Mechanisms of Defective Interfering RNA Replication and Interference with Helper Virus Infections

Previous work on RNA replication, a central process in viral pathogenesis, has focused on promoter elements. The discovery by the PI and others of novel cis-acting elements, such as RNA replication enhancers that up-regulate RNA synthesis and RNA replication silencers that down-regulate RNA synthesis, changes our view of RNA virus replication. These RNA replication enhancer and silencer elements may play major roles not only in replication, but they may also affect the fitness and evolution of viruses and the symptoms they cause in infected hosts. Defective interfering (DI) RNAs, due to their simple genomes and robust replication are attractive model systems for understanding the role of cis-acting elements in RNA replication. DI RNAs are highly competitive, parasitic RNAs associated with many virus infections. DI RNAs compete against their helper (parental) viruses, resulting in a decreased replication rate of the helper virus and frequently leading to symptom attenuation in the host. The hypothesis to be tested in this research is that the superior competitiveness of the DI RNAs relative to the helper virus is due to (i) favorable positioning of cis-acting elements, such as promoters and replication enhancers in DI RNAs, and/or (ii) the absence of some replication silencer elements in DI RNAs that may have been deleted during DI RNA formation. This research uses tombusviruses, model (+)-strand RNA viruses of plants, to: (i) characterize replication enhancer and silencer elements by using a recently developed cell-free replicase system; (ii) elucidate the mechanism of enhancer-mediated stimulation and silencer-mediated inhibition of RNA transcription through testing the direct effect of RNA enhancer and silencer elements on template binding by tombusvirus replicase proteins expressed in E. coli. These studies will lead to a better understanding of regulation of viral RNA synthesis by sequences on the RNA being copied. The studies will also elucidate the ability of certain incomplete viral variants to attenuate infections by the original, complete viruses from which they were derived. These variants are called defective interfering RNA's. They are derived from viruses that infect a wide range of organisms, including plants.