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Potato leafroll virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational 'readthrough' of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5-GFP lost its nucleolar localization in the presence of replicating PLRV.

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed 'monomer' VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT-PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5'-terminal untranslated sequence of the virus RNA and suggest that if there is an 'origin of assembly' motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.

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.

Coat protein gene sequences of eight isolates of potato mop-top virus from the Peruvian Andes and of three isolates from Scotland were compared. Despite wide geographical separation, there was little sequence variation among all isolates.

Four potato clones with host gene-mediated resistance to potato leafroll virus (PLRV) multiplication were transformed with the PLRV coat protein (CP) gene. Plants of lines expressing high levels of transcript were highly resistant to PLRV multiplication; virus concentration was only 20-40 ng/g of leaf, which is approximately 1% of the concentration reached in susceptible cultivars. The effects of the transgenic and host-derived resistance genes appear to be additive.

Potato leafroll luteovirus (PLRV) is transmitted naturally by aphids, but two isolates (15 and V) are known to be only poorly transmissible (PAT); these isolates are also distinct in that their particles lack an epitope present in transmissible (HAT) isolates of PLRV (Tamada et al., Ann. Appl. Biol. 104, 107-116, 1984; Massalski and Harrison, J. Gen. Virol. 68, 1813-1821, 1987). Virus cultures propagated vegetatively for several years in potato plants from the source of isolate V were shown still to be poorly transmissible by Myzus persicae. Moreover, when isolate V was transmitted by aphids, the subisolates obtained (V4 and V31) were no more readily transmissible than was the stock culture PLRV-V. The subisolates were also unchanged antigenically by the transmission. Thus the inefficient transmission had not restored the properties of HAT isolates. Sequence comparisons among the coat proteins and the readthrough domains of HAT and PAT Scottish isolates suggested that either or both of two amino acid changes in the C-terminal part of the readthrough protein are responsible for the poor transmission and the loss of an epitope. The readthrough protein is therefore thought to play a role in the circulative aphid transmission of PLRV.

The 5'-most open reading frame of the c.4kb RNA-2 of Indian peanut clump furovirus (IPCV) encodes a protein of 208 amino acids. This protein is thought to be the coat protein of IPCV because its amino acid composition and M(r) closely resemble those reported for IPCV coat protein and because its amino acid sequence is 61% identical to that of the coat protein of peanut clump virus (PCV) from West Africa. The extent of the sequence identity between IPCV and PCV coat proteins confirms previous conclusions that the viruses are distinct rather than strains of one virus. The sequences of the coat proteins of IPCV and PCV were between 18% and 26% identical to those of other furoviruses and those of unrelated tobamoviruses and tobraviruses. In contrast, the coat protein sequences were 37% (IPCV) and 36% (PCV) identical to that of the coat protein of barley stripe mosaic hordeivirus (BSMV). This similarity between the coat proteins of viruses from different groups (= genera) is unusual but is consistent with previous reports of sequence relatedness in various genes between certain furoviruses and BSMV.

The presence of a mobile element in the coat protein of pepper ringspot tobravirus (PRV) has been established by 1H n.m.r. spectroscopy; two-dimensional correlation spectroscopy (COSY) measurements show that this element consists of alanine, asparagine, glycine, proline, serine and threonine only. By reference to the amino acid sequence of the coat protein, it is concluded that the mobile element is associated with the C-terminal region and consists of between 11 and 38 residues.

Parsnip yellow fleck and rice tungro spherical viruses, with monopartite ss RNA genomes, resemble picornaviruses in the polymerase and NTP-binding domains of their encoded polyproteins. Though in separate genera, they may comprise a new family.

The nucleotide sequence of raspberry ringspot nepovirus (RRV) RNA-2 consists of 3928 nucleotides and a poly(A) tract at the 3' end. RNA-2 contains one open reading frame which encodes a polypeptide of M(r) 123508 (123K). Edman degradation located the N terminus of the coat protein 514 residues from the C-terminal end of the 123K protein, which suggests that the coat protein is released from the polyprotein by cleavage of a C-A bond. The RRV coat protein has some sequence similarities with the coat proteins of other nepoviruses, but is no more like any one nepovirus than another. In contrast, the portion of the 123K protein to the N-terminal side of the coat protein is similar in sequence to the corresponding parts of the polyproteins of tomato black ring and grapevine chrome mosaic nepoviruses, though not to those of other nepoviruses.

Northern blot analysis of nucleic acid from potato plant tissues and tobacco protoplasts infected with a Scottish isolate of potato leafroll luteovirus (PLRV) detected the 6 kb genomic RNA and one subgenomic RNA species of about 2.7 kb; RNA extracted from virus particles contained only the genomic species. Blotting with small defined probes suggested that the location of the 5' end of the subgenomic RNA was between 2380 and 2510 nucleotides from the 3' end of the PLRV genome (between 3370 and 3500 nucleotides from the 5' end of PLRV Dutch isolate RNA). When RNA extracted from PLRV-infected or mock-inoculated protoplasts was used as the template for primer extension using primers complementary to the sequence at, or upstream of, the initiation codon of the coat protein gene, a single major infection-specific product was detected. A primer complementary to the sequence between 162 and 179 nucleotides upstream of the coat protein AUG yielded a product of 56 nucleotides. Thus, the subgenomic RNA has a leader sequence of 212 nucleotides, is 2505 nucleotides in length and starts at a position equivalent to 3376 nucleotides from the 5' end of the PLRV-Dutch genome, 11 nucleotides upstream of the termination codon of the putative polymerase gene. The nucleotide sequence immediately downstream of this position closely resembles that of the 5' end of the PLRV genomic RNA.

An antiserum was raised against a fusion protein containing part of the 56K polypeptide (P5) encoded by the open reading frame (ORF) at the 3' end of the genome of potato leafroll virus (PLRV). This antiserum reacted specifically with 80K and 90K polypeptides in PLRV-infected protoplasts, with a 90K polypeptide in infected potato tissue and with a 53K polypeptide in protein extracted from purified particles of PLRV. Monoclonal antibodies raised against purified PLRV particles also reacted with these polypeptides, as well as with the 23K coat protein. Virus particles partially purified from infected protoplasts contained some 90K polypeptide as well as the major 23K coat protein. The ORFs of the 23K coat protein and P5 are contiguous and in frame. The results suggest that the P5 polypeptide of PLRV occurs in infected cells as part of a readthrough protein comprising the 23K coat protein joined to the P5 amino acid sequence. Moreover the readthrough protein can be assembled into virus particles as a minor component together with the main 23K component. The P5 protein may thus contribute to properties of PLRV determined by its virus particle surface.

Coat protein of raspberry ringspot nepovirus (RRV) migrated during electrophoresis in a discontinuous buffer system as two components which were capable of interchanging during this procedure. However, only one component was formed during electrophoresis in a continuous buffer system. Treatment of the protein before electrophoresis with iodoacetamide, which alkylates sulfhydryl groups, prevented the formation of the faster migrating electrophoretic component. Thus the faster migrating component is probably an artifact caused by S-S bond formation. Although the coat proteins of two other nepoviruses did not behave in this way, the results obtained with RRV emphasize the need for caution in interpreting electrophoretic heterogeneity in virus proteins. The estimated molecular weight of RRV coat protein was 57,000.