1.11.5
INTERACTIONS BETWEEN TOMBUSVIRUSES AND TWO NEWLY DESCRIBED SATELLITE RNAS: A DETERMINANT FOR EFFICIENT SAT RNA ACCUMULATION MAPS TO ORF 1 OF THE VIRAL GENOME

A CELIX1, J BURGYAN2 and E RODRIGUEZ-CEREZO1

1Centro Nacional de Biotecnología (CSIC), Cantoblanco, 28049 Madrid, Spain; 2Agricultural Biotechnology Center, Gödöllo, Hungary

Background and objectives
A recent survey of 60 field isolates of tomato bushy stunt virus (TBSV) obtained from eggplant and tomato plants showed that four isolates contained either a 822-nt-long satellite RNA (sat RNA B1) or a 614-nt-long satellite RNA (sat RNA B10). TBSV sat RNAs B1 and B10 are linear RNA molecules with no apparent coding capacity. This was the first report of sat RNAs associated to tombusvirus natural infections [1]. In this work we have constructed infectious clones of both sat RNAs to study their interaction with different tombusviruses in different host plants.

Materials and methods
Full-length cDNA clones of TBSV sat-RNAs B1 and B10 were obtained by RT-PCR and cloned downstream of the T7 RNA polymerase promotor to obtain in vitro RNA transcripts. Four tombusviruses were used as helper viruses in the form of in vitro transcripts derived from full-length clones: cymbidium ringspot virus (CymRSV), carnation italian ringspot virus (CIRV) and the Cherry (TBSV-Ch) and P (TBSV-P) strains of TBSV. Viral genomic RNAs with or without sat RNA were inoculated into a natural host (eggplant) and two experimental hosts (Nicotiana clevelandii and N. benthamiana). Total RNA extracts were obtained from inoculated leaves and analysed by Northern blot with virus- and sat RNA-specific probes.

Results and conclusions
In vitro transcripts of both sat RNAs were infectious. The presence of sat RNA B10 in the inoculum resulted in a marked attenuation of symptoms regardless of the host plant or helper virus used in the assay. In contrast, sat RNA B1 did not modify symptomatology in any of the helper virus-host combinations tested. When both sat RNas were included in the inoculum, the attenuating effect was dominant although both sat RNA species were detected in the plant. In all cases, the attenuating sat RNA B10 dramatically reduced the accumulation of viral genomic RNA, while sat RNA B1 either had no effect or slightly decreased the levels of viral genomic RNA. In spite of this, for each particular helper virus, both sat RNAs reached similar levels in infected tissues. An interesting exception to this was the observation that CIRV was a poor helper for sat RNA B1, but maintained sat RNA B10 to high levels. A collection of chimaeric viruses derived from the CIRV and CymRSV genomes [2] was assayed in N. benthamiana in an attempt to identify a determinant specific for high accumulation of sat-RNA B1. This determinant has been mapped to the C-terminus of the ORF 1 of the tombusvirus genome. The product of ORF 1, a 33-kDa protein, is involved in virus replication, although its precise role in this process has not been established. Experiments in protoplasts are in progress to confirm that this region of ORF 1 affects replication of sat-RNA B1, and that other processes such as virus transport are not affected.

References
1. Célix A, Rodríguez-Cerezo E, García-Arenal F, 1997. Virology 239, 277-284.
2. Burgyan J, Rubino L, Russo M, 1996. Journal of General Virology 77, 1967-1974.