Viral genome-linked protein (VPg) controls accumulation and phloem-loading of a potyvirus in inoculated potato leaves

The viral protein covalently linked to the 5' end of the plus-sense, single-stranded RNA genome of potyviruses (genus Potyvirus) can be an avirulence determinant in incompatible potyvirus-host combinations in which the resistance prevents systemic virus infection. The mechanism is not well known. This study shows that virus strain-specific resistance to systemic infection with Potato virus A (PVA) in Solanum commersonii is overcome by a single amino acid (aa) substitution, His118Tyr, in the viral genome-linked protein (VPg). Virus localization and other experiments revealed that Tyr118, controls phloem loading of PVA. The critical boundary may be constituted in phloem parenchyma, companion cells, or both. Tyr118 also controls the cellular level of virus accumulation in infected leaves, including phloem cells. Amino acid substitutions at three additional positions of the central part (aa 116) and C terminus (aa 185) of the VPg and of the N terminus of the 6K2 protein (aa 5) affect virus accumulation and rate of systemic infection but are not sufficient for phloem loading of PVA. These data, together with previous studies, indicate that the PVA VPg aa residues crucial for systemic infection are host specific. Also, our data and previous studies on other potyvirus-host species combinations indicate that the central part of the VPg is a domain with universal importance to virus-host interactions required for systemic invasion of plants with potyviruses.     

The 6K2 protein and the VPg of potato virus A are determinants of systemic infection in Nicandra physaloides

Infection with the isolate PVA-M of potato virus A (PVA; genus Potyvirus) is restricted to the inoculated leaves of Nicandra physaloides (Solanaceae), whereas the isolate PVA-B11 infects plants systemically by 10 days post inoculation. Resistance to systemic infection was shown to develop during plant growth. A recombinant virus (B11-M) in which a 1,208-nucleotide sequence of the full-length cDNA clone of PVA-B11 was replaced with the corresponding sequence from PVA-M displayed a phenotype similar to that of PVA-M. The replaced sequence contained four amino acid differences between the two isolates: one in the 6K2 protein and three in the viral genome-linked protein (VPg). Site-directed mutagenesis of the cDNA clones and inoculation of the mutants to N. physaloides indicated that the amino acid substitutions of Met5Val in the 6K2 protein or Leu185Ser in the VPg permitted vascular movement and systemic infection. However, resistance was only partially overcome by these changes, since systemic infection proceeded at a slower rate than with PVA-B11. The amino acid substitution Val116Met in the VPg alone was sufficient to overcome resistance and recover the phenotype of the isolate PVA-B11. These data indicated that both the 6K2 protein and the VPg were avirulence determinants of PVa-M in N. physaloides and suggested a possibly coordinated function of them in the vascular movement of PVA.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

Detection of the potyviral genome-linked protein VPg in virions and its phosphorylation by host kinases

The multifunctional genome-linked protein (VPg) of Potato virus A (PVA; genus Potyvirus) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species Solanum commersonii and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of S. commersonii differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.     

Detection of a natural point mutation in Potato virus A that overcomes resistance to vascular movement in Nicandra physaloides., and studies on seed-transmissibility of the mutant virus

Isolate M of Potato virus A (PVA‐M; genus Potyvirus) is avirulent in Nicandra physaloides L. (family Solanaceae). The inoculated leaves are infected but no systemic infection is observed. Forty plants of ‘Black Pod’ (BP) and ‘Black Pod Alba’ (BPA), two variants of N. physaloides described in this study, were inoculated with PVA‐M. Two plants of BP and one plant of BPA were systemically infected. Mosaic, blistering and dark green islands developed on the systemically infected leaves, and flowers showed colour‐break symptoms. PVAprogeny were sequence‐characterised for the 6K2 protein and viral genome‐linked protein (VPg) encoding regions known to control the long distance movement of PVA in N. physaloides. All virus progeny (designated as PVA‐Mm) in the systemically infected leaves of the plants inoculated with PVA‐M contained only a single amino acid substitution (Vail 16Met) in the central part of VPg due to a nucleotide substitution G6033A, as compared to PVA‐M. Other PVA isolates that infected N. physaloides systemically also contained Metll6 in VPg. In a previous study using chimeric viruses, Metl16 in VPg was shown to be a major determinant for vascular movement of PVA in N. physaloides, and this study reveals that the mutation for Metl16 can occur in vivo during replication of the avirulent PVA‐M in infected plants. Immunolocalisation studies on BP and BPA plants showed that the pods (berries) and seed coat contained PVA‐Mm in the developing seeds, but no virus was detected in embryons. Up to 27% of the mature seeds contained PVA‐Mm but no transmission to seedlings was observed in a total of 450 seeds tested, and no test plants were infected following mechanical inoculation with extracts prepared from the seeds.     

