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The genomes of umbraviruses differ from those of most other viruses in that they do not encode a coat protein, and thus no virus particles are formed in infected plants. Protection of umbraviral RNA outside the host plant, during vector transmission, utilizes the coat protein of an assistor luteovirus, but this review focuses on the mechanisms that compensate for the lack of a coat protein in processes within the host plant. As well as an RNA-dependent RNA polymerase, umbravirus genomes encode two other proteins from almost completely overlapping open reading frames. One of these is a cell-to-cell movement protein that can mediate the transport of homologous and heterologous viral RNAs through plasmodesmata without the participation of a coat protein. The other, the ORF3 protein, binds to viral RNA to form filamentous ribonucleoprotein particles that have elements of helical structure. It serves to stabilize the RNA and facilitates its transport through the vascular system of the plant. It may also be involved in protection of the viral RNA from the plant's defensive RNA-silencing response, although it is not a suppressor of silencing. The ORF3 protein also enters the cell nucleus, specifically targeting the nucleolus. Although the function of this localization is unknown, the ORF3 protein may provide a valuable tool for investigating plant nucleolar function.

The proteins encoded by open reading frame 3 (ORF3) of the umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed., (Copyright 2001 Academic Press.)

Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed., (Copyright 1999 Academic Press.)

Detection of the three agents of groundnut rosette disease (groundnut rosette assistor virus, groundnut rosette virus and its satellite RNA) in plants and vector aphids by reverse transcription-polymerase chain reaction (RT-PCR) is reported. Three procedures for extraction of total RNA from groundnut were tested, of which two were found to be useful in giving RNA of sufficient quality for RT-PCR. Of these two, the total RNA extraction kit supplied by Qiagen was found to be the most versatile for extraction of all three agents from individual vector aphids (Aphis craccivora). Both groundnut rosette assistor virus and groundnut rosette virus could be detected from total RNA extracted from a single aphid that had been exposed to either green or chlorotic rosette-infected groundnut plants. They could be detected in aphids stored in 70% ethanol for up to 30 days at room temperature. However, satellite RNA could be amplified only when total RNA extracted from two or more aphids was used. Groundnut rosette assistor virus, groundnut rosette virus and its satellite RNA were detected by RT-PCR in aphids that had been exposed only to groundnut rosette diseased plants containing all three agents. The potential of RT-PCR in studying certain key issues of rosette disease epidemiology is discussed.

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Isolates of groundnut rosette umbravirus (GRV) contain a satellite RNA (sat-RNA), about 900 nucleotides (nt) in length, different variants of which are responsible for the symptoms of different forms of rosette disease in groundnuts and, in the particular instance of sat-RNA YB3b, for the production of yellow blotch symptoms in Nicotiana benthamiana. Sat-RNA YB3b does not affect the accumulation of GRV genomic or subgenomic RNAs in infected plants. Replication of sat-RNA YB3b and induction of yellow blotch symptoms do not require the production of any sat-RNA-encoded proteins. Experiments with deletion mutants identified three functional untranslated elements in sat-RNA YB3b. One (designated R) comprises nt 47-281, is essential for sat-RNA replication and appears to be cis-acting. The other two (designated A and B) comprise nt 280-470 and 629-849, respectively, are both involved in yellow blotch symptom production and can act in trans. Element A contains the determinant that is unique to sat-RNA YB3b. The process of symptom induction by sat-RNA YB3b apparently involves a novel type of specific interaction of two untranslated RNA elements, which can complement each other, with a host factor or factors.

