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Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.

Previously, transmission of poleroviruses has relied solely on the use of their aphid vectors. Biolistic inoculation allowed for the first time the mechanical transmission of Beet western yellows virus (BWYV) and Potato leafroll virus (PLRV) to several host plants. Inoculation with purified preparations and viral RNA extracts of PLRV resulted in 30-50% systemically infected Nicotiana occidentalis P1 plants and 15-30% infected Nicotiana clevelandii plants. Particle bombardment was also used successfully to infect N. clevelandii plants with in vitro RNA transcripts of full-length cDNA of BWYV.

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.

A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.

Virions of beet western yellows luteovirus contain a major capsid protein (P22.5) and a minor readthrough protein (P74), produced by translational readthrough of the major capsid protein sequence into the neighboring open reading frame, which encodes the readthrough domain (RTD). The RTD contains determinants required for efficient virus accumulation in agroinfected plants and for aphid transmission. The C-terminal halves of the RTD are not well conserved among luteoviruses but the N-terminal halves contain many conserved sequence motifs, including a proline-rich sequence separating the rest of the RTD from the sequence corresponding to the major coat protein. To map different biological functions to these regions, short in-frame deletions were introduced at different sites in the RTD and the mutant genomes were transmitted to protoplasts as transcripts and to Nicotiana clevelandii by agroinfection. Deletions in the nonconserved portion of the RTD did not block aphid transmission but had a moderate inhibitory effect on virus accumulation in plants and abolished symptoms. Deletion of the proline tract and the junction between the conserved and nonconserved regions inhibited readthrough protein accumulation in protoplasts by at least 10-fold. The mutants accumulated small amounts of virus in plants, did not induce symptoms, and were nontransmissible by aphids using agroinfected plants, extracts of infected protoplasts, or purified virus as a source of inoculum. Other deletions in the conserved portion of the RTD did not markedly diminish readthrough protein accumulation but abolished its incorporation into virions. These mutants accumulated to low levels in agroinfected plants and elicited symptoms, but could not be aphid-transmitted. A preliminary map has been produced mapping these functions to different parts of the RTD.

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.

In order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots. In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins. The one most readily detected has an M(r) of 63K, and was identified as symbionin. This is the predominant protein synthesized by the bacterial endosymbiont of the aphid and is released into the haemolymph. Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin. Inhibition of prokaryotic protein synthesis by feeding M. persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus transmission by more than 70%. The major coat protein of the virus was found to be degraded in the antibiotic-treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss of infectivity. For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction.