Viruses use non-coding RNAs to manipulate many different types of cellular machines. An interesting and medically important example of this behavior was recently discovered to take place during infections caused by tick and mosquito-borne flaviviruses, including those responsible for causing Dengue, West Nile and Yellow Fever. During the course of flaviviral infection, ~300-500nt sub-genomic viral RNAs accumulate in a cell and have been shown to determine viral cytopathicity and pathogenesis. These subgenomic flavivirus RNAs, or 'sfRNAs', are produced by incomplete degradation of the viral genome carried out by the 5'3'host-cell exonuclease Xrn1. After catalyzing 5'3'decay of the preceding 95% (>10kb) of the viral genome, Xrn1 becomes stalled at one of several conserved secondary structures located in the flaviviral 3'untranslated region (UTR). This process leaves the majority of the viral 3'UTR intact and capable of interfering with aspects of the cellular immune response. The molecular mechanisms that underlie this phenomenon, including the biophysical characteristics of the RNA structures that resist degradation by Xrn1, are almost entirely mysterious. A full molecular understanding of these viruses is important and depends on understanding how sfRNAs are formed. Accordingly, the first specific aim of this proposal will test the hypothesis that the Xrn1-resistat RNA structures (xrRNAs) resist the activity of Xrn1 based on the formation an exceptionally stable structure. To test this hypothesis I will characterize the temperature dependent folding of several xrRNAs and related constructs in order to determine if xrRNAs are somehow abnormally resilient to thermal denaturation. Using a combination of mutagenesis strategies, functional assays and temperature dependent chemical probing experiments I will further characterize the thermostability of specific aspects of xrRNA structure. Using the data produced in these experiments I will learn whether or not xrRNAs operate due to extraordinary physical stability. The second specific aim of this proposal is to determine the structure of an Xrn1-resistant RNA using X-ray crystallography. Current models of xrRNA secondary structure cannot readily account for how these RNAs escape degradation by Xrn1. Through the iterative design, screening and optimization of flaviviral xrRNA constructs I intend to produce diffraction quality crystals of one of these RNAs and solve its structure. Obtaining this structure will be an important milestone in reconstructing the molecular-scale collision that takes place between Xrn1 and these structures and will provide fundamental insight into the molecular basis of viral disease.
This proposal describes research aimed at understanding a class of RNA structures that are critical in the lifecycle of the human pathogenic viruses that cause Dengue, West Nile and Yellow Fever. It includes research that will interrogate the unique biophysical and structural properties of these RNAs as they pertain to their role in viral disease.