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Contigs with the highest sequence similarity (73%) to Apricot pseudo-chlorotic leaf spot virus (genus Trichovirus, family Betaflexiviridae) were identified by high-throughput sequencing from a symptomless sweet cherry accession. The complete genome sequence of this new virus is 7460 nucleotides, excluding the 3' poly(A) tail. Its genome organization is very similar to several trichoviruses infecting fruit trees, with three open reading frames encoding putative replicase, movement protein and coat protein (CP). The virus shares amino acid sequence identities of 60-73% at replicase and 53-76% at CP with other trichoviruses. Phylogenetic analyses group it and other trichoviruses in a cluster. These results support that this virus, which is tentatively named cherry latent virus 1, should be considered a new member in the genus Trichovirus.

Petunia plants are used for urban landscaping in many parts of the world, including South Korea. In this study, we aimed to investigate the occurrence of petunia vein clearing virus (PVCV) infection in petunia plants in Seoul, South Korea. PVCV was detected from 23 of 79 petunia samples collected from Seoul. We obtained the complete genome sequences of the Korean isolates in this study (called PVCV-Kr, Kr2, and Kr3), which were compared with the genome sequence of the USA isolate of the virus (PVCV-USA). The genomic DNA of the three PVCV isolates was found to comprise 7210-7267 nucleotides (nts), which is 4-15 nts longer than the PVCV-USA genome. The genomes of the Kr and Kr2 isolates encode a large polyprotein of 252 kDa (2180 amino acids (aa)). The genome of the Kr3 isolate encodes a large polyprotein of 255 kDa (2203 aa). The polyprotein has six protein domains: a movement protein (MP; 72 aa), a coiled-coil domain (CC; 33 aa), an RNA-binding domain (RB; 18 aa), a protease (PR; 21 aa), a reverse transcriptase (RT; 196 aa), and an RNase H (RH; 121 aa). The large polyprotein and six domains of the three isolates showed 93.9-100.0% sequence homology with those of PVCV-USA. Furthermore, the polymerase polyprotein gene (PR, RT, and RH) of the four PVCV isolates containing the USA isolate grouped with those of Rice tungro bacilliform virus and Soybean chlorotic mottle virus, which belong to the same family (Caulimoviridae). Our findings suggested that the Korean isolates represent a new isolate of PVCV. To our knowledge, this is the first report of PVCV detection in South Korea.

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Maize Iranian mosaic virus (MIMV; Mononegavirales, Rhabdoviridae, Nucleorhabdovirus) infects maize and several other poaceous plants. MIMV encodes six proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). In the present study, MIMV gene expression and genetic polymorphism of an MIMV population in maize were determined. N, P, P3, and M protein genes were more highly expressed than the 5' terminal G and L genes. Twelve single nucleotide polymorphisms were identified across the genome within a MIMV population in maize from RNA-Seq read data pooled from three infected plants indicating genomic variations of potential importance to evolution of the virus. MIMV N, P, and M proteins that are known to be involved in rhabdovirus replication and transcription were characterized as to their intracellular localization and interactions. N protein accumulated exclusively in the nucleus and interacted with itself and with P protein. P protein accumulated in both the nucleus and cell periphery and interacted with itself, N and M proteins in the nucleus. M protein was localized in the cell periphery and on endomembranes, and interacted with P protein in the nucleus. MIMV proteins show a distinctive combination of intracellular localizations and interactions.

Rice stripe virus (RSV) infects rice and causes great yield reduction in some Asian countries. In this study, rice cDNA library was screened by a Gal4-based yeast two-hybrid system using pc4, a putative movement protein of RSV, as the bait. A number of positive colonies were identified and sequence analysis revealed that they might correspond to ten independent proteins. Two of them were selected and further characterized. The two proteins were a J protein and a small Hsp, respectively. Interactions between Pc4 and the two proteins were confirmed using coimmunoprecipitation. Implications of the findings that pc4 interacted with two chaperone proteins were discussed.

