The overarching goal of this fellowship application is to provide support for work which will focus on dissecting the structure-function relationships in ribonucleoprotein (RNP) complexes that mediate post-transcriptional gene regulation in viruses with double-strandedA (dsRNA) genomes. Mammalian reoviruses, an established model system of dsRNA virus replication, will be used for these studies. The central hypothesis is that the mechanism of reovirus RNA regulation involves specific cis-acting elements in viral RNA with cellular and viral RNA binding proteins.
Two specific aims are proposed to obtain mechanistic insight into post-transcriptional control of reovirus RNA.
In Specific Aim 1, the role of the RNA-editing enzyme, ADAR1, in the reovirus infectious cycle will be determined using ADAR1-deficient transformed cells, murine embryonic fibroblasts and primary cultures of explanted rat neurons. Experiments will be performed to determine whether viral RNAs serve as editing substrates and to assess the effect of reovirus infection on editing cellular RNAs.
In Specific Aim 2, viral RNA translational regulatory elements will be defined using chimeric reporter RNAs containing the luciferase open reading frame fused to conserved sequences from the 5'and 3'terminal regions of viral RNAs. The activity of reporter RNAs will be studied using cell-free translation systems, transformed cells, and primary neuronal cultures. Viruses containing mutations in translational control elements will be rescued by plasmid-based reverse genetics, and effects of these changes on viral protein synthesis and replication will be determined by infecting transformed cells, murine embryonic fibroblasts, and explanted neurons. Health relevance- the work proposed in this application has great potential to advance our understanding of virus-cell interactions that promote cell injury and disease in the host. The ability to conduct research about the molecular biology of viruses will lead to a better understanding of pathogenesis and immune responses to viral infection. This work will also foster the development of viral preventative, diagnostic, and therapeutic modalities against pathogenic RNA viruses.