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-Like Potato virus A in Nicotiana Species

The multifunctional nuclear inclusion protein a (NIa) of potyviruses (genus Potyvirus; Potyviridae) accumulates in the nucleus of virus-infected cells for unknown reasons. In this study, two regions in the viral genome-linked protein (VPg) domain of NIa in Potato virus A (PVA) were found to constitute nuclear and nucleolar localization signals (NLS) in plant cells (Nicotiana spp). Amino acid substitutions in both NLS I (residues 4 to 9) and NLS II (residues 41 to 50) prevented nuclear localization, whereas mutations in either single NLS did not. Mutations in either NLS, however, prevented nucleolar localization and prevented or diminished virus replication in protoplasts, accumulation in infected plant tissues, and/or systemic movement in plants. One NLS mutant was partially complemented by the wild-type VPg expressed in transgenic plants. Furthermore, NLS I controlled NIa accumulation in Cajal bodies. The VPg domain interacted with fibrillarin, a nucleolar protein, and depletion of fibrillarin reduced PVA accumulation. Overexpression of VPg in leaf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereas NLS I and NLS II mutants, which exhibited reduced nuclear and nucleolar localization, showed no such activity. These results demonstrate that some of the most essential viral functions required for completion of the infection cycle are tightly linked to regulation of the NIa nuclear and nucleolar localization.     

Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation

RNA granules are cellular structures, which play an important role in mRNA translation, storage, and degradation. Animal (+)RNA viruses often co-opt RNA granule proteins for viral reproduction. However, the role of RNA granules in plant viral infections is poorly understood. Here we use Potato virus A (PVA) as a model potyvirus and demonstrate that the helper component-proteinase (HCpro), the potyviral suppressor of RNA silencing, induces the formation of RNA granules. We used confocal microscopy to demonstrate the presence of host RNA binding proteins including acidic ribosomal protein P0, argonaute 1 (AGO1), oligouridylate-binding protein 1 (UBP1), varicose (VCS) and eukaryotic initiation factor iso4E (eIF(iso)4E) in these potyvirus-induced RNA granules. We show that the number of potyviral RNA granules is down-regulated by the genome-linked viral protein (VPg). We demonstrated previously that VPg is a virus-specific translational regulator that co-operates with potyviral RNA granule components P0 and eIF(iso)4E in PVA translation. In this study we show that HCpro and varicose, components of potyviral RNA granules, stimulate VPg-promoted translation of the PVA, whereas UBP1 inhibits this process. Hence, we propose that PVA translation operates via a pathway that is interrelated with potyviral RNA granules in PVA infection. The importance of these granules is evident from the strong reduction in viral RNA and coat protein amounts that follows knock down of potyviral RNA granule components. HCpro suppresses antiviral RNA silencing during infection, and our results allow us to propose that this is also the functional context of the potyviral RNA granules we describe in this study.     

Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg

Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn(2+) than Mg(2+) and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [alpha-(32)P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses.     

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.     

VPg gene amplification correlates with infective particle formation in foot-and-mouth disease virus.

In order to analyze the function of VPg amplification in aphthoviruses, we have undertaken the first mutational analysis of the repetitive VPg-coding region using an improved foot-and-mouth disease virus (FMDV) cDNA clone from which infective viral RNA was synthesized. A set of VPg mutants was constructed by site-directed mutagenesis which includes different VPg deletion mutations, a VPg insertion mutation, and amino acid residue replacement mutations that interfere with binding of the VPg protein to the viral RNA and with its proteolytic processing. Our results revealed that an amazing flexibility in the number of VPgs is tolerated in FMDV. Optimal viability is given when three VPgs are encoded. Deletion as well as insertion of one VPg gene still resulted in infective particle production. Infective particle formation was observed as long as one VPg remained intact. No obvious differences in the individual VPg molecules with regard to their promoting viral RNA synthesis were observed, indicating that all three VPgs can act equally in FMDV replication. Mutant polyprotein processing was comparable to that of the wild-type virus. However, VPg mutants showed reduced viral RNA synthesis levels after infection. The levels of viral RNA synthesis and infective particle formation were found to correlate with the number of functional VPgs left in the mutant virus. These findings suggest a direct VPg gene dosage effect on viral RNA synthesis, with a secondary effect on infective particle formation.     