Symptom production in groundnut plants infected with groundnut rosette virus (GRV) depends on the presence of satellite RNA (sat-RNA) in the GRV culture, and sat-RNA variants that induce only mild symptoms are known. One such variant drastically diminished the replication of GRV genomic RNA in infected Nicotiana benthamiana plants. This down-regulating ability did not involve either of the two open reading frames in the sat-RNA but was controlled by a region near its 5' end, which is required for sat-RNA replication. When N. benthamiana plants were inoculated with GRV and the mild satellite and challenged by inoculation with a GRV isolate (YB) containing a sat-RNA that induces yellow blotch symptoms, no symptoms appeared and little GRV genomic RNA or sat-RNA was detected in the plants, provided the two inoculations were no more than 2 days apart. A GRV isolate containing a sat-RNA that neither induces symptoms in N. benthamiana nor affects genomic RNA accumulation also provided protection against yellow blotch symptom production if inoculated before or up to 2 days after isolate YB. However, in this case protection ws incomplete and both GRV RNA and sat-RNA accumulated to normal levels. It is suggested that sequences from the mild sat-RNA may provide a novel source of resistance against rosette disease.

Pea enation mosaic virus (PEMV) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two RNA species, one of which is related to the genomes of luteoviruses and the other to those of umbraviruses. In both complexes, these viral RNA components may be supplemented with satellite RNAs that are dependent on the umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. Sequence analysis identified regions of similarity between the satellites of groundnut rosette virus (GRV) and PEMV, particularly at the 5' and 3' termini and around duplicate sequence repeats present in each satellite RNA. The umbravirus GRV and the umbravirus-like PEMV RNA-2 were each able to support the replication and systemic spread of homologous and heterologous satellites. The presence of the PEMV satellite in infections with GRV had no effect on symptom expression in Nicotiana spp. or in Arachis hypogaea. Likewise, in Pisum sativum, the GRV satellite had no effect on the symptoms induced by PEMV. However, the intense yellow blotch symptoms induced in Nicotiana benthamiana by the YB3 GRV satellite in conjunction with GRV were also manifested when PEMV was the helper. Although PEMV RNA-1 was capable of supporting the encapsidation and aphid transmission of the GRV satellite, no evidence was obtained that the essential role of the GRV satellite in the aphid transmission of GRV could be supplied by the PEMV satellite. These data further strengthen the hypothesis of an evolutionary relationship between PEMV and the luteovirus-umbravirus complexes.

Complementary DNA clones representing the entire genome of groundnut rosette umbravirus (GRV) were obtained and sequenced. GRV RNA comprises 4019 nucleotides and contains four large open reading frames (ORFs). The second ORF from the 5' end includes sequences that encode motifs characteristic of viral RNA-dependent RNA polymerases and is probably expressed by a -1 frameshift mechanism as a fusion protein with the product of the 5'-most ORF. The other two ORFs are almost completely overlapping in different reading frames, and are probably expressed from subgenomic RNA. One of the putative products has significant sequence similarity with viral movement proteins. None of the putative proteins encoded by GRV RNA seems to be a structural protein. In genome organization and in the amino acid sequences of its potential products, the RNA of GRV is similar to that of carrot mottle mimic umbravirus, and to the umbravirus-like RNA-2 of pea enation mosaic virus.

The particle proteins of tobraviruses and tobamoviruses share six sequence motifs, two of which are also present in furoviruses and hordeiviruses. Analyses of four different polyclonal antisera to tobacco rattle tobravirus by Pepscan revealed that the C-terminal region of the particle protein was immunodominant. The N-terminal region and a central region (residues 110-121) were more weakly immunogenic. These results suggest that these regions are exposed externally on the assembled virus particle. Papain digestion showed that the C terminus can be removed without apparent structural damage to the particle. The external location of the C-terminal region along the sides of the particle could explain some transmission properties of the rod-shaped viruses.

