A Novel Interaction Network Used by Potyviruses in Virus-Host Interactions at the Protein Level.

Host proteins that are central to infection of potyviruses (genus Potyvirus; family Potyviridae) include the eukaryotic translation initiation factors eIF4E and eIF(iso)4E. The potyviral genome-linked protein (VPg) and the helper component proteinase (HCpro) interact with each other and with eIF4E and eIF(iso)4E and proteins are involved in the same functions during viral infection. VPg interacts with eIF4E/eIF(iso)4E via the 7-methylguanosine cap-binding region, whereas HCpro interacts with eIF4E/eIF(iso)4E via the 4E-binding motif YXXXXLΦ, similar to the motif in eIF4G. In this study, HCpro and VPg were found to interact in the nucleus, nucleolus, and cytoplasm in cells infected with the potyvirus potato virus A (PVA). In the cytoplasm, interactions between HCpro and VPg occurred in punctate bodies not associated with viral replication vesicles. In addition to HCpro, the 4E-binding motif was recognized in VPg of PVA. Mutations in the 4E-binding motif of VPg from PVA weakened interactions with eIF4E and heavily reduced PVA virulence. Furthermore, mutations in the 4G-binding domain of eIF4E reduced interactions with VPg and abolished interactions with HCpro. Thus, HCpro and VPg can both interact with eIF4E using the 4E-binding motif. Our results suggest a novel interaction network used by potyviruses to interact with host plants via translation initiation factors.

Differential Requirement of the Ribosomal Protein S6 and Ribosomal Protein S6 Kinase for Plant-Virus Accumulation and Interaction of S6 Kinase with Potyviral VPg.

Ribosomal protein S6 (RPS6) is an indispensable plant protein regulated, in part, by ribosomal protein S6 kinase (S6K) which, in turn, is a key regulator of plant responses to stresses and developmental cues. Increased expression of RPS6 was detected in Nicotiana benthamiana during infection by diverse plant viruses. Silencing of the RPS6 and S6K genes in N. benthamiana affected accumulation of Cucumber mosaic virus, Turnip mosaic virus (TuMV), and Potato virus A (PVA) in contrast to Turnip crinkle virus and Tobacco mosaic virus. In addition, the viral genome-linked protein (VPg) of TuMV and PVA interacted with S6K in plant cells, as detected by bimolecular fluorescence complementation assay. The VPg-S6K interaction was detected in cytoplasm, nucleus, and nucleolus, whereas the green fluorescent protein-tagged S6K alone showed cytoplasmic localization only. These results demonstrate that the requirement for RPS6 and S6K differs for diverse plant viruses with different translation initiation strategies and suggest that potyviral VPg-S6K interaction may affect S6K functions in both the cytoplasm and the nucleus.

Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Silencing suppressor protein VPg of a potyvirus interacts with the plant silencing-related protein SGS3.

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families.

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.

Mutation of a Short Variable Region in HCpro Protein of Potato virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco.

Helper component proteinase (HCpro) is a multifunctional protein of potyviruses (genus Potyvirus). HCpro of Potato virus A (PVA) interacts with the microtubule-associated protein HIP2 in host cells, and depletion of HIP2 reduces virus accumulation. This study shows that HCpro of Potato virus Y and Tobacco etch virus also interact with HIP2. The C-proximal portion of PVA HCpro determines the interaction with HIP2 and was found to contain a stretch of six residues comprising a highly variable region (HVR) in potyviruses. Mutations in HVR reduced PVA accumulation in tobacco plants and induced necrotic symptoms novel to PVA. Microarray and quantitative reverse transcription polymerase chain reaction analyses revealed induction of many defense-related genes including ethylene- and jasmonic acid-inducible pathways in systemically infected leaves at necrosis onset. Salicylic acid-mediated signaling was dispensable for the response. Genes related to microtubule functions were down-regulated. Structural modeling of HCpro suggested that all mutations in HVR caused conformational changes in adjacent regions containing functionally important motifs conserved in potyviruses. Those mutations, which also caused conformational changes in HVR, led to the greatest reduction of fitness. Our results implicate HVR in the regulation of HCpro conformation and virus-host interactions and suggest that mutation of HVR induces host defense.

Interaction of the microtubule-associated host protein HIP2 with viral helper component proteinase is important in infection with potato virus A.

Microtubules (MT) outline and maintain the overall shape of cells and can reorganize cellular membranes to serve as sites of RNA virus replication. Here, we provide data on involvement of an MT-associated protein in infection of plants with a potyvirus, Potato virus A (PVA), representing the largest family of plant-infecting RNA viruses. Our results showed that helper-component proteinase (HCpro)-interacting protein 2 (HIP2) of potato (Solanum tuberosum) is an MT-associated protein similar to Arabidopsis SPR2. Virus-induced silencing of HIP2 in Nicotiana benthamiana resulted in a spiral-like growth phenotype, similar to the Arabidopsis spr2 mutant, and the spr2 phenotype in Arabidopsis was complemented with potato HIP2. HCpro of PVA interacted with HIP2 of potato and tobacco (Nicotiana tabacum). The interaction was detected by bimolecular fluorescence complementation in PVA-infected leaves on MT and MT intersections at the cell cortex. HIP2-HCpro interaction was determined by the C-proximal α-helix-rich domain of HIP2, whereas the N-proximal putative TOG domain and the central coiled-coil domain of HIP2 controlled HIP2 dimerization and binding to MT. Accumulation of PVA was significantly reduced in the HIP2-silenced leaves of N. benthamiana, which indicates that HIP2-HCpro interactions are important for virus infection.

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.

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants.

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.

HandyGun: An improved custom-designed, non-vacuum gene gun suitable for virus inoculation.

Particle bombardment with a non-vacuum gene gun is an efficient method for transfection of plant cells with cloned viruses and initiation of virus infection. The HandyGun developed in this study is an improved version of a non-vacuum gene gun. Bombardment parameters were studied by inoculating an infectious, 35S promoter-driven cDNA of Potato virus A (PVA; Potyvirus) to the potato clone 'A6', Nicotiana benthamiana and N. tabacum as plasmid DNA coated on microprojectiles (gold particles). The large number of initial infection sites (necrotic local lesions) observed on inoculated 'A6' leaves and the high percentage of Nicotiana plants which were infected systemically with PVA following inoculation with HandyGun were not particularly sensitive to variation in the parameters tested (helium pressure and the amounts of plasmid DNA and gold particles). Data showed that HandyGun is a robust and reliable tool for obtaining high infection rates in plants reproducibly. It is easy and inexpensive to use and can be constructed from parts commonly available.

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.

Elimination of antiviral defense by viral RNase III.

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro.

Human papillomavirus type 16 entry: retrograde cell surface transport along actin-rich protrusions.

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30-60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing.

A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions.

The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584-594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2-4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.

Localization of a potyvirus and the viral genome-linked protein in wild potato leaves at an early stage of systemic infection.

The upper noninoculated 'sink' leaves of the wild potato species, Solanum commersonii, were studied for distribution of Potato virus A (PVA) at an early stage of systemic infection. Viral RNA was detected by in situ hybridization, and five viral proteins were localized using immunohistochemical staining in leaf sections. Initial systemic infection foci were found at the vicinity of major and minor veins. In these infection foci, the viral coat protein, cylindrical inclusion protein, and helper component-proteinase colocalized with viral RNA in parenchyma and mesophyll cells, but none of these were detected in companion cells (CC). In contrast, VPg, which is the N-proximal half of the NIa protein (separated from the C-terminal proteinase domain, NIapro, by an autocatalytic cleavage) and acts as a viral genome-linked protein, was detected in CC in the infection foci, but only at an early stage of virus unloading. Outside the infection foci, conspicuous signals for VPg were readily and exclusively detected in CC of many veins in all vein classes in the absence of signals for NIapro, other viral proteins, and viral RNA. Taken together, our data indicate that both major and minor veins may unload PVA in the sink leaves of potato. The data suggest that VPg is translocated from inoculated source leaves to the sink leaves, where it accumulates in CC at an early stage of systemic infection. These findings suggest that VPg may be a 'phloem protein' that specifically acts in CC in the sink leaves to facilitate virus unloading.

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.

Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells.

Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse-chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The CI-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of CI-6K2 was found in the membrane fraction, in contrast to fully processed CI. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from CI, but did not affect processing of VPg, NIaPro, NIb or CP from the polyprotein. The 6K2-encoding sequence could be removed without any detectable effect on polyprotein processing. However, deletion of either the 6K1 or 6K2 protein-encoding regions rendered PVA non-infectious. Mutations at the 6K2/VPg cleavage site reduced virus infectivity in plants, but had a less pronounced, albeit detectable, effect on proteolytic processing in the baculovirus system. The results of this study indicate that NIaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/CI and NIb/CP sites can also be processed in trans. Both 6K peptides are indispensable for virus replication, and proteolytic separation of the 6K2 protein from the adjacent proteins by NIaPro is important for the rate of virus replication and movement.