Helper Component-proteinase Protein Research Articles

Potato virus Y HC-Pro Reduces the ATPase Activity of NtMinD, Which Results in Enlarged Chloroplasts in HC-Pro Transgenic Tobacco.

Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.

Potyviral VPg and HC-Pro Proteins and the Cellular Translation Initiation Factor eIF(iso)4E Interact with Exoribonuclease Rrp6 and a Small α-Heat Shock Protein

The helper-component proteinase (HC-Pro) of potyvirus is a multifunctional protein involved in many mechanisms of viral life cycle. In addition, HC-Pro protein was the first identified suppressor of RNA silencing in plants. However, the identities and functions of direct targets toward the pathways of RNA-silencing suppression mediated by HC-Pro are still to be determined. Here, a yeast two-hybrid search for potyviral HC-Pro interacting tobacco proteins was done to identify host partners and potential silencing suppressors. Two interacting cDNA clones were isolated. One of them encodes an Rrp6-like protein, a subunit of the exosome complex that belongs to the RNase D family of the DEDD superfamily of 3′–5′ hydrolytic exoribonucleases. The other clone codes for a small α-heat shock protein (α-Hsp). The interactions were validated by cross interaction assays with other potyviral HC-Pro proteins. Moreover, both identified clones also interacted with pathogenic viral protein-linked genomes (VPgs) and with translation eukaryotic initiation factors (iso) 4E (eIF(iso)4E) which are host determinants of resistance or susceptibility to potyvirus infections. All together, these findings emphasize the role of the potyviral HC-Pro and VPg proteins and the translation initiation factor eIF(iso)4E, as key players of the plant–virus interplay, where the exoribonuclease Rrp6 and a small α-heat shock protein appear as novel sharing targets.

Turnip mosaic potyvirus probably first spread to Eurasian brassica crops from wild orchids about 1000 years ago.

Turnip mosaic potyvirus (TuMV) is probably the most widespread and damaging virus that infects cultivated brassicas worldwide. Previous work has indicated that the virus originated in western Eurasia, with all of its closest relatives being viruses of monocotyledonous plants. Here we report that we have identified a sister lineage of TuMV-like potyviruses (TuMV-OM) from European orchids. The isolates of TuMV-OM form a monophyletic sister lineage to the brassica-infecting TuMVs (TuMV-BIs), and are nested within a clade of monocotyledon-infecting viruses. Extensive host-range tests showed that all of the TuMV-OMs are biologically similar to, but distinct from, TuMV-BIs and do not readily infect brassicas. We conclude that it is more likely that TuMV evolved from a TuMV-OM-like ancestor than the reverse. We did Bayesian coalescent analyses using a combination of novel and published sequence data from four TuMV genes [helper component-proteinase protein (HC-Pro), protein 3(P3), nuclear inclusion b protein (NIb), and coat protein (CP)]. Three genes (HC-Pro, P3, and NIb), but not the CP gene, gave results indicating that the TuMV-BI viruses diverged from TuMV-OMs around 1000 years ago. Only 150 years later, the four lineages of the present global population of TuMV-BIs diverged from one another. These dates are congruent with historical records of the spread of agriculture in Western Europe. From about 1200 years ago, there was a warming of the climate, and agriculture and the human population of the region greatly increased. Farming replaced woodlands, fostering viruses and aphid vectors that could invade the crops, which included several brassica cultivars and weeds. Later, starting 500 years ago, inter-continental maritime trade probably spread the TuMV-BIs to the remainder of the world.

Attenuated mutants of Potato virus Y necrotic strain produced by nitrous acid treatment and mutagenesis-in-tissue culture methods

We produced attenuated mutants of Potato virus Y necrotic strain not only by nitrous acid treatment but also by a novel method, probably unique to plant viruses, which we call the “mutagenesis-in-tissue culture method”. This relies on the natural or experimental generation of virus sequence variants within an infected plant, and then isolating the mutants by serially cloning them in plants. A total of fifteen attenuated mutants were obtained and studied. Nucleotide and amino acid sequences of the genomes of the attenuated mutant populations were compared with those parental severe isolates, and the amino acid changes in relevant genomic regions for viral attenuation were inferred. Many of the mutations were located in the 5’ half of the genome; 65 % were located in the protein 1 (P1) and helper component proteinase protein (HC-Pro) encoding regions. Amino acid changes mostly involved simultaneous changes in two or more protein encoding regions, one of which was often in the HC-Pro encoding region. The attenuated mutants M-MY10 and N-NA10 were effective in cross-protection against the original severe isolate NTND6.

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.