Murine monoclonal antibodies (MAbs) were produced against a nematode non-transmissible isolate of tobacco rattle tobravirus (TRV-PLB). Two of the MAbs (SCR 80 and 81) did not react with the serologically closely related isolate TRV-PPK20 (both isolates belong to the PRN serotype), which is readily transmissible by nematodes. When further isolates of the PRN serotype were tested, all the non-transmissible ones reacted with these two MAbs, but so also did some of those that are readily transmissible. SCR 80 and 81 were able to trap TRV-PLB particles onto electron microscope grids and reacted with metatopes located near the C terminus of the particle protein. The epitope recognized by SCR 81 was discontinuous. The MAbs that reacted with both TRV-PLB and TRV-PPK20 recognized either continuous cryptotopes (SCR 78) or discontinuous neotopes (SCR 79 and 82).

Ten cDNA clones, representing the satellite-like RNA-3 associated with five different isolates of groundnut rosette virus, were prepared and sequenced. The sequences are at least 87% identical, but differences associated both with symptom differences and with geographical origin were found. The sequences contain up to five potential open reading frames (ORFs), of which three are in the strand that is most abundant in infected plants and two are in the complementary strand. Only one of these ORFs is present in all 10 sequences. Neither the sequence of RNA-3 nor that of its potential translation products had significant similarities to sequences in the databases.

Tobravirus isolate I6 is an anomalous strain of tobacco rattle virus (TRV), which has biological properties typical of TRV, but which is serologically related to pea early-browning virus (PEBV). Its RNA-2 species is a natural recombinant molecule which contains internal sequences, including the particle protein gene, resembling those of PEBV, but with some TRV-like sequences at each end. Sequencing of RNA-2 of isolate I6 showed that, at the 3' end, it is almost identical to RNA-2 of PEBV strain SP5, and TRV-like sequences are limited to the 25 residues that are common to both viruses. However, unlike PEBV RNA-2, I6 RNA-2 can be replicated by TRV RNA-1-coded enzymes. Thus, specific recognition of RNA-2 by TRV replicase seems not to involve sequences at the 3' end. About 275 residues at the 5' end of I6 RNA-2 are similar to those in RNA-2 of TRV strain TCM, and are jointed to the PEBV-SP5-like subgenomic promoter and coding sequences by about 100 residues of uncertain origin. These results support the idea that I6 RNA-2 originated by recombination between TRV and PEBV RNA-2 molecules, although the exact position of the junction and therefore the mechanism involved are unclear.

Transgenic Samsun NN tobacco plants expressing the coat protein of tobacco rattle virus were exposed to mechanical leaf inoculation with tobacco rattle virus and to viruliferous trichodorid vector nematodes. Whereas plants were resistant to mechanical inoculation the vector nematodes successfully transmitted tobacco rattle virus to the roots as well as to the leaves of these plants. It is suggested that transgenic resistance is overcome either because vector nematodes inject relatively large numbers of virus particles into a cell or because they inject destabilized particles. The results indicate that coat protein-mediated resistance is unlikely to be of value for controlling tobacco rattle virus in field crops.

Pseudorecombinant isolates produced from an efficiently transmitted and a non-transmitted isolate of tobacco rattle tobravirus were tested for their transmissibility by trichodorid vector nematodes. Isolates with RNA-2 originating from the non-transmissible isolate were not transmitted by the vector, whereas isolates with RNA-2 originating from the efficiently transmitted isolate were transmitted efficiently. It is therefore concluded that the factor determining vector transmissibility is located on the RNA-2 genome segment of TRV. Because the serological properties of the isolates also correlate with transmissibility, it is likely that the virus particle protein is involved in the transmission process.

An assay involving reverse transcription followed by polymerase chain reaction specifically detected tobacco rattle virus RNA in extracts of infected plants. It detected a wide range of serological variants and typical non-particle producing NM-type isolates. The sensitivity of the method was sufficient to detect TRV RNA in 10 ng total nucleic acid from an infected plant, or in a relatively crude nucleic acid preparation from 60 micrograms infected leaf tissue.

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.

