Structure and function of the Powassan Virus 3' untranslated region

PROJECT SUMMARY Flaviviruses are a genus of single stranded (+)-sense RNA viruses containing a multitude of important human pathogens. These viruses share a genomic architecture (10-11kb in length) comprised of a single open reading frame flanked by 5' and 3' untranslated regions. During a flaviviral infection, noncoding RNAs (ncRNAs) derived from a portion of the 3' untranslated region (UTR) are formed through the partial degradation of the viral genome by the host 5'→3' exoribonuclease Xrn1. These ncRNAs are called subgenomic flaviviral RNAs (sfRNAs) and are important for viral pathogenesis and evasion of host/vector immune responses. These ncRNA are formed through a complexly folded RNA structural motif found at the beginning of the 3' UTR called exonuclease resistant RNA (xrRNA). Currently, flaviviral xrRNAs are grouped into three structural classes, the Class 1a (MBFV), Class 1b, and Class2, based on their overall architecture and the stabling intramolecular interactions needed for resistance. Most of the work on xrRNA have been done with the Class1a and Class1b xrRNA, which are found in mosquito borne, insect specific and no known vector flaviviruses, with a major lack of understanding of the Class2 xrRNA that are typically found in tick borne flaviviruses. Currently we only have cursory knowledge of these Class2 xrRNA, how they confer resistance, and how they fit in with the overall flaviviral 3'UTR architecture. Powassan Virus (POWV) is the only tick borne flavivirus endemic to in North America and has caused a growing concern. Predictive models based on comparative sequence alignment have identified two Class2 xrRNA in the 3'UTR of POWV coupled with an interesting overall 3'UTR architecture. Our predictive models show minimal space between each predicted structural motif present in the 3'UTR, suggesting that this untranslated region folds into a compact rod-like structure. This proposed rod like structure makes for an ideal target for structural studies not only of the Class 2 xrRNA but of the overall POWV 3'UTR architecture. I hypothesize that the two Class 2 xrRNAs present in the POWV 3'UTR have specific structures that are critical for sfRNA generation, and that the overall architecture of the 3'UTR folds into a defined rod-like structure which is critical for the pathogenesis of the virus. The goal of the project is to use a combination of biochemical, structural, and virological methods to examine the structures of the Class2 xrRNA in the context of the full POWV 3'UTR, while testing the effects of these structures in vivo and the effects on viral pathogenesis. Aim1. In this aim I will use structural and biochemical techniques to investigate the two proposed class 2 xrRNA and the full 3'UTR in both 2D and 3D. These experiments will generate an accurate secondary structure, and the first high resolution 3D model of the class 2 xrRNA and the full flaviviral 3'UTR. Aim2. I will use biochemistry and classical virology to begin to understand the effects other class2 xrRNA in the context of viral pathogenesis in multiple cell lines. This aim will begin to define the importance of these structures in viral pathogenesis and their effect on sfRNA biogenesis.