Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Ultrastructural study of acquisition and retention of potato leafroll luteovirus in the alimentary canal of its aphid vector, Myzus persicae Sulz.

The chronology of PLRV acquisition and retention by Myzus persicae was investigated using electron microscopy. Examination demonstrated a rapid translocation of the virus through the intestine into the haemocoel. Indeed, viral particles could be observed in the intestinal epithelial cells, then in the haemocoel, 4 and 8 h, respectively, after their arrival in the lumen of the alimentary canal. However, the virus accumulated in the intestinal epithelial cells. In these cells, the first viral particles were seen enclosed in isometric or tubular isolated vesicles; a few hours later, they were present in tubular aggregated vesicles and also in lysosomes or multivesicular bodies. After a 40 h acquisition period, all studied intestinal epithelial cells exhibited high numbers of viral particles which were consistently distributed throughout these cell structures. When aphids were removed from viral source, viral particles were detected in intestinal lumen for a further three days and in intestinal epithelial cells for a total of eight days. Virus content in these cells began to decrease from the second day. Areas with tubular aggregated vesicles were maintained for seven days following aphid removal from viral source, but progressively became smaller and fewer. The accumulation and the persistence of PLRV in the intestine are discussed.

Tagging potato leafroll virus with the jellyfish green fluorescent protein gene.

A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthamiana protoplasts electroporated with plasmid DNA containing this cDNA behind the 35S RNA promoter of Cauliflower mosaic virus became infected with the recombinant virus (PLRV-GFP). Up to 5% of transfected protoplasts showed GFP-specific fluorescence. Progeny virus particles were morphologically indistinguishable from those of wild-type PLRV but, unlike PLRV particles, they bound to grids coated with antibodies to GFP. Aphids fed on extracts of these protoplasts transmitted PLRV-GFP to test plants, as shown by specific fluorescence in some vascular tissue and epidermal cells and subsequent systemic infection. In plants agroinfected with PLRV-GFP cDNA in pBIN19, some cells became fluorescent and systemic infections developed. However, after either type of inoculation, fluorescence was mostly restricted to single cells and the only PLRV genome detected in systemically infected tissues lacked some or all of the inserted GFP cDNA, apparently because of naturally occurring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stages of PLRV infection.

Posterior midgut and hindgut are both sites of acquisition of Cucurbit aphid-borne yellows virus in Myzus persicae and Aphis gossypii.

Members of the family Luteoviridae ('luteovirids') rely strictly on aphid vectors for plant-to-plant transmission. This interaction operates according to a persistent and circulative manner, which implies that the virions are being endocytosed and exocytosed across two epithelial barriers (alimentary tract and accessory salivary glands) in the vector's body. In several luteovirid-aphid vector species combinations, the route of virions in the insect has been investigated ultrastructurally by transmission electron microscopy (TEM). Here, we used TEM to follow the route of Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus) in its two efficient vector species, Myzus persicae and Aphis gossypii. We demonstrated that CABYV particles are acquired from the gut lumen to the haemocoel through two different sites in both aphid species, i.e. the posterior midgut (as for Beet western yellows virus in M. persicae) and the hindgut (as for Barley yellow dwarf virus complex in cereal aphids). This 'dual' tissue specificity of CABYV represents an original situation among viruses in the family Luteoviridae examined so far by TEM. A variety of virion-containing structures (e.g. clathrin-coated and tubular vesicles, endosome-like bodies) are found in intestinal cells of both types in both aphids. Release of virus particles from midgut and hindgut cells into the haemolymph was confirmed by immunotrapping using CABYV-specific antibodies. In accessory salivary glands, transport of CABYV virions across the cells was similar in each aphid species, and occurred by a transcytosis mechanism involving formation of tubular and coated vesicles before release of free virions in the salivary canal.

Purification and coat protein gene sequence of a Montana RMV-like isolate of barley yellow dwarf virus.

A Montana barley yellow dwarf virus (BYDV) isolate, BYDV-RMV-MT, is serologically identical to the New York RMV type isolate (RMV-NY) but differs in aphid transmission phenotype. A purification procedure for BYDV-RMV-MT was developed and cDNAs encompassing the entire coat protein gene and a portion of the putative polymerase gene of both RMV-MT and RMV-NY were cloned and sequenced. Diameters of RMV-MT virions averaged 24.7 nm. Average virus yield was 4.2 mg/kg plant tissue. There was 81% sequence identity between the clones of MT and NY RMV isolates at the nucleotide level. At the amino acid level the polymerase genes were 91% identical to each other and 74% homologous with that of beet western yellow virus. The coat protein amino acid sequences of the two RMV isolates were only 81% identical and, compared to other sequenced luteoviruses, both were most similar to cucurbit aphid-borne yellows virus.

Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus.

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.

Assessment of the autonomy of replicative and structural functions encoded by the luteo-phase of pea enation mosaic virus.

The genome of pea enation mosaic virus (PEMV) is composed of two taxonomically unrelated RNAs, interacting to create what has traditionally been considered a bipartite virus. The cohesiveness of this interaction was assessed by examining the autonomy of each RNA in viral replication, coat protein expression and systemic invasion. Using a pea protoplast system, in vitro transcripts of RNA1 were found to be capable of initiating RNA2-independent replication, including the formation of the distinctive nuclear membrane-based replication complex associated with wild-type PEMV infection. Western blotting and electron microscopic analysis demonstrated that the synthesis of the RNA1-encoded coat protein, as well as virion assembly, was also independent of RNA2-directed functions. Mechanical inoculations with transcripts of RNA1 failed to establish a systemic RNA1 infection, whereas inoculations with RNA2 were able to establish a largely asymptomatic systemic infection. Combined inoculum containing RNA1 and RNA2 transcripts were able to recreate wild-type PEMV symptomatology, demonstrating the dependence of RNA1 on RNA2 for mechanical passage. With the notable exception of the adaptation of PEMV to establish a true systemic invasion, these data further strengthen the analogy between PEMV and the helper-dependent complexes associated with members of the luteovirus group.

Beet western yellows luteovirus capsid proteins produced by recombinant baculoviruses assemble into virion-like particles in cells and larvae of Bombyx mori.

We subcloned various constructs of the beet western yellows luteovirus (BWYV) 3' proximal genes into Bombyx mori nuclear polyhedrosis virus (BmNPV) transfer vectors and constructed recombinant BmNPVs. The recombinant BmNPVs were used to infect BmN cells and B. mori larvae. Protein expression was analyzed by SDS-PAGE and immunoblot analysis, and the BWYV-encoded capsid protein (CP), P19, readthrough protein (RT), and a modified capsid-readthrough protein (CP-RT*) were detected in BmN cells infected by the respective BmNPVs. However, we detected only the BWYV CP and P19 in BmN cells infected with a recombinant BmNPV containing all three BWYV 3' proximal genes. BmN cells and fat body cells from B. mori larvae infected with recombinant BmNPVs were analyzed by transmission electron microscopy and immunogold labeling. Particles which morphologically and serologically resembled BWYV virions were detected in the nuclei of BmN cells and B. mori larval fat body cells infected with recombinant BmNPVs producing either the BWYV CP alone or BWYV CP plus a BWYV CP-RT*. Similar particles were not detected in cells infected with a control BmNPV or recombinant BmNPV producing only CP-RT*.