Functional Analysis of Cymbidium Ringspot Virus Genome

Dalmay, Tamás; Rubino, Luisa; Burgyán, József; Kollàr, Àgnes; Russo, Marcello

doi:10.1006/viro.1993.1310

Cited by 97 publications

(132 citation statements)

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“…For instance, all tombusvirus DI RNAs (as well as the genomic RNAs from which they are derived) terminate at the 39 end with -GCAGCCC, whereas the CymRSV sat RNA terminates with -CAACCC . Mutational studies have shown that genomic and DI RNAs tolerate only deletion of the terminal sequence -CCC, whereas deletion of the G residue at position 24, or even substitution with an A residue, which does not alter the secondary structure of the 39 region, abolishes infectivity (Dalmay et al, 1993a;Pantaleo et al, 2003). In fact, it was suggested that minus-strand synthesis may initiate at the 24 position (Dalmay et al, 1993b).…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Several small tissue pieces showing clear signs of infiltration were excised after 24-48 h and nucleic acids were extracted as described previously (Rubino et al, 2001). Preparation of protoplasts from fully expanded leaves, transfection and RNA analysis were done as previously described (Dalmay et al, 1993a;Rubino et al, 2001). Protoplasts were transfected with full-length CIRV genomic RNA (Burgyan et al, 1996) or deletion mutants DB/H (see above) or DB/N (obtained by restriction of the CIRV full-length clone with BamHI and NcoI at nt 3870, at the beginning of ORF4), together with DI (Burgyan et al, 1992) or sat (Rubino et al, 1990) RNA in vitro transcripts.…”

Section: Methodsmentioning

confidence: 99%

Section: The Replication Of Cymrsv Sat Rna With Defective Helper Genomesmentioning

confidence: 99%

“…Approximately 10 6 cells were transfected with 50 ng of in vitro-synthesized DI or sat RNA together or not with the helper genome (200 ng) or with water, as described previously (Dalmay et al, 1993a). Protoplasts were incubated for 24 h at room temperature (22-25 uC) under continuous fluorescent light, then disrupted and extracted with phenol.…”

Section: The Replication Of Cymrsv Sat Rna With Defective Helper Genomesmentioning

confidence: 99%

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Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

Rubino¹,

Pantaleo²,

Navarro³

et al. 2004

Journal of General Virology

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Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer. INTRODUCTIONTombusviruses are small isometric plant viruses belonging to the genus Tombusvirus, family Tombusviridae. Their genome is a monopartite, single-stranded, positive-sense RNA of 4800 nt containing five ORFs. ORF1 and -2 encode the replicase proteins of 33 or 36 kDa (p33 or p36), depending on the species, and the corresponding readthrough products, p92 and p95. ORF3 encodes the coat protein (CP), and two nested ORFs (ORF4 and -5) encode two proteins involved in virus movement and pathogenesis Silhavy et al., 2002). Tombusvirus infections are often associated with small parasitic RNAs, which can be either defective interfering (DI) or satellite (sat) RNAs. DI RNAs are shortened forms of genomic RNA deprived of all viral genes required for replication, encapsidation and movement of the viral genome. They are generated by errors of the viral replicase, which bypasses local intramolecular base-paired regions of genomic RNA, thus leading to the synthesis of deletion mutants. All described tombusvirus DI RNA molecules contain segments of viral RNA, derived from the 59 leader sequence, the replicase and movement protein genes and the 39 noncoding sequence. Tombusviruses can support the replication of homologous or heterologous DI RNA in plant (Havelda et al., 1998) or yeast cells (Pantaleo et al., 2003(Pantaleo et al., , 2004Panavas & Nagy, 2003). Tombusvirus DI RNAs have been widely used for the analysis of cis-acting factors directing the replication of viral RNA both in vivo Chang et al., 1995;Ray & White, 1999White & Morris, 1994;Wu & White, 1998;Wu et al., 2001) and in vitro (Nagy & Pogany, 2000; Panavas et al., 2002a, b;Panavas & Nagy, 2003).The molecular biology of tombusvirus sat RNAs has been much less studied. So far, the best characterized sat RNA is the 621 nt sat RNA of Cymbidium ringspot virus (CymRSV) whose cis-acting sequence requirements essential for replication and the significance and ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: The Replication Of Cymrsv Sat Rna With Defective Helper Genomesmentioning

confidence: 99%

Section: The Replication Of Cymrsv Sat Rna With Defective Helper Genomesmentioning

confidence: 99%

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Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

Rubino¹,

Pantaleo²,

Navarro³

et al. 2004

Journal of General Virology

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The Enigma of pX: A Host-Dependentcis-Acting Element with Variable Effects on Tombusvirus RNA Accumulation

Scholthof

Jackson

1997

Virology

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Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.

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Nucleotide Sequence and Infectivity of a Full-Length cDNA Clone of Panicum Mosaic Virus

et al. 1998

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The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.

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Functional Analysis of Cymbidium Ringspot Virus Genome

Cited by 97 publications

References 0 publications

Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

The Enigma of pX: A Host-Dependentcis-Acting Element with Variable Effects on Tombusvirus RNA Accumulation

Nucleotide Sequence and Infectivity of a Full-Length cDNA Clone of Panicum Mosaic Virus

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