Formation of multimers of cucumber mosaic virus satellite RNA (original) (raw)
Related papers
Nucleic Acids Research, 1983
Sat-RNA is one of several replicating satellite RNAs which have been isolated from RNA encapsidated in cucumber mosaic virus (CMV) and which are totally dependent on CMV for replication. The 336 residue sequence of Sat-RNA obtained using the dideoxynucleotide chain termination and partial enzymic digestion procedures shows only a few short stretches (up to 11 residues) of sequence homology with one of the three CMV genomal RNAs so far sequenced. Sat-RNA has 88% sequence homology with another, previously sequenced, satellite RNA of CMV, CARNA 5. Analysis of partial digests of 5'or 3'-32P-Sat-RNA with nuclease S1 or RNase T1 under non-denaturing conditions showed that only about 10% of the residues in Sat-RNA were cleaved. Further data on base-paired segments of Sat-RNA were obtained using digestion with RNase T1 followed by electrophoretic fractionation of the resulting fragments under both non-denaturing and denaturing conditions. On the basis of this data, a complete secondary structure model is proposed for Sat-RNA with 52% of its residues involved in base pairs. A prominent hairpin at the 3'-terminus of Sat-RNA shows considerable sequence and structural homology with parts of the 3'-terminal tRNA-like structure of the CMV genomal RNAs. I NTRODUCT ION Several satellite RNAs have been found encapsidated with viral RNA in some, but not all, isolates of cucumber mosaic virus (CMV) (1-3). These satellite RNAs are small (about 335 residues) linear molecules and are much smaller than the three CMV genomal RNAs of approximately 4,000, 3,400 and 2,200 residues (RNAs 1 to 3, respectively; ref. 4) with which they have essentially no sequence homology detectable by hybridization analysis with complementary DNA (cDNA) (2,5). They are totally dependent on the helper virus for their replication, encapsidation and transmission. Sequence and structural studies of such satellite RNAs are important for several reasons. They may provide information about their possible origins and, when compared to known sequence and structural features of the helper virus RNAs, about the minimum requirements for viral RNA
Emergence of a new satellite RNA from cucumber mosaic virus isolate P1
Journal of Zhejiang University SCIENCE A, 2003
The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT-PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat-Pl-1 and Sat-P1-2) . Sat-Pl-1 contained 335 nucleotides, and Sat-P1-2 contained 394 nucleotides. These two satRNAs shared 64 % overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat-Pl-1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat-P1-2 represents the emergence of a new satellite ( necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.
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.
2012
Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.
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.
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.
Host-specific encapsidation of a defective RNA 3 of Cucumber mosaic virus
Journal of General Virology, 2004
Defective (D) RNAs were generated in tobacco upon passage of two isolates of Cucumber mosaic virus (CMV) initially derived from RNA transcripts of cDNA clones. In both cases, the D RNA was derived by a single in-frame deletion of either 339 or 411 nt within the 3a gene of Fny-CMV RNA 3 or M-CMV RNA 3, respectively. The generation of D RNAs was rare and occurred with two CMV isolates, the virions of which were known to differ in physico-chemical properties. The Fny-CMV D RNA 3, designated D RNA 3-1, was maintained by passage together with Fny-CMV in tobacco, but was lost by passage in squash. D RNA 3-1 accumulated in the inoculated squash cotyledons but not in upper, systemically infected leaves. Virions purified from infected squash cotyledons or leaf mesophyll protoplasts did not contain D RNA 3-1. Therefore, the failure of D RNA 3-1 to accumulate in squash leaves systemically infected by CMV was due to a lack of encapsidation of the D RNA 3-1 and movement out of the inoculated leaves.
Journal of General Virology, 1995
A cucumber mosaic virus (CMV-Ix) from Ixora is unusual in that it does not support the accumulation of some well-characterized CMV satellite RNAs in plants. CMV-Ix can support a particular satellite RNA variant which causes lethal tomato necrosis when inoculated with other CMV strains but not when inoculated with CMV-Ix. This difference in ability to support accumulation of specific satellite variants is apparent even when their sequences differ by only 10 nucleotides. Electroporation of tomato protoplasts with combinations of CMV-Ix or CMV-1 RNA plus the same satellite variants showed similar differences in accumulation, indicating a defect in satellite RNA replication and not movement or encapsidation. Pseudorecombinant virus infections between CMV-1 and CMV-Ix indicated that the genomic determinants responsible for this phenotype reside on RNA 1 since only combinations with CMV-Ix RNA 1 failed to replicate satellite RNA. The complete genome of CMV-Ix was cloned, sequenced and compared with the genomes of other cucumoviruses. CMV-Ix is most similar in RNA and protein sequence to subgroup 1 CMV-Fny and CMV-Y but slightly less similar than they are to each other. CMV-Ix and all cucumovirus strains sequenced thus far share a domain in the 3' untranslated portion of their genomic RNAs in which 39 of 40 bases are completely conserved.
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.