Detection and subcellular localization of the turnip yellow mosaic virus 66K replication protein in infected cells.

Turnip yellow mosaic virus (TYMV) encodes a 206-kDa (206K) polyprotein with domains of methyltransferase, proteinase, NTPase/helicase, and RNA-dependent RNA polymerase (RdRp). In vitro, the 206K protein has been shown to undergo proteolytic processing, giving rise to the synthesis of 140-kDa (140K) and 66-kDa (66K) proteins, the latter comprising the RdRp protein domain. Antibodies were raised against the 66K protein and were used to detect the corresponding viral protein in infected cells; both leaf tissues and protoplasts were examined. The antiserum specifically recognized a protein of approximately 66 kDa, indicating that the cleavage observed in vitro is also functional in vivo. The 66K protein accumulates transiently during protoplast infection and localizes to cellular membrane fractions. Indirect immunofluorescence assays and electron microscopy of immunogold-decorated ultrathin sections of infected leaf tissue using anti-66K-specific antibody revealed labeling of membrane vesicles located at the chloroplast envelope.

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system.

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.

Molecular cloning and characterization of the Arabidopsis thaliana alpha-subunit of elongation factor 1B.

Using a PCR-based approach, we have isolated two Arabidopsis thaliana cDNA clones (alpha1 and alpha2) encoding the alpha-subunit of translation elongation factor 1B (eEF1Balpha). They encode open reading frames of 228 and 224 amino acids respectively, with extensive homology to eEF1Balpha subunits from different organisms, particularly in the C-terminal half of the protein. They both lack a conserved phosphorylation site that has been implicated in regulating nucleotide exchange activity. Using a plasmid shuffling experiment, we demonstrated that both alpha1 and alpha2 clones are able to complement a mutant yeast strain deficient for the eEF1Balpha subunit. This provides evidence that Arabidopsis encodes at least two functional isoforms of this subunit, termed eEF1Balpha1 and eEF1Balpha2. A third cDNA clone was isolated that appeared to result from an alternative splicing event of the eEF1Balpha1 gene.

Genetic analysis of the monopartite tomato yellow leaf curl geminivirus: roles of V1, V2, and C2 ORFs in viral pathogenesis.

Tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted geminivirus with a monopartite genome. We have investigated the functions of the V1, V2, and C2 ORFs by mutational analysis. We analyzed the ability of TYLCV mutants containing disrupted ORFs V1, V2, or C2 to replicate, spread, and cause symptoms in Nicotiana benthamiana and tomato plants. All the mutants retained the capability of autonomous replication in protoplast-derived cells of tomato and leaf discs of N. benthamiana, although both V1 and V2 gene products appeared to play a role in the accumulation of viral single-stranded DNA. In contrast, none of the mutants was able to systemically infect tomato plants, demonstrating that the V1, V2, and C2 gene products are all required for a successful infection process in this host. The effect of the mutation in ORF C2 appeared to be host-specific, since N. benthamiana plants were systemically infected, although symptom development was attenuated.

Activation of the CaMV as-1 cis-element by salicylic acid: differential DNA-binding of a factor related to TGA1a.

Transcription from the as-1 element of the cauliflower mosaic virus is induced by salicylic acid (SA), an endogenous signal involved in plant defence responses. Electrophoretic mobility shift assays demonstrated that the binding of a tobacco cellular factor, named SARP, correlates with the SA-induced activation of transcription. SARP was shown to contain proteins immunologically related to TGA1a, a transcription factor previously cloned for its ability to bind to the as-1 element. The molecular mass of SARP was estimated to be 40 kDa by South-Western experiments. Treatment of the extracts with dissociating agents led to an increase in the DNA binding activity, suggesting the presence of an inhibitor that sequesters SARP. The DNA binding activity appeared sensitive to phosphatase treatment, suggesting a role for phosphorylation in the SA-induced gene activation. These results represent an analysis of immediate early response to SA and potentially elucidate the events of the SA signal transduction pathway.

DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus.

The in vitro DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus (TYLCV) was studied following its expression in Escherichia coli as a fusion protein with an His tag N-terminal extension (His-C2). Southwestern blotting experiments demonstrated that the C2 protein is able to bind both single-stranded and double-stranded DNA probes. In electrophoretic mobility shift assays performed using purified protein and single-stranded DNA probes several shifted complexes were formed. The presence of NaCl (up to 800 mM) did not substantially affect binding profiles, demonstrating a stable interaction. His-C2 appeared to bind single-stranded DNA in a sequence-nonspecific manner, with a preference for single-stranded compared to double-stranded DNA. Deletion mutants demonstrated that the central core of C2 (amino acids 33 to 104), which contains a Cys-His rich region, is sufficient for conferring binding activity. The potential significance of this DNA-binding activity with respect to possible biological functions of TYLCV C2 protein is discussed.

Identification of the nicking tyrosine of geminivirus Rep protein.

The replication initiator (Rep) proteins of geminiviruses perform a DNA cleavage and strand transfer reaction at the viral origin of replication. As a reaction intermediate, Rep proteins become covalently linked to the 5' end of the cleaved DNA. We have used tomato yellow leaf curl virus Rep protein for in vivo and in vitro analyses. Isolating a covalent peptide-nucleotide complex, we have identified the amino acid of Rep which mediates cleavage and links the protein to DNA. We show that tyrosine-103, located in a conserved sequence motif, initiates DNA cleavage and is the physical link between geminivirus Rep protein and its origin DNA.

DNA replication specificity of TYLCV geminivirus is mediated by the amino-terminal 116 amino acids of the Rep protein.

Geminiviruses are plant DNA viruses replicating by a rolling circle mechanism. We have investigated the specificity of replication origin recognition of two different isolates of tomato yellow leaf curl virus (TYLCV). Here, we show that TYLCV-Sardinian and -Israeli replication proteins display a high degree of specificity for their respective origins. The DNA sequences recognized are located on the left part of the intergenic region whereas the amino-terminal 116 amino acids of the Rep protein determine the specificity of origin recognition.

Geminivirus replication: genetic and biochemical characterization of Rep protein function, a review.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'replication initiator protein' (Rep) which is the sole viral protein essential for replication. Rep protein catalyzes multiple reactions during the reproductive cycle of the virus. Here we summarize the recent advances of in vivo and in vitro analyses of the various Rep protein activities in a model for replication initiation and termination. In addition, the position of the geminivirus Rep protein within a general context of bacterial and mammalian replication initiator proteins is discussed.

Movement of tomato yellow leaf curl geminivirus (TYLCV): involvement of the protein encoded by ORF C4.

Tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted geminivirus with a monopartite genome. We have investigated the ability of a TYLCV DNA mutant containing a disrupted ORF C4 to infect Nicotiana benthamiana and tomato plants. The mutant retained the capability of autonomous replication in protoplast-derived cells of tomato and was able to infect N. benthamiana plants systemically although DNA levels were reduced and symptom development was attenuated. However, when tomato plants were inoculated, the virus was unable to move systemically unless a second site mutation or a reversion in planta restored the integrity of ORF C4. The infected plants remained asymptomatic or showed very mild symptoms. The results strongly suggest that the ORF C4 encodes a protein involved in virus movement, a novel finding for whitefly-transmitted geminiviruses. The involvement of a C4 protein in symptom determination is discussed.

Two proteins encoded by beet necrotic yellow vein virus RNA 3 influence symptom phenotype on leaves.