HC-Pro, a potyvirus RNA silencing suppressor, cancels cycling of Cucumber mosaic virus in Nicotiana benthamiana plants

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses--Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)--synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.

Discriminating Mutations of HC-Pro ofZucchini yellow mosaic viruswith Differential Effects on Small RNA Pathways Involved in Viral Pathogenicity and Symptom Development

Helper component-proteinase (HC-Pro), the gene-silencing suppressor of Potyvirus spp., interferes with microRNA (miRNA) and short-interfering RNA (siRNA) pathways. Our previous studies showed that three mutations of highly conserved amino acids of HC-Pro, R(180)I (mutation A), F(205)L (B), and E(396)N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R(180)I/E(396)N, induces transient leaf mottling in host plants followed by recovery. This mutant confers complete cross protection against subsequent infection by the parental ZYMV (ZG) strain. Here, we sought to obtain molecular evidence on the roles of the three highly conserved amino acids of HC-Pro in miRNA and siRNA pathways using transgenic Arabidopsis plants expressing comparable levels of wild-type and mutant HC-Pro proteins. We demonstrated that amino acid residues 180, 205, and 396 of HC-Pro are critical for suppression of miRNA, trans-acting siRNA (ta-siRNA), and virus-induced gene silencing (VIGS) pathways but not for sense-post transcriptional gene silencing (s-PTGS). Because the HC-Pro double mutant (R(180)I/E(396)N) does not interfere with miRNA and ta-siRNA pathways, the ZGAC mutant virus elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the HC-Pro double AC mutant. Thus, through manipulating these three conserved amino acids on HC-Pro, symptom severity of diseases caused by Potyvirus spp. can be modulated to generate useful cross protectants for field application. Although some of our mutated HC-Pro proteins do not interfere with miRNA and ta-siRNA pathways, they still retain the ability to suppress s-PTGS.

The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development

The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV(FRNK) caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA "passenger" strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV(FINK) did. HC-Pro(FRNK) specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro(FINK). Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC(FINK) mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.

HC-Pro Protein of Potato Virus Y Can Interact with Three Arabidopsis 20S Proteasome Subunits In Planta

The multifunctional protein helper component proteinase (HC-Pro) is thought to interfere with the activity of the 20S proteasome; however, no sites of interaction have been identified for either protein. Here, we first show that the Potato virus Y (PVY) HC-Pro protein can interact with three Arabidopsis 20S proteasome subunits (PAA, PBB, and PBE), using a yeast two-hybrid system and the bimolecular fluorescence complement assay. In addition, yeast two-hybrid analysis of the interaction between several mutant subunits of the 20S proteasome and PVY HC-Pro confirmed that residues 81 to 140 of PAA, 1 to 80 of PBB, and 160 to 274 of PBE are necessary for binding PAA, PBB, and PBE to PVY HC-Pro, respectively. Deletion mutant analysis of PVY HC-Pro showed that the N terminus (residues 1 to 97) is necessary for its interaction with three Arabidopsis 20S proteasome subunits. The ability of HC-Pro to interact and interfere with the activity of the 20S proteasome may help explain the molecular basis of its multifunctional character.

Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing

Helper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.

Functional replacement of Wheat streak mosaic virus HC-Pro with the corresponding cistron from a diverse array of viruses in the family Potyviridae

Helper component-proteinase (HC-Pro) of Wheat streak mosaic virus strain Sidney 81 (WSMV-Sidney 81) was systematically replaced with the corresponding cistron derived from four strains of WSMV (Type, TK1, CZ, and El Batán 3), the tritimovirus Oat necrotic mottle virus (ONMV), the rymoviruses Agropyron mosaic virus (AgMV) and Hordeum mosaic virus (HoMV), or the potyviruses Tobacco etch virus (TEV) and Turnip mosaic virus (TuMV). These HC-Pro proteins varied in amino acid sequence identity shared with HC-Pro of WSMV-Sidney 81 from high (strains of WSMV at ∼86–99%) to moderate (ONMV at 70%) to low (rymoviruses and potyviruses at ∼15–17%). Surprisingly, all chimeric viral genomes examined were capable of systemic infection of wheat upon inoculation with RNA transcripts produced in vitro. HC-Pro replacements derived from tritimoviruses did not alter host range relative to WSMV-Sidney 81, as each of these chimeric viruses was able to systemically infect wheat, oat, and corn line SDp2. These results indicate that differences in host range among tritimoviruses, including the inability of ONMV to infect wheat or the inability of WSMV strains Type and El Batán 3 to infect SDp2 corn, are not determined by HC-Pro. In contrast, all chimeric viruses bearing HC-Pro replacements derived from rymoviruses or potyviruses were unable to infect SDp2 corn and oat. Collectively, these results indicate that HC-Pro from distantly related virus species of the family Potyviridae are competent to provide WSMV-Sidney 81 with all functions necessary for infection of a permissive host (wheat) and that virus–host interactions required for systemic infection of oat and SDp2 corn are more stringent. Changes in symptom severity or mechanical transmission efficiency observed for some chimeric viruses further suggest that HC-Pro affects virulence in WSMV.

