Characterization and localization of the viral RNA replication complex in tobacco plants infected with cucumber mosaic virus (original) (raw)
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FEBS letters, 1991
Both positive [(+)] and negative [(-)] sense versions of two satellite RNA (satRNA) genes from cucumber mosaic virus (CMV), the necrogenic I17N and the nonnecrogenic R, have been introduced into the genome of tobacco plants. On infection with satRNA-free CMV, satRNA was amplified in plants expressing each of the four genes. All four genes confer protection against CMV infection. However, co-inoculation of plants with viral RNA and CMV satRNA transcripts synthesized in vitro showed that (-) sense transcripts were less active than the corresponding (+) sense transcripts. This is the first report that (-) sense CMV satRNA transcripts can serve as a template for satRNA replication.
Japanese Journal of Phytopathology, 1990
Cucumber mosaic virus (CMV) RNA replication complex was prepared from a crude membrane fraction of infected tobacco leaves and was further purified by a zwitterionic detergent, Zwittergent, and Sepharose 4B column chromatography. The active fraction was found at void volume of the column and in the precipitate after following ultracentrifugation. In vitro products synthesized by the enzyme fraction as well as by the crude enzyme were full-length viral RNAs 1-4, suggesting that the characteristic of the enzyme essentially unchanged by the Zwittergent treatment. Analysis of proteins labeled with radioactivity in CMV-infected tobacco leaf disks showed that the enzyme fraction contained three infection-specific proteins, Mr 105,000, Mr 32,000 and Mr 25,000, which comigrated with viral 1a, 3a and coat proteins, respectively, synthesized in vitro. However, a protein corresponding to 2a protein encoded by CMV RNA 2 was not identified in in vitro translation system nor in the enzyme fraction. Considering our previous results that inoculation of RNAs 1 and 2 is necessary to induce replicase activity, this result implies that at least la protein is closely related to the replicase activity, while the role of 2a protein in RNA replication is yet to be solved.
Journal of Virology, 2002
The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco ( Nicotiana tabacum ) and cucumber ( Cucumis sativus ). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, h...
RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves
The EMBO Journal, 1984
*To whom reprint requests should be sent Communicated by M. Yaniv Cauliflower mosaic virus (CaMV) is a plant DNA virus with an 8-kb circular double-stranded genome. CaMV-specific DNA and RNA molecules present in infected Brassica cells share some structural features with DNAs and RNAs of retroviruses and hepatitis B virus. This led to the hypothesis that CaMV replication occurs via reverse transcription of an RNA intermediate. Here we report the first characterization of a new DNA polymerase activity, specific to CaMVinfected tissues. A subcellular fraction of infected cells shows capacity to copy poly(C) and the heteropolymeric regions of natural mRNAs. Chromatographic isolation of the poly(C)dependent activity clearly establishes that it is distinct from the classical 'y-like DNA polymerases previously described in plant cells. The significant homology observed between defined regions of the Moloney murine leukemia virus (MMLV) polymerase and CaMV unassigned gene V product favors the idea that the reverse transcriptase-like DNA polymerase detected in infected cells is a virus-encoded enzyme. Key words: RNA-dependent DNA polymerase/replication/ CaMV/unassigned gene V/homology CaMV-MMLV DNA animal virus whose genome shares some structural features with the genome of CaMV, can replicate by reverse transcription of an RNA intermediate (Summers and Mason, 1982). This led to the proposal that replication of CaMV might also occur by reverse transcription (Guilley et at., 1983; Hull and Covey, 1983b; Pfeiffer and Hohn, 1983). The following observations support this model: (i) the major polyadenylated virus-specific transcript (8.2-kb RNA), complementary to the ca strand, spans the entire genome with a terminal redundancy of 180 nucleotides (Covey et at., 1981; Guilley et al., 1982); (ii) small single-stranded DNA molecules complementary to the major transcript extend counterclockwise from the discontinuity Al in CaMV DNA to the 5' end of this large viral 8.2-kb RNA. They also bear ribonucleotides at their 5' ends (Covey et al., 1983; Guilley et al., 1983) and other unencapsidated molecules can also be taken as evidence consistent with the reverse transcription model (Hull and Covey, 1983c); (iii) there is perfect complementarity between the 14 nucleotides just beyond Al and the 3' end of wheat and bean initiator tRNAMet (Guilley et al., 1983; Hull and Covey, 1983b; Pfeiffer and Hohn, 1983); (iv) nucleoprotein complexes extracted from infected plants support CaMV DNA synthesis, which is partially resistant to actinomycin D and partially impaired by RNase treatment (Pfeiffer and Hohn, 1983). Preliminary data we obtained had suggested the existence of a peculiar DNA polymerase activity in subcellular extracts from infected cells (Modjtahedi, 1982). Here, the characterization of an RNA-dependent DNA polymerase present only in CaMV-infected cabbage cells is reported. We have also examined the possibility that this enzyme could be encoded by the unassigned reading frame V of the CaMV genome.