Mapping of viral genomic regions important in cross-protection between strains of a potyvirus

Cross-protection was tested between potato and tobacco strains of Potato virus A, a member of the genus Potyvirus (PVA), in tobacco plants. Cross-protection was effective only at the initiation of infection. The potato strains provided only weak cross-protection against the tobacco strain, whereas the tobacco strain provided strong cross-protection against potato strains. The tamarillo strain (TamMV) showed cross-protection phenotypes mostly resembling those of the potato strains. Chimera of the PVA strains were utilized to map viral genomic regions important for cross-protection. The coat protein (CP) encoding region and the helper component proteinase (HCpro) affected cross-protection and virus accumulation. An amino acid substitution at the CP N-terminus reduced virus accumulation and the ability to overcome cross-protection, whereas amino acid substitutions introduced to the HCpro increased virus accumulation and the ability to overcome cross-protection. Closer sequence relatedness between the protector and challenger isolate, as determined by the CP-encoding sequence, was correlated with an increased cross-protection ability. Cross-protection was not overcome by inoculation with nonencapsidated viral RNA. Thus, the differences in cross-protection abilities between PVA strains and chimera were not explained with the "re-encapsidation model" described for strains of Tobacco mosaic tobamovirus but may be associated with a virus infection-induced RNA silencing mechanism.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.     

Natural resistance to Clover yellow vein virus in beans controlled by a single recessive locus

We characterized the resistance of the common bean cv. Jolanda to Clover yellow vein virus no. 30 (ClYVV). After inoculation, the virus was detected in neither inoculated nor upper leaves, suggesting that the resistance operates at either the viral replication or cell-to-cell movement level. To analyze the mechanism of resistance, we developed a green fluorescent protein (GFP)-tagged ClYVV, and monitored GFP fluorescence at sites of infection on ClYVV-inoculated leaves. No GFP fluorescence was detected in Jolanda, whereas its expression in single cells and spread on inoculated leaves were observed clearly in susceptible cultivars. ClYVV-introduced Jolanda cells were found to be still viable; therefore, it is unlikely that the restriction of multiplication was due to rapid cell death. Genetic analysis indicated that a single recessive locus controlled the resistant phenotype of Jolanda. We designated this locus desc (determinant of susceptibility to ClYVV). Meanwhile, a spontaneous mutant virus that overcomes the resistance (ClYVV-Br) was isolated. Inoculation assays using chimeric viruses suggested that a viral genome-linked protein (VPg) might be the avirulence determinant. The resistance mechanism may be associated with the role of VPg in the viral infection cycle.     

Abiotic stress responses promote Potato virus A infection in Nicotiana benthamiana

The effect of abiotic stress responses on Potato virus A (PVA; genus Potyvirus) infection was studied. Salt, osmotic and wounding stress all increased PVA gene expression in infected Nicotiana benthamiana leaves. According to the literature, an early response to these stresses is an elevation in cytosolic Ca(2+) concentration. The infiltration of 0.1 m CaCl(2) into the infected leaf area enhanced the translation of PVA RNA, and this Ca(2+) -induced effect was more profound than that induced solely by osmotic stress. The inhibition of voltage-gated Ca(2+) channels within the plasma membrane abolished the Ca(2+) effect, suggesting that Ca(2+) had to be transported into the cytosol to affect viral gene expression. This was also supported by a reduced wounding effect in the presence of the Ca(2+) -chelating agent ethylene glycol tetraacetic acid (EGTA). In the absence of viral replication, the intense synthesis of viral proteins in response to Ca(2+) was transient. However, a Ca(2+) pulse administered at the onset of wild-type PVA infection enhanced the progress of infection within the locally infected leaf, and the virus appeared earlier in the systemic leaves than in the control plants. This suggests that the cellular environment was thoroughly modified by the Ca(2+) pulse to support viral infection. One message of this study is that the sensing of abiotic stress, which leads to cellular responses, probably via Ca(2+) signalling, associated with enhanced virus infection, may lead to higher field crop losses. Therefore, the effect of abiotic stress on plant viral infection warrants further analysis.     

A dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral movement encoded by potato virus M

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.     

Protein composition of 6K2‐induced membrane structures formed during Potato virus A infection

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein‐induced membranous structures from Potato virus A (PVA)‐infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N‐terminal Twin‐Strep‐tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non‐tagged Cerulean‐6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep‐tag‐based affinity chromatography was developed. Both (+)‐ and (–)‐strand PVA RNA and viral protein VPg were co‐purified specifically with the affinity tagged PVA‐SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA‐SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2‐induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co‐purified with PVA‐derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.