Okra mosaic virus (OkMV) is a tymovirus infecting members of the family Malvaceae. Early infections in okra (Abelmoschus esculentus) lead to yield losses of 12-19.5%. Besides intensive biological characterizations of OkMV only minor molecular data were available. Therefore, we determined the complete nucleotide sequence of a Nigerian isolate of OkMV. The complete genomic RNA (gRNA) comprises 6,223 nt and its genome organization showed three major ORFs coding for a putative movement protein (MP) of M r 73.1 kDa, a large replication-associated protein (RP) of M r 202.4 kDa and a coat protein (CP) of M r 19.6 kDa. Prediction of secondary RNA structures showed three hairpin structures with internal loops in the 5'-untranslated region (UTR) and a 3'-terminal tRNA-like structure (TLS) which comprises the anticodon for valine, typical for a member of the genus Tymovirus. Phylogenetic comparisons based on the RP, MP and CP amino acid sequences showed the close relationship of OkMV not only to other completely sequenced tymoviruses like Kennedya yellow mosaic virus (KYMV), Turnip yellow mosaic virus (TYMV) and Erysimum latent virus (ErLV), but also to Calopogonium yellow vein virus (CalYVV), Clitoria yellow vein virus (CYVV) and Desmodium yellow mottle virus (DYMoV). This is the first report of a complete OkMV genome sequence from one of the various OkMV isolates originating from West Africa described so far. Additionally, the experimental host range of OkMV including several Nicotiana species was determined.

Two tospoviruses, Chrysanthemum stem necrosis virus (CSNV) and Zucchini lethal chlorosis virus (ZLCV), cause economical losses in several ornamental and vegetable crops in Brazil. The nucleocapsid gene and movement protein sequences had already been reported for both viruses, though the glycoprotein precursor gene sequence was not available. In this study, cDNA fragments (ca. 4 kb) of the M RNA 3' portion of CSNV (isolate Chry-1) and ZLCV (isolate 1038), including the complete glycoprotein precursor gene, partial NSm gene, and the entire intergenic and 3' untranslated regions, were cloned and sequenced. The sequences were assembled with the corresponding 5' region sequence (NSm gene and 5'UTR) of the same isolates to build up the complete sequence of the M RNA segment of both species. The M RNA of CSNV was 4,828 nucleotide-long, while of ZLCV 4,836 nucleotides. Both M RNA molecules comprised two ORFs in an ambisense arrangement. The vcRNA coded for the viral glycoprotein (Gn/Gc) precursor gene of CSNV and ZLCV (both with 127.5 kDa). Comparison of deduced amino acids of the CSNV and ZLCV glycoprotein precursor genes with those of other tospoviruses showed the highest identity with that of Tomato spotted wilt virus (86%) and with that of CSNV (82%), respectively. However, the nucleotide sequence of the intergenic and 3' untranslated regions of CSNV and ZLCV shared lower identities with other tospoviruses. The glycoprotein precursor gene is thought to be a good candidate as additional classification parameter for Tospovirus taxonomy. The presence of the RGD motif in both Gc proteins indicated that they are typical American tospoviruses, which was confirmed by phylogenetic analysis. The membrane topology of both glycoproteins is discussed.

The complete genome of a tymovirus infecting Solanum violaefolium was sequenced. The genome comprised 6284 nt, with a 5′-UTR of 137 nt and a comparatively longer 3′-UTR of 121 nt. Sequence analysis confirmed three ORFs encoding a movement protein, a polyprotein, and a coat protein (CP). The isolate was considered to be the Tomato blistering mosaic virus (ToBMV) based on a CP amino acid sequence identity of 95.3 %. The nucleotide sequence of the complete genome of the S. violaefolium isolate, however, differed markedly from the other two reported ToBMV isolates, with identities of 76.6 and 76.3 %, below one of the demarcation criteria of the genus Tymovirus (overall genome identity of 80 %). No recombination signals were detected in the genome of this isolate. The high identity of the CP amino acid sequence and similar host responses suggest that the S. violaefolium isolate belongs to the same species as the Tomato blistering mosaic virus. The sequence analysis of this ToBMV isolate thus suggests that the demarcation criterion of 80 % overall genome sequence identity in the genus Tymovirus may require revision.

