Cucumovirus satellite RNA: Molecular structure, replication and viral symptom modulation

This dissertation focuses on the cucumoviral satellites, which in this laboratory carry the designation CARNA 5 and PARNA 5 (for cucumber mosaic virus (CMV) satellites and peanut stunt virus (PSV) satellites, respectively). In the first part it deals with how certain features of the molecular structure of these satellites relate to their viral symptom-modulating properties. Via quantitative bioassay of select CARNA 5 recombinant and mutant transcripts it was demonstrated that within the satellite structure more than one sequence element, which may be widely separated along the length of the molecule, is needed for optimal expression of a symptom such as lethal tomato necrosis. These sequence elements probably constitutes a three-dimensional requirement facilitating coordinated action or juxtapositioning of different sequence elements with respect to their interaction site(s) for necrosis induction. These and other experiments on tomato necrosis expression revealed the necessity not only to carefully select each partner of the triad satellite-virus-host plant, but also to control additional variables such as environmental temperature that might affect their trilateral interactions and thereby dramatically influence biological expression.; In the second part of this dissertation the main focus has been on the crucial importance of satellite replication competition in the process of viral symptom modulation. For the first time it was demonstrated that viral replicase isolated from cucumovirus-infected plants can catalyze the complete replication of the cucumovirus-associated satellites CARNA 5 or PARNA 5 with high specificity. This has validated the basic premise of a previously proposed biochemical mechanism which rationalizes the disease regulatory properties of satellites by their competition with the helper viral RNA for the shared but virus-encoded replicase. An apparent replication advantage of CARNA 5 over viral RNA was shown to exist in an in vitro experiment with CMV replicase, while the linkage of such a replication advantage to symptom attenuation followed from experiments with different PARNA variants and PSV replicase. With the experiments involving (+) as well as ({dollar}-{dollar}) stranded CARNA 5 templates, a beginning was also made with the study of recognition signals at the termini of the satellite molecule that facilitate the replication process. In addition, preliminary experiments using chimeric CARNA 5/PARNA 5 templates provided some insight into the location of the promoter structures for ({dollar}-{dollar}) strand synthesis of these satellites.