Complementary DNA (cDNA) probes, prepared by nick-translation or by oligolabelling of a 520 bp fragment representing residues 4741 to 5261 in the potato leafroll luteovirus (PLRV) sequence, were equally sensitive, with a detection limit equivalent to sap from about 600 micrograms of infected potato or Nicotiana clevelandii leaf tissue and to RNA from about 120 micrograms tissue. Increasing the concentration of oligolabelled probe gave similar results with shorter autoradiographic exposures, but also resulted in positive signals with sap extracts from healthy plants. In contrast, a complementary RNA (cRNA) probe made by in vitro transcription of the cDNA insert could be used at higher concentration without giving rise to reactions with healthy plant extracts, and had a detection limit equivalent to 5 micrograms tissue/spot. Five oligolabelled probes representing different regions of the PLRV genome detected PLRV equally well. A probe that represented a portion of the particle protein gene also detected beet western yellows luteovirus (BWYV), with which it has 69% nucleotide sequence homology, and an English strain of the RPV form of barley yellow dwarf luteovirus, and reacted weakly with extracts from plants infected with groundnut rosette assistor luteovirus or carrot red leaf luteovirus. Probes for regions on either side of the particle protein gene also detected RPV, but not any of the other luteoviruses tested, in agreement with earlier suggestions that RPV is more closely related to PLRV than are BWYV or the other luteoviruses tested. An attempt to improve the detection of weak heterologous reactions by using a cRNA probe was unsuccessful, perhaps because tests using cRNA are more affected by mismatching than tests using cDNA probes.

An antiserum, elicited by a synthetic peptide coupled to bovine serum albumin, reacted specifically with the non-structural 16K protein of tobacco rattle virus. The protein was detected in extracts of systemically infected Nicotiana clevelandii leaves, but only in those made with the aid of SDS, urea and 2-mercaptoethanol. Immunogold labelling of ultrathin sections showed that the protein was mainly associated with nuclei, but was also present in the cytoplasm. These observations suggest that the 16K protein binds to macromolecular components of infected cells, especially in nuclei, but do not clarify its function.

Patterns of variation are examined in four groups of plant viruses, with special reference to their particle proteins and to changes in vector transmissibility and specificity. In the nepoviruses and potyviruses, non-circulative transmission, by nematodes and aphids respectively, seems dependent on structural features on the surface of the virus particles. The N-terminal part of the particle protein may play the key role in potyviruses. Similarly in the luteoviruses, and possibly in the geminiviruses, specificity of circulative transmission by aphids, whiteflies and leafhoppers is linked to the antigenic specificity of the virus particles. Among naturally occurring isolates of the same virus, variation seems often to be discontinuous, and is predominantly of two sorts. Minor variations, characterized by loss of an epitope or substitutions of a few amino acids, can be associated with loss of transmissibility in luteoviruses and potyviruses, or have no effect. Major variations are associated with differences in vector specificity and seem likely to involve radical genetic changes that have evolved over long periods. The adaptation of virus particle proteins for transmission by vectors probably results in conservation of the genes that encode them, and in greater conservation of some parts of these genes than of others.

The molecular weights of proteins from several tobacco rattle viruses (or tobraviruses) have been determined by electrophoresis with 'marker' proteins in acrylamide gels of different strengths. Plotting KR, a measure of the effect of varying the gel strength, against molecular weight gave a value of 23,000 +/- 2,800 for tobacco rattle virus (TRV) strain CAM and similar values for other strains of TRV and pea early-browning virus. A value of 21,700 +/- 700 was obtained for TRV strain PRN by sedimentation to equilibrium in 6 M guanidine hydrochloride. These estimates are similar to those made by a statistical reappraisal of published data on amino acid composition, and a value of about 22,000 is proposed for TRV coat proteins.