Maize rayado fino virus (MRFV) possesses an open reading frame (ORF43) predicted to encode a 43 kDa protein (p43) that has been postulated to be a viral movement protein. Using a clone of MRFV (pMRFV-US) from which infectious RNA can be produced, point mutations were introduced to either prevent initiation from three potential AUG initiation codons near the 5'-end of ORF43 or prematurely terminate translation of ORF43. Inoculation of maize seed via vascular puncture inoculation (VPI) resulted in plants exhibiting symptoms typical of MRFV infection for all mutants tested. Furthermore, corn leafhoppers (Dalbulus maidis) transmitted the virus mutants to healthy plants at a frequency similar to that for wild-type MRFV-US. Viral RNA recovered from plants infected with mutants both prior to and after leafhopper transmission retained mutations blocking ORF43 expression. The results indicate that ORF43 of MRFV is dispensable for both systemic infection of maize and transmission by leafhoppers.

Mungbean yellow mosaic virus-Vigna (MYMV-Vig) is a bipartite geminivirus that causes a severe yellow mosaic disease in blackgram. An assay was developed to study MYMV-Vig replication by agroinoculation of tobacco leaf discs with partial dimers of the virus. This assay, in a non-host model plant, was used to evaluate pathogen-derived resistance contributed by MYMV-Vig genes in transgenic plants. Viral DNA accumulation was optimum in tobacco leaf discs cultured for 10 days after infection with Agrobacterium tumefaciens strain Ach5 containing partial dimers of both DNA A and DNA B of MYMV-Vig. Transgenic tobacco plants with MYMV-Vig genes for coat protein (CP), replication-associated protein (Rep)-sense, Rep-antisense, truncated Rep (T-Rep), nuclear shuttle protein (NSP) and movement protein (MP) were generated. Leaf discs from transgenic tobacco plants, harbouring MYMV-Vig genes, were agroinoculated with partial dimers of MYMV-Vig and analyzed for viral DNA accumulation. The leaf discs from transgenic tobacco plants harbouring CP and MP genes supported the accumulation of higher levels of MYMV-Vig DNA. However, MYMV-Vig accumulation was inhibited in one transgenic plant harbouring the Rep-sense gene and in two plants harbouring the T-Rep gene. Northern analysis of these plants revealed a good correlation between expression of Rep or T-Rep genes and inhibition of MYMV-Vig accumulation.

A study was undertaken to measure the rate of coat protein (CP) independent cell-to-cell movement of cowpea chlorotic mottle bromovirus (CCMV) in three different host plants. A CCMV RNA3 variant in which the CP gene was substituted with enhanced green fluorescent protein (C3/DeltaCP-EGFP) was coinoculated to three different host plants with transcripts of wild type RNAs 1 and 2. Comparative analysis of cell-to-cell movement monitored by the EGFP expression at various days post inoculation revealed that the rate of spread varied with the type of host species inoculated: fastest movement was observed in Nicotiana benthamiana while the rate spread was significantly slower in the natural host cowpea. When CP was expressed as EGFP fusion (C3/CP:EGFP) the rate of spread in N. benthamiana and C. quinoa was slower than that was observed in the absence of CP and remained subliminal in cowpea. Analysis of infection foci by confocal laser scanning microscope revealed that localization of CP:EGFP fusion was distinct in N. benthamiana and C.quinoa and accumulated as fluorescent inclusions at the cell periphery. Additional experiments involving coinoculation of either C3/DeltaCP-EGFP or C3/CP:EGFP with heterologous brome mosaic bromovirus (BMV) genomic RNAs 1 and 2 revealed that, in addition to movement protein and CP, viral replicase also influences cell-to-cell spread. The significance of these results in relation to the mechanism of bromovirus movement is discussed.

