cleavage occurs after the Gln residue). The amino acid residue (Gln) predicted to occupy the -1 position relative to the scissile bond has been substituted, by mutagenesis of cloned cDNA, at each of four cleavage sites. The altered sites were not cleaved by the 49-kDa proteinase. A series of synthetic polyproteins that contained the 49-kDa proteinase linked to adjoining proteins via defective cleavage sites were expressed, and their proteolytic activities were analyzed. As part of a polyprotein, the proteinase was found to exhibit cis (intramolecular) and trans (intermolecular) activity.     

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein

We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.     

A plant virus vector for systemic expression of foreign genes in cereals

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.     

Functions of the tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa).

The NIb protein of tobacco etch potyvirus (TEV) possesses several functions, including RNA-dependent RNA polymerase and nuclear translocation activities. Using a reporter protein fusion strategy, NIb was shown to contain two independent nuclear localization signals (NLS I and NLS II). NLS I was mapped to a sequence within amino acid residues 1 to 17, and NLS II was identified between residues 292 and 316. Clustered point mutations resulting in substitutions of basic residues within the NLSs were shown previously to disrupt nuclear translocation activity. These mutations also abolished TEV RNA amplification when introduced into the viral genome. The amplification defects caused by each NLS mutation were complemented in trans within transgenic cells expressing functional NIb, although the level of complementation detected for each mutant differed significantly. Combined with previous results (X. H. Li and J. C. Carrington, Proc. Natl. Acad. Sci. USA 92:457-461, 1995), these data suggest that the NLSs overlap with essential regions necessary for NIb trans-active function(s). The fact that NIb functions in trans implies that it must interact with one or more other components of the genome replication apparatus. A yeast two-hybrid system was used to investigate physical interactions between NIb and several other TEV replication proteins, including the multifunctional VPg/proteinase NIa and the RNA helicase CI. A specific interaction was detected between NIa and NIb. Deletion of any of five regions spanning the NIb sequence resulted in NIb variants that were unable to interact with NIa. Clustered point mutations affecting the conserved GDD motif or NLS II within the central region of NIb, but not mutations affecting NLS I near the N terminus, reduced or eliminated the interaction. The C-terminal proteinase (Pro) domain of NIa, but not the N-terminal VPg domain, interacted with NIb. The effects of NIb mutations within NLS I, NLS II, and the GDD motif on the interaction between the Pro domain and NIb were identical to the effects of these mutations on the interaction between full-length NIa and NIb. These data are compatible with a model in which NIb is directed to replication complexes through an interaction with the Pro domain of NIa.     

Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.

The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.     

Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification.

A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPg-proteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase active-site residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential.     

Co-ordinated expression of multiple enzymes in different subcellular compartments in plants

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the Escherichia coli acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea rbcS gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP-NIa-ACK-CAT or TP-ACK-CAT-NIa failed to yield chloroplast-localized ACK and CAT proteins, although the latter did give rise to a chloroplast-localized ACK-CAT polyprotein. These results indicate that the NIa proteinase acts in cis more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK-NIa-TP-CAT and TP-ACK-NIa-TP-CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.     

Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing.

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus-encoded proteinases (NIa and HC-Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N-terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC-Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC-Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC-Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC-Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC-Pro had no effect on N-terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC-Pro N-terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.     

A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.     

Functional Analysis of the Interaction between VPg-Proteinase (NIa) and RNA Polymerase (NIb) of Tobacco Etch Potyvirus, Using Conditional and Suppressor Mutants

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.     

Comparison of the substrate specificity of two potyvirus proteases

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.     

Structural determinants of tobacco vein mottling virus protease substrate specificity

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac‐RETVRFQSD) at 1.7‐Å resolution. As observed in several crystal structures of TEV protease, the C‐terminus (～20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ～10‐fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k_cat and K_m for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.