Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes.

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

The open reading frame 1-encoded ('36K') protein of Carnation Italian ringspot virus localizes to mitochondria.

The localization of the 36 kDa ('36K') protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K-GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata.

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

Role of the beet western yellows virus readthrough protein in virus movement in Nicotiana clevelandii.

Luteoviruses such as beet western yellows polerovirus (BWYV) are confined to and multiply within the phloem compartment of their hosts. The readthrough domain (RTD) of the minor BWYV capsid protein P74 is required for efficient virus accumulation in Nicotiana clevelandii. Experiments were carried out to determine if the low virus titres observed following agro-inoculation of whole plants with certain RTD mutants are due to a defect in virus multiplication in the nucleate cells of the phloem compartment or to inefficient virus movement to new infection sites. Immuno-localization of wild-type and an RTD-null mutant virus in thin sections of petioles and in phloem cells of leaf lamina, as well as electron microscopy observations, were all consistent with the conclusion that the RTD is not essential for efficient virus multiplication in the nucleate phloem cells but intervenes in virus movement to increase the rate at which new infection foci are established and expand.

Protein 2A of grapevine fanleaf nepovirus is implicated in RNA2 replication and colocalizes to the replication site.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

The first triple gene block protein of peanut clump virus localizes to the plasmodesmata during virus infection.

The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata.

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao.

Cacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin 19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

In situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus RNA 3.

The in situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus (BNYVV) RNA 3 and of the BNYVV coat protein (CP) was studied by immunoelectron microscopy in infected leaf and root cells of Chenopodium murale and C. quinoa. The CP was detected in the cytoplasm of all cell types except xylem, sieve elements, and companion cells. P25 was detected in the cytoplasm and nuclei of the same cell types. The intensity of CP labelling varied depending upon the stage of infection of the cell, whereas the P25 labelling intensity was similar in newly infected cells and in cels at later stages of infection. These results suggest that P25 may be synthesized at an earlier stage of infection than CP. Its presence in the nuclei of newly infected cells may be related to the reported effect of P25 on leaf symptom development.

Detection by immunogold labelling of P75 readthrough protein near an extremity of beet necrotic yellow vein virus particles.

RNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5'-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.

Immunodetection of grapevine fanleaf virus satellite RNA-encoded protein in infected Chenopodium quinoa.

An antiserum was raised against a fusion protein containing the C-terminal half of the protein (P3) encoded by the satellite RNA of grapevine fanleaf virus (GFLV; F13 isolate) and the N-terminal portion of the CI repressor of phage lambda. This antiserum specifically recognized P3 synthesized in the in vitro wheatgerm translation system and also in infected Chenopodium quinoa plants. In these plants, the amount of virus increased for 10 days, then remained constant for up to 21 days, whereas P3 was detected transiently, reaching its maximum on day 10.

An N-proximal sequence of the alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants.

We have made transgenic tobacco plants (Nicotiana tabacum, cv. Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (A1MV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3 delta[1-12] and P3 delta[1-77]). The same proteins were expressed in recombinant yeast. By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by A1MV-infected tobacco plants. P3 delta[1-12] behaved similarly but P3 delta[1-77] was found only in the cytoplasm. It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls.

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.

Nucleic acid binding properties of the 92-kDa replicase subunit of alfalfa mosaic virus produced in yeast.

The 92-kDa non-structural protein of alfalfa mosaic virus (one of the replicase subunits) was synthesized by Saccharomyces cerevisiae transformed with a recombinant expression vector. The yeast-expressed protein had the immunological and size characteristics of the naturally made viral protein. It was partially purified and its nucleic acid binding properties were tested by gel-retardation electrophoresis and nitrocellulose adsorption. The protein interacted with single-stranded RNA, double-stranded RNA and double-stranded DNA in a salt-dependent manner, with a slight preference for RNA. These properties may be related to its putative function as a core RNA polymerase.

Replication of alfalfa mosaic virus RNA: evidence for a soluble replicase in healthy and infected tobacco leaves.

Soluble RNA-dependent RNA polymerases from healthy and alfalfa mosaic virus (AMV)-infected leaves were purified more than 400-fold from the 100,000g supernatant of leaf homogenates, using ammonium sulfate precipitation, Sephadex G-100 filtration, and chromatography on DEAE Sephadex A25 and P11-phosphocellulose. The DEAE-chromatography step eliminated host poly(U)polymerase-like activity, cellular RNases, and endogenous RNA, yielding a typical RNA-dependent RNA polymerase ("DEAE-enzyme") which was dependent on exogenous RNA. On nondenaturing 4-30% gradient slab gels, the activity of the enzyme was recovered in two peaks containing proteins of 340K and 160K. RNA products were synthesized in the presence of the "DEAE-enzyme" from healthy and infected leaves: When an excess of AMV RNA was introduced into the reaction mixture, only synthesis of minus strand RNA was detected. However, when limiting amounts of AMV RNA were used as template some small plus strands were detected, suggesting that synthesized minus strands may in turn serve as template. In both cases, the products were partially double stranded, and of very heterogenous size. Their size depended on the length of the RNA template which in turn varied according to the degree of RNase contamination of the enzyme fractions. These results are the first demonstration that both healthy and AMV-infected tobacco leaves contain a soluble replicase, although its possible in vivo role in viral replication is yet to be demonstrated.