Journal of General Virology, 1994
Both positive [(+)] and negative [(-)] sense versions of two satellite RNA (satRNA) genes from cucumber mosaic virus (CMV), the necrogenic I17N and the nonnecrogenic R, have been introduced into the genome of tobacco plants. On infection with satRNA-free CMV, satRNA was amplified in plants expressing each of the four genes. All four genes confer protection against CMV infection. However, co-inoculation of plants with viral RNA and CMV satRNA transcripts synthesized in vitro showed that (-) sense transcripts were less active than the corresponding (+) sense transcripts. This is the first report that (-) sense CMV satRNA transcripts can serve as a template for satRNA replication.
Journal of General Virology, 1995
A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl //-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS--polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also tem-plate-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.
Virology, 2013
Satellite RNAs (satRNA) associated with Cucumber mosaic virus (CMV) have been shown to generate multimers during replication. We have discovered that multimers of a CMV satRNA generated in the absence of its helper virus (HV) are characterized by the addition of a hepta nucleotide motif (HNM) at the monomer junctions. Here, we evaluated the functional significance of HNM in HV-dependent replication by ectopically expressing wild type and mutant forms of satRNA multimers in planta either in (+) or (−)-strand polarity. Comparative replication profiles revealed that (−)-strand multimers with complementary HNM (cHNM) are the preferred initial templates for HV-dependent replication than (−)-strand monomers and multimers lacking the cHNM. Further mutational analyses of the HNM accentuate that preservation of the sequence and native length of HNM is obligatory for efficient replication of satRNA. A model implicating the significance of HNM in HV-dependent production of monomeric and multimeric forms of satRNA is presented.► SatRNA mutilmers with hepta nucleotide motif (HNM) are produced in the absence of its helper virus. ► (−)-strand multimers with HNM are better templates than (−)-strand monomers. ► Preservation of the HNM sequence and length is obligatory for efficient replication.
Formation of multimers of cucumber mosaic virus satellite RNA
The Journal of general virology, 1997
Double-stranded RNA multimers of cucumber mosaic virus (CMV) satellite RNA were detected in CMV-infected plants. RT-PCR showed that plus-sense and minus-sense monomers and plus-sense multimers of satellite RNA were present. Multimeric minus-sense RNA was not present except in the form of multimeric dsRNA. Sequence analysis of 52 cloned junction regions in head-to-tail repeats of unit-length satellite RNA indicated that about 35% of the junction sequences were precise fusions of monomer units, 56% lacked sequence of the 5' component, and 10% lacked sequence of both 3' and 5' components. No junction contained additional nucleotides. Deletions at the junction regions may have accumulated during CMV multiplication in inoculated plants. These data suggest that replicase is not released from the template during synthesis of multimeric molecules of satellite RNA.
Journal of General Virology, 2005
The genome of Cucumber mosaic virus consists of three single-stranded RNA molecules, RNAs 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome and have been implicated in movement of the viral RNAs in plants. The 3a movement protein (MP), encoded by RNA 3, is essential for transferring the RNA genomes from infected cells to adjacent cells across the plasmodesmata. Far-Western analysis demonstrated that bacterially expressed 2a polymerase protein directly interacted with the MP. Interaction was confirmed in a yeast two-hybrid assay, and co-immunoprecipitation analysis showed that the MP interacted only with the 2a polymerase protein. A yeast three-hybrid assay showed that the 1a-2a protein interaction relevant for replicase complex formation was not affected by the MP. Although the MP has no affinity for the 1a protein, it interacted indirectly with the 1a protein via the 2a polymerase protein. These results suggest that the replicase complex may be involved in movement through its interaction with the MP.
Journal of virology, 1997
A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein...