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L Rubino

Publications and source records attributed to L Rubino.

27 records · Page 2Linked to original sources
PubMed1993

Functional analysis of cymbidium ringspot virus genome.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5' proximal ORFs encode proteins of 33 and 92 kDa and the three 3' proximal ORFs encode proteins of 41, 22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3' nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF5 (19-kDa protein) and ORF6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

Base Sequence↗
PubMed1993

Resistance to cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing the virus coat protein gene.

Transgenic Nicotiana benthamiana plants expressing the coat protein gene of cymbidium ringspot virus (CyRSV) were tested for resistance against infection with CyRSV. Transgenic plants showed resistance to infection only when the purified virions concentration in the inoculum was as low as 0.05 micrograms/ml. No protection was observed in transgenic plants inoculated with virion concentrations of 0.5 and 5.0 micrograms/ml or when the inoculum was in vitro synthesized genomic RNA.

Base Sequence↗
PubMed1992

Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNA.

A full-length DNA copy of cymbidium ringspot virus (CyRSV) satellite RNA was cloned downstream of the bacteriophage T7 RNA polymerase promoter. In vitro transcripts were biologically active in plants when coinoculated with the helper virus or its RNA. Although the transcripts contained 7 or 29 extra nucleotides at the 3' end, the proper 3' terminus was restored in the satRNA progeny. Full-length cDNA clones of CyRSV satRNA under the control of the cauliflower mosaic virus 35S promoter and terminator were used to transform Nicotiana benthamiana plants. Integration of CyRSV satRNA sequence in the plant genome was tested by PCR amplification of DNA extracts from transformed plants and by detection of satRNA-related transcripts in total RNA extracts. Inoculation of transgenic plants with the helper virus induced replication of satRNA of the same size as the native molecule. Sequence analysis of the satRNA progeny showed that it was identical to natural CyRSV satRNA. Infected transgenic plants were not protected from apical necrosis and death by the presence of satRNA sequences. Rather, replication of satRNA was found to suppress accumulation of defective interfering RNA, which acts in the absence of satRNA as an attenuator of virus replication and disease.

Base Sequence↗
PubMed1992

The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules.

A DNA copy of DI RNA of cymbidium ringspot tombusvirus was cloned downstream of a phage T7 promoter. In vitro-transcribed RNA replicated in Nicotiana clevelandii when co-inoculated with full-length viral genomic RNA transcripts and protected plants from apical necrosis. Artificial deletion mutants derived from the DI RNA clone showed that most of the central sequence block is necessary for replication. Hybrid DI RNA-satRNA clones were prepared and in vitro-synthesized RNA was inoculated to plants in the presence of helper viral RNA. There was replication only of in vitro transcripts derived from hybrid clones where satRNA sequences were inserted upstream or downstream from the central block, but not of those derived from clones where satRNA sequence replaced the central block. Progeny RNA of biologically active clones was either full-length or showed deletions depending on the insertion of satRNA sequences in DI RNA. DI RNA-satRNA constructs having part of the 5' region exchanged were not replicated.

Base Sequence↗
PubMed1991

De novo generation of cymbidium ringspot virus defective interfering RNA.

Nicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low Mr RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.

Base Sequence↗
PubMed1990

Sequence analysis of cymbidium ringspot virus satellite and defective interfering RNAs.

The nucleotide sequences of cloned cDNA copies of satellite and defective interfering (DI) RNAs of cymbidium ringspot virus were determined. DI RNA is 499 nucleotides long and is composed of six stretches of sequence derived from CyRSV genomic RNA. Four of these stretches share common 5' sequences and the 5' and 3' ends are identical to those of genome RNA. Satellite RNA is 621 nucleotides long. In some regions the sequence is very similar (60% to 100% identical) to genomic RNA. A consensus sequence is proposed to be involved in the replication of genomic, DI and satellite RNAs.

Base Sequence↗
PubMed1990

Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus.

The two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of Mr 39,636 resembling in composition those of other large nepovirus satellite RNAs, a 5' leader sequence of 16 nucleotides and a 3' non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.

Amino Acid Sequence↗
PubMedDate not supplied

Replication and movement of a coat protein mutant of cymbidium ringspot tombusvirus.

The spread of cymbidium ringspot tombusvirus (CyRSV) in host tissue was studied by using a coat protein gene mutant with a six-nucleotide deletion; the deletion removes two amino acids from the shell domain (S) of the capsid protein. Mutated protein subunits were synthesized in infected cells but could not assemble into virus particles. Virions were formed, however, with inoculation of mutated RNA in transgenic plants expressing normal CyRSV coat protein. The mutant is restricted in long-distance movement in Nicotiana clevelandii, whereas it spreads systemically in N. benthamiana. These results suggest that tombusviruses may spread either as complete virions or in a nonvirion form, depending on the host plant species.

Base Sequence↗