RNA 3 of the beet necrotic yellow vein virus (BNYVV) quadripartite RNA genome is not essential for virus multiplication on leaves of Tetragonia expansa but has dramatic effects on symptom expression. Virus isolates containing RNA 3 produce bright yellow local lesions while isolates lacking RNA 3 produce much milder symptoms. Using directed mutagenesis of cDNA clones followed by in vitro synthesis of biologically active transcripts, a 25 kDa open reading frame (ORF) of RNA 3 was shown to be responsible for the yellow local lesion phenotype. In addition, two deletion mutants of RNA 3 were found to elicit the appearance of severe necrotic local lesions. Analysis of one of these mutants revealed that necrosis was due to the overexpression of a second short ORF, N, overlapping the 3'-terminal portion of the 25 kDa ORF. As shown by gene fusion studies, gene N is not detectably expressed from full-length RNA 3 but is translationally activated by deletion of upstream sequences. Introduction of gene N into the genome of the unrelated DNA virus, cauliflower mosaic virus, elicits a necrotic response instead of the typical mosaic symptoms, demonstrating that gene N can induce necrosis outside of the context of a BNYVV infection.

Shortened forms of beet necrotic yellow vein virus RNA-3 and -4: internal deletions and a subgenomic RNA.

Beet necrotic yellow vein virus RNA-3 and RNA-4, produced as full-length biologically active transcripts in vitro, can undergo spontaneous internal deletions when inoculated onto Chenopodium quinoa leaves along with RNA-1 and -2. The deletion process is specific, giving rise to only a few major species, and can be rapid; deleted forms appear after only one or two passages in leaves. In one of the shortened forms of RNA-4, the deletion precisely eliminated one copy of a 15 nucleotide (nt) direct sequence repeat from the full-length prototype sequence, suggesting that 'copy-choice' switching of the replicase-template complex from one repeat to the other during RNA replication was responsible for the generation of this deletion. The deletion found in a major shortened form of RNA-3, on the other hand, did not occur near sequence repeats but began with GU and ended with AG like a nuclear intron sequence. Thus it is possible that the deleted sequence has been removed by splicing. However, two other deletions that were characterized were not associated with either of these types of sequence feature. An approximately 600 nt 5'-terminally truncated non-encapsidated form of RNA-3 was also detected in infected plant tissue. The evidence suggests that it is a subgenomic RNA derived from RNA-3.

Mapping sequences required for productive replication of beet necrotic yellow vein virus RNA 3.

Of the four genome components of beet necrotic yellow vein virus only RNAs 1 and 2 are essential for viral replication in leaves. We have mapped cis-regulatory elements on RNA 3 by introducing deletions into expressible cDNA clones and inoculating leaves with the altered transcripts along with RNAs 1 and 2. Transcripts carrying internal deletions extending to within 69 residues of the 3' poly(A) tail or to within about 300 residues of the 5' terminus were efficiently amplified and encapsidated in vivo. The 3' terminal cis-essential domain can be folded into a secondary structure which is conserved among all four genomic RNAs and which probably contains the minus-strand promoter. RNA 3 transcripts with 75% of the central core of the sequence deleted or replaced by the beta-glucuronidase (GUS) gene were also viable. GUS activity was detected in infected tissue in the latter case.

Multiplication of beet necrotic yellow vein virus RNA 3 lacking a 3' poly(A) tail is accompanied by reappearance of the poly(A) tail and a novel short U-rich tract preceding it.

Beet necrotic yellow vein virus RNAs 1 and 2 but not RNAs 3 and 4 are required for viral multiplication in Chenopodium quinoa leaves. Elimination of the 3' poly(A) tail from RNA 3 transcripts markedly attenuated their ability to be amplified when co-inoculated with RNAs 1 and 2 to this host. Successful multiplication of the tailless RNA 3 was accompanied by the reappearance of new 3' poly(A) tails on the progeny. The evidence suggests that the newly acquired poly(A) sequence results from the action of a poly(A) polymerase rather than recombination with the homologous 3' terminal domains of RNAs 1 or 2. An unexpected feature of these progeny RNA 3 molecules was the presence of a novel short heterogenous U-rich tract separating the poly(A) tail from the 3' end of the heteropolymeric RNA 3 sequence proper.