Mapping replication and silencing suppression elements in the RCNMV genome

Viruses infect all Kingdoms of life on Earth. Their life cycle represents a constant struggle for survival in hostile cellular environments. To survive the virus must both avoid the host response and replicate in a timely manner. The following dissertation includes investigations into both of these aspects of viral infection focusing on plant-infecting viruses.;When infected by viruses, plants respond by initiating defense pathways. One of these pathways is known as RNA silencing. In this pathway host proteins target double-stranded viral RNA intermediates for cleavage. The product of this cleavage, a short-interfering RNA (siRNA), is incorporated into a protein complex that guides sequence specific cleavage of viral RNA. To cause a productive infection, the virus must devise countermeasures to this targeting. They accomplish this by suppressing the RNA silencing pathway by encoding proteins known as viral suppressors of RNA silencing (VSRs). Identifying these VSRs is critical to the understanding of how any plant virus survives in its host. Commonly used assays for identifying VSRs all use reporters expressed in the nucleus. These nuclear-based DNA reporters are being used to assay for the RNA silencing suppression activity of RNA virus proteins. In this thesis I describe the development of a new VSR identification assay that uses a disarmed RNA virus as a reporter, which should be an accurate predictor for VSR activity of other RNA viruses. The reporter is the plant-infecting virus Turnip crinkle virus (TCV) with its previously characterized VSR, the coat protein, replaced with sGFP resulting in a construct termed TCV-sGFP. After validating the use of TCV-sGFP as a reporter for RNA silencing suppression activity TCV-sGFP was used to identify a previously uncharacterized VSR for the plant-infecting virus Red clover necrotic mosaic virus (RCNMV). RCNMV's replication complex was previously implicated in the suppression of RNA silencing, while the new TCV-sGFP assay allowed us to detect a second suppressor, a protein known to be involved in viral movement, known simply as the movement protein (MP). Domains of MP were analyzed and it was found that the amino acid residues between positions 122 and 277 were required for suppression activity.;The RNA silencing pathway has two principal components, siRNAs and host proteins. To disrupt the pathway the MP of RCNMV must interfere with one or both of these components. In this thesis the possibility that the MP suppresses RNA silencing by binding to siRNAs is addressed by employing electrophoretic mobility shift assays to examine MP's ability to bind to siRNAs. MP was found to have no siRNA binding capability, indicating that it's mode of action likely relies on binding to or modifiying host proteins.;Finally, initial studies into the temporal regulation of RCNMV replication were undertaken. As mentioned, for a productive infection to take place the virus must go beyond evading the host defense response, it must also properly regulate the timing of expression for its many genes. Using real time PCR, the timing of various replication events in the RCNMV life cycle were mapped to more fully understand how RCNMV replicates inside the cell. These time course studies have revealed that virion formation has little effect on RCNMV's ability to replicate, and that RNA-1 replicates to a slightly higher degree in the absence of RNA-2. Additionally several uncharacterized negative sense replication intermediates that exist during a wildtype infection were identified. This work forms the foundation for future studies into the temporal regulation of RCNMV replication.