The complete nucleotide sequence of tobacco rattle virus (TRV) strain SYM RNA-1 was determined from a series of overlapping cDNA clones. cDNA prepared by primer extension was used to determine the exact 5' terminus. The RNA sequence was 6791 nucleotides in length and contained four open reading frames (ORFs). The ORF nearest the 5' terminus coded for a polypeptide of predicted mol. wt. 134,000 (134K) and terminated at an opal (UGA) stop codon. Readthrough of this stop codon would result in the production of a protein of 194K. The gene for a 29K polypeptide started one base beyond the 194K stop codon and, in turn, was followed by the gene for a 16K protein at the 3' end of RNA-1. Amino acid comparisons of the 194K protein with the putative replicase of tobacco mosaic virus showed three regions of strong homology, suggesting that the 134K and 194K proteins were similarly involved in virus replication. The 5' terminal sequences of both genome RNA species of TRV strains ORY, N5 and PRN together with that of SYM RNA-2 were also determined. Alignments of these sequences showed that there was a 22 base repeated sequence close to the 5' terminus in all these RNA species. It was also shown that the 5' terminus of RNA-1 was homologous with the same region in RNA-2.

NM isolates of tobacco rattle virus (TRV) are perpetuated by the larger genome nucleic acid (RNA-1) but do not produce nucleoprotein particles; their identification therefore presents special problems. Assays of the infectivity of nucleic acid extracts, and of previously frozen sap, suggested that such isolates occur in potato plants with stem-mottle disease. These isolates were identified by two tests. In the first, TRV nucleoprotein particles were produced in plants inoculated with a mixture of nucleic acid from diseased potato leaves and non-infective RNA-2 of an authentic culture of TRV, showing that the TRV genome had been reconstituted. In the second test, TRV RNA was identified in nucleic acid from diseased potato leaves by hybridization to 3H-labelled DNA complementary in nucleotide sequence to RNA-1 of the PRN strain of TRV. This cDNA test consistently identified TRV in stem-mottle affected potato plants grown in the glasshouse or in field crops. Although particle-producing isolates of TRV have been reported in potato, stem-mottle disease is apparently usually caused by infection with NM isolates.

A sequence of 5987 nucleotides is reported for the RNA of potato leafroll luteovirus (PLRV). The sequence contains six large open reading frames, and non-coding regions of 174 nucleotides at the 5' end, 141 nucleotides at the 3' end and 197 nucleotides between two large blocks of coding sequences. The 5' coding region encodes two polypeptides of 28,000 (28K) and 70K which overlap in different reading frames and circumstantial evidence suggests that the third open reading frame in the 5' block is translated by frameshift readthrough near the end of the 70K polypeptide to give a 118K polypeptide. The C-terminal part of the 118K protein contains the consensus sequence for RNA-dependent RNA polymerases. In vitro translation of PLRV RNA resulted in the synthesis mainly of 28K and 70K polypeptides and the largest product made was about 125K; these sizes are similar to those predicted for the translation products of the 5' block of coding sequence. The 3' block of coding sequence codes for three polypeptides: a 23K coat protein, a 17K polypeptide which is encoded in a different frame, and a 53K polypeptide which immediately follows the coat protein coding sequence, and is in the same reading frame. Circumstantial evidence suggests that the 53K polypeptide is translated by readthrough of the amber termination codon of the coat protein gene. The amino acid sequences encoded by the 3' block of coding sequence show many similarities with analogous polypeptides translated from the nucleotide sequences of RNA of barley yellow dwarf virus, PAV strain (BYDV) and, in particular, beet western yellows virus (BWYV). The 118K polypeptide has some similarities with the putative polymerase of southern bean mosaic virus and much more extensive similarities with the corresponding BWYV polypeptide but almost none with that of BYDV. In contrast, the amino acid sequence of the 28K polypeptide is not like that of proteins of the other luteoviruses or of viruses in other groups. The nucleotide sequences reported will appear in the EMBL, GenBank and DDBJ databases under the accession number X14600.

Discusses variations in whitefly-transmitted geminiviruses or the begomovirus. Description of geminiviruses; Serological relationships; Antigenic variation based on geography; Details on begomovirus genomes.