Structural Characterization of HC-Pro, a Plant Virus Multifunctional Protein

The helper component proteinase (HC-Pro) is a key protein encoded by plant viruses of the genus Potyvirus. HC-Pro is involved in different steps of the viral cycle, aphid transmission, replication, and virus cell-to-cell and systemic movement and is a suppressor of post-transcriptional gene silencing. Structural knowledge of HC-Pro is required to better understand its multiple functions. To this aim, we purified His-tagged wild-type HC-Pro and a N-terminal deletion mutant (DeltaHC-Pro) from plants infected with recombinant potyviruses. Biochemical analysis of the recombinant proteins confirmed that HC-Pro is a dimer in solution, that the N terminus is not essential for self-interaction, and that a large C-terminal domain is highly resistant to proteolysis. Two-dimensional crystals of the recombinant proteins were successfully grown on Ni2+-chelating lipid monolayers. Comparison of projection maps of negatively stained crystals revealed that HC-Pro is composed of two domains separated by a flexible constriction. Cryo-electron crystallography of DeltaHC-Pro allowed us to calculate a projection map at 9-A resolution. Our data from electron microscopy, biochemical analysis, and secondary structure predictions lead us to suggest a model for structure/function relationships in the HC-Pro protein.

Pathogen derived resistance to Potato virus Y: mechanisms and risks

Since the concept of pathogen derived resistance (PDR) was proposed in 1985, genetic transformation of plants to express virus-derived sequences has been used to engineer resistance to many viruses. This paper reviews PDR approaches to Potato virus Y (PVY, type member of the genus Potyvirus). PDR to viruses operates often through RNA-mediated resistance mechanisms that do not require protein expression. Studies on the RNA-mediated resistance have led to the discovery of post-transcriptional gene silencing (PTGS), a mechanism that controls gene expression in eukaryotic cells and provides natural protection against virus infections. Viruses, in turn, can suppress the PTGS with some of their proteins, such as the helper component-proteinase protein of PVY. Expression of PVY proteins in transgenic plants entails a risk for heterologous encapsidation or synergism with viruses that infect the PVY-resistant transgenic plant. These risks are avoided using RNA-mediated resistance, but a risk still exists for recombination between the transgene transcript and the RNA genome of the infecting virus, which may create a virus with altered properties. The harmful consequences can be limited to some extent by removing functional motifs from the viral sequence used as a transgene.;

A viral suppressor of gene silencing in plants.

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5'-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Requirement for HC-Pro Processing during Genome Amplification of Tobacco Etch Potyvirus

The helper component-proteinase (HC-Pro) of tobacco etch potyvirus (TEV) is a multifunctional protein with several known activities. The N-terminal region is required for aphid transmission and efficient genome amplification, the central region is required for long-distance movement in plants, and the C-terminal domain is a cysteine-type proteinase that autocatalytically cleaves between itself and the P3 protein. To investigate the requirement for HC-Pro-mediated proteolysis during viral replication, a variety of mutations resulting in amino acid substitutions and insertions in the proteolytic domain and cleavage site motif were introduced into the viral genome. Mutations affecting the active site residues, His722 and Cys649, or the cleavage site P1′ residue, Gly764, inhibited proteolytic activity of HC-Pro. Mutant genomes containing these modifications were amplification-defective in protoplasts and plants. Mutants with substitutions affecting several conserved, but non-active site, residues (Ser610, Cys694, and Asp715) within the proteinase domain encoded active HC-Pro proteinases and were similar to parental virus in protoplasts and plants. To determine if the replication defect of the proteinasedebilitated mutants was due to inactivation of HC-Pro proteolytic activity or simply to the inability of HC-Pro and P3 protein to separate, a sequence coding for a heterologous cleavage site recognized by the TEV NIa proteinase was inserted between the HC-Pro and P3 coding regions of an active site mutant. This cleavage site was functional in vitro using purified NIa proteinase. However, this modification was insufficient to restore amplification activity to the mutant. In addition, the active site mutant was not complemented by wild-type HC-Pro supplied in trans by transgenic plants. These results suggest that an active HC-Pro proteinase is required in cis for TEV genome amplification.