The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.

The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C. Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein beta-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 x g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.     

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.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

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.     

Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications.

Tobacco etch virus (TEV) encodes three proteinases that catalyze processing of the genome-encoded polyprotein. The P1 proteinase originates from the N terminus of the polyprotein and catalyzes proteolysis between itself and the helper component proteinase (HC-Pro). Mutations resulting in substitution of a single amino acid, small insertions, or deletions were introduced into the P1 coding sequence of the TEV genome. Deletion of the N-terminal, nonproteolytic domain of P1 had only minor effects on virus infection in protoplasts and whole plants. Insertion mutations that did not impair proteolytic activity had no measurable effects regardless of whether the modification affected the N-terminal nonproteolytic or C-terminal proteolytic domain. In contrast, three mutations (termed S256A, F, and delta 304) that debilitated P1 proteolytic activity rendered the virus nonviable, whereas a fourth proteinase-debilitating mutation (termed C) resulted in a slow-infection phenotype. A strategy was devised to determine whether the defect in the P1 mutants was due to an inactive proteinase domain or due simply to a lack of proteolytic maturation between P1 and HC-Pro. Sequences coding for a surrogate cleavage site recognized by the TEV NIa proteinase were inserted into the genome of each processing-debilitated mutant at positions that resulted in NIa-mediated proteolysis between P1 and HC-Pro. The infectivity of each mutant was restored by these second-site modifications. These data indicate that P1 proteinase activity is not essential for viral infectivity but that separation of P1 and HC-Pro is required. The data also provide evidence that the proteinase domain is involved in additional, nonproteolytic functions.     

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.     

Potyviral NIa proteinase, a proteinase with novel deoxyribonuclease activity

The NIa proteinase from pepper vein banding virus (PVBV) is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. It accumulates in the nucleus of the infected plant cell and forms inclusion bodies. The function of this protein in the nucleus is not clear. The purified recombinant NIa proteinase was active, and the mutation of the catalytic residues His-46, Asp-81, and Cys-151 resulted in complete loss of activity. Most interesting, the PVBV NIa proteinase exhibited previously unidentified activity, namely nonspecific double-stranded DNA degradation. This DNase activity of the NIa proteinase showed an absolute requirement for Mg(2+). Site-specific mutational analysis showed that of the three catalytic residues, Asp-81 was the crucial residue for DNase activity. Mutation of His-46 and Cys-151 had no effect on the DNase activity, whereas mutant D81N was partially active, and D81G was completely inactive. Based on kinetic analysis and molecular modeling, a metal ion-dependent catalysis similar to that observed in other nonspecific DNases is proposed. Similar results were obtained with glutathione S-transferase-fused PVBV NIa proteinase and tobacco etch virus NIa proteinase, confirming that the DNase function is an intrinsic property of potyviral NIa proteinase. The NIa protein present in the infected plant nuclear extract also showed the proteinase and the DNase activities, suggesting that the PVBV NIa protein that accumulates in the nucleus late in the infection cycle might serve to degrade the host DNA. Thus the dual function of the NIa proteinase could play an important role in the life cycle of the virus.     

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     

An Ry-mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y

Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry-mediated resistance against PVY in potato, we expressed each of the PVY-encoded proteins in leaves of PVY-resistant (Ry) and -susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry-mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry-mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host-encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry-mediated resistance response.     

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.     

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.     

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.     

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.     

VPg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement.

The V20 cultivar of Nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (TEV). In V20, both TEV-HAT and TEV-Oxnard strains are capable of genome amplification and cell-to-cell movement, but only TEV-Oxnard is capable of systemic infection by vasculature-dependent long-distance movement. To investigate the basis for host-specific movement of TEV, chimeric virus genomes were assembled from TEV-HAT and TEV-Oxnard. Viruses containing the TEV-Oxnard coding regions for HC-Pro and/or capsid protein (CP), two proteins that are known to be essential for TEV long-distance movement, failed to infect V20 systemically. In contrast, chimeric viruses encoding the TEV-Oxnard VPg domain of NIa were able to infect V20 systemically. The critical region controlling the infection phenotype in V20 was mapped to a 67-nucleotide segment containing 10-nucleotide differences, but only five amino acid differences, between TEV-HAT and TEV-Oxnard. In V20 coinfection experiments, a restricted strain had no effect on systemic infection by a long-distance movement-competent chimeric strain, suggesting that the restricted strain was not inducing a generalized systemic resistance response. These data suggest that the VPg domain, which is covalently attached to the 5' end of genomic RNA, interacts either directly or indirectly with host components to facilitate long-distance movement.     

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.     

Immunocytology Shows the Presence of Tobacco Etch Virus P3 Protein in Nuclear Inclusions

Intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). Immunocytological studies localized many of the tobacco etch virus (TEV)-encoded proteins in infected cells. We used antiserum against the nonstructural P3 protein of TEV to determine the subcellular location of the P3 protein in ultrathin sections of virus-infected cells. Immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or NIs. Absorption of antiserum with purified NIs or P3 protein resulted in no labeling. TEV NIs are known to contain a bifunctional genome-linked protein–viral proteinase (NIa–VPg) and RNA-dependent RNA polymerase (NIb). It appeared that the TEV P3 protein was a third nonstructural viral protein of NIs of TEV if the NIa–VPg is considered one protein. The presence of P3 in NIs was also supported by Western blot assays. P3 protein in the nucleolus and nucleus could indicate that it, too, is involved in early stages of viral replication.     

A protein interaction map of soybean mosaic virus strain G7H based on the yeast two-hybrid system

A protein interaction map of Soybean mosaic virus (SMV) strain G7H was generated by the yeast two-hybrid system. Clones encoding the genes P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and CP were fused downstream of the GAL4 binding domain (GAL4-BD) and of the GAL4 activation domain (GAL4-AD). The GAL4-BD and GAL4-AD fusion derivatives of each gene were co-transformed into yeast and transformants in which interaction took place were identified on selective media. Interacting fusion proteins were extracted from the yeast cells, run on SDS-PAGE gels and finally checked by Western blotting with GAL4 polyclonal antibodies. Strong interactions were detected between the pairs CP/CP, HC-Pro/HC-Pro, NIa/NIa, and CP/HC-Pro. Relatively weak but significant interaction was detected between VPg and NIa. Although not all of the protein-protein interactions previously reported in other potyviruses were detected, the interactions revealed here were, in general, similar to those reported previously.