Plant virus HC-Pro is a determinant of eriophyid mite transmission

The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, approximately 16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5'-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids.     

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.     

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.     

Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication.     

Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes. Here, we investigate the effects of overexpressing DCL1, one of four Dicers in Arabidopsis thaliana, on P1/HC-Pro-induced defects in development and small RNA metabolism. Expression of a DCL1 cDNA transgene (35S:DCL1) produced a mild gain-of-function phenotype and largely rescued dcl1 mutant phenotypes. The 35S:DCL1 plants were competent for virus-induced RNA silencing but were impaired in transgene-induced RNA silencing and in the accumulation of some miRNAs. Ectopic DCL1 largely alleviated developmental anomalies in P1/HC-Pro plants but did not correct the P1/HC-Pro-associated defects in small RNA pathways. The ability of P1/HC-Pro plants to suppress RNA silencing and the levels of miRNAs, miRNA*s, and miRNA target messages in these plants were essentially unaffected by ectopic DCL1. These data suggest that P1/HC-Pro defects in development do not result from general impairments in small RNA pathways and raise the possibility that DCL1 participates in processes in addition to miRNA biogenesis.     

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.     

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 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.     

Adaptation of Soybean mosaic virus avirulent chimeras containing P3 sequences from virulent strains to Rsv1-genotype soybeans is mediated by mutations in HC-Pro

In Rsv1-genotype soybean, Soybean mosaic virus (SMV)-N (an avirulent isolate of strain G2) elicits extreme resistance (ER) whereas strain SMV-G7 provokes a lethal systemic hypersensitive response (LSHR). SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-N is avirulent whereas SMV-G7 and SMV-G7d are both virulent. Exploiting these differential interactions, we recently mapped the elicitor functions of SMV provoking Rsv1-mediated ER and LSHR to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively. The phenotype of both SMV-G7 and SMV-G7d were rendered avirulent on Rsv1-genotype soybean when the part of the genome encoding the N-terminus or the entire P3 cistron was replaced with that from SMV-N; however, reciprocal exchanges did not confer virulence to SMV-N-derived P3 chimeras. Here, we describe virulent SMV-N-derived P3 chimeras containing the full-length or the N-terminal P3 from SMV-G7 or SMV-G7d, with or without additional mutations in P3, that were selected on Rsv1-genotype soybean by sequential transfers on rsv1 and Rsv1-genotype soybean. Sequence analyses of the P3 and helper-component proteinase (HC-Pro) cistrons of progeny recovered from Rsv1-genotype soybean consistently revealed the presence of mutations in HC-Pro. Interestingly, the precise mutations in HC-Pro required for the adaptation varied among the chimeras. No mutation was detected in the HC-Pro of progeny passaged continuously in rsv1-genotype soybean, suggesting that selection is a consequence of pressure imposed by Rsv1. Mutations in HC-Pro alone failed to confer virulence to SMV-N; however, reconstruction of mutations in HC-Pro of the SMV-N-derived P3 chimeras resulted in virulence. Taken together, the data suggest that HC-Pro complementation of P3 is essential for SMV virulence on Rsv1-genotype soybean.     

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.     

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.     

Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro

In soybean, Rsv1, a single dominant resistance gene, invokes extreme resistance (ER) against most Soybean mosaic virus (SMV) strains, including SMV-N, but not SMV-G7, which provokes a virulent lethal systemic hypersensitive response (LSHR). The elicitor functions of the two viruses provoking Rsv1-mediated ER and LSHR have been mapped to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively, which differ by nine residues between the two strains. To identify amino acids of P3 from SMV-N provoking Rsv1-mediated ER, the unique residues of SMV-G7 were substituted with those of SMV-N. Of the mutants tested on Rsv1-genotype soybean, only SMV-G7(I788R) and SMV-G7(T948A) lost virulence. However, substitution of amino acids of SMV-N, individually or in combination, with the reciprocal residues from SMV-G7 at these two positions failed to confer virulence to SMV-N. In the search for additional virulence determinants, a series of SMV-N chimeras was generated in which fragments within a region from near the middle of the helper-component proteinase (HC-Pro) cistron to the 5' end of the cytoplasmic inclusion cistron, nucleotides 1,605 to 3,787, were replaced with those of SMV-G7. Only SMV-N-derived chimeras harboring the 3' region of HC-Pro, at least from nucleotide 2,013, and the entire 5' end of P3 (nucleotides 2,430 to 3,237) from SMV-G7 were virulent whereas reciprocal exchanges resulted in loss of SMV-G7 virulence. This region of HC-Pro differs by three amino acids between SMV-N and SMV-G7. Analyses of SMV-G7-derived HC-Pro site-directed mutants showed that only SMV-G7(M683R) lost virulence on Rsv1-genotype soybean; however, SMV-N(R682M) failed to gain virulence. Nevertheless, an SMV-N derived mutant with three concurrent substitutions, R682M+R787I+A947T, gained virulence. The data indicate that both P3 and HC-Pro are involved in virulence of SMV on Rsv1-genotype soybean.     

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.     

High Levels of Resistance in Agropyron Species to Barley Yellow Dwarf and Wheat Streak Mosaic Viruses

Several Agropyron species were tested for new sources of resistance to barley yellow dwarf virus (Bydv ) and wheat streak mosaic virus (WSMV). With BYDV strain PAV, 11 of the 17 Agropyron species showed no virus transmission when plants were given access feed by viruliferous Rhopalosiphum padi. Similar trials with BYDV strain RMV (vectored by R. maidis) indicated that all plants, except susceptible control plants, remained virus free. Virus status was confirmed by enzyme-linked immunosorbent assays. When plants were mechanically inoculated with WSMV, 11 Agropyron species failed to express symptoms, while five other species showed a segregating response or had some accessions segregating and some resistant. Test results suggest that resistance to BYDV and WSMV in Agropyron species does not appear to be correlated with any specific genome of Agropyron species although most of the Agropyron species containing S genome were resistant to BYDV and WSMV.