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5). This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain. P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids. In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding. These mutations were inserted into the full-length infectious clone. It has been shown that ORF 5 mutations do not affect replication of GVA-RNA. However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.

The 3a protein of Cucumber mosaic virus is essential for the cell-to-cell movement of the viral RNA through plasmodesmata. We have introduced an epitope peptide before the stop codon of the 3a protein and cloned the tagged ORF into a binary vector for Agrobacterium-mediated transformation. The established transgenic tobacco lines produced the 3a protein, which was specifically detected with anti-3a and anti-epitope antisera. Metabolic labeling and subsequent immunoprecipitation revealed that [32P]-orthophosphate was incorporated into the 3a protein. The phosphoamino acid analysis indicated that the 3a protein contained phosphoserine but not phosphothreonine or phosphotyrosine. This is the first demonstration of the 3a protein phosphorylation in planta.

The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA.

Resistance to potato virus X (PVX) is determined by the product of a host resistance gene and a viral determinant specifying either virulence (resistance-breaking ability) or avirulence (resistance sensitivity). The viral coat protein is the determinant of resistance mediated by the host Nx gene while the 25 kDa movement protein is the determinant of Nb-mediated resistance. Group 1 and group 4 strains of PVX are avirulent or virulent respectively for both these determinants while group 2 and group 3 strains are virulent for one but avirulent for the other determinant. There are two alternative evolutionary mechanisms by which the various strain groups might have evolved: either by recombination between strains carrying virulence (or avirulence) determinants that evolved once only, or alternatively, by independent evolution of at least one virulence (or avirulence) determinant in distinct phylogenetic branches. These alternative hypotheses were investigated by (i) determining the complete genomic sequence of a group 1 and a group 4 strain and (ii) comparing the completely sequenced genomes of six isolates representative of the four strain groups. The analysis revealed the same phylogeny for all five PVX genes. Thus, there is no evidence that the PVX strain groups evolved by recombination.

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host. Fil: Trucco, Verónica Milagros. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: de Breuil, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Bejerman, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Lenardon, Sergio Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; Argentina Fil: Giolitti, Fabián José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.

The 3'-terminal sequence of citrus tatter leaf virus lily isolate (CTLV-L) was determined from cloned cDNA. The sequence contains two open reading frames (ORFs). ORF1 encodes a protein that contains consensus sequences associated with the RNA-dependent RNA polymerase. ORF2, which is in a different reading frame within ORF1, can encode a 36 kD protein, putatively identified as a movement protein. CTLV-L coat protein (CP) was found to be located in the C-terminal region of the polyprotein encoded by ORF1. Evolutionary relationships and classification of capilloviruses is discussed.

Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.

N20-RNA 3L, a large form of RNA 3 associated with Alfalfa mosaic virus (AMV) strain N20 comprises 2281 nt and has approximately 97% overall sequence similarity to the longest previously described RNA 3 of AMV strain YSMV (YSMV-RNA 3; 2188 nt). Compared with YSMV-RNA 3, N20-RNA 3L contains an additional 97 nt in the 5' leader upstream of the open reading frames for movement protein (MP) and coat protein (CP). Two overlapping unidentified reading frames (URF1 and URF2) result from this modification, each of which code for putative translation products of 21 amino acids. The URF1 putative peptide has a hydrophilic N-terminus and a hydrophobic C-terminus, indicating a possible association with both host cell membrane and cytosol whereas the putative URF2 product is predominantly hydrophobic. A further structural modification found in N20-RNA 3L is a new tandem repeat of 243 nts which overlaps with the MP open reading frame.

The complete sequence of RNA 3 of the Blencowe, British (B) isolate of tomato aspermy virus (TAV) is presented. The RNA 3 of TAV-B is dicistronic, encoding the putative movement protein 3a and the capsid protein.