Hepatitis C Virus (HCV) infection represents a worldwide pandemic and is currently the leading cause of cirrhosis and liver transplantation. After transplantation recurrence is almost universal with 96% of patients testing positive for viral RNA and exhibiting histological evidence of infection within the first year. Type I interferons (IFN) and interferon inducible genes are responsible for the innate antiviral state and single nucleotide polymorphisms (SNPs) within these genes may affect the patients ability to respond post-transplantation. We hypothesize the elucidation of associations between SNPs in Type-I Interferon and Interferon inducible genes and HCV recurrence post-liver transplantation might help to identify HCV patients with different prognosis and improve liver transplant recipient selection. 100 HCV positive patients were genotyped using Allelic Discrimination on an ABI Prism 7700 sequence detector (Applied Biosystems) for SNPs in IFNB1, OAS-1, and ISG-15 to establish a relationship between SNPs and clinical complications post-transplantation. Quantitative real-time polymerase chain reaction (QPCR) was also run to determine the relationship between SNPs or disease state and the level of RNA expression. Results were collected and analyzed using Fishers exact test, Kaplan-Meir method, and the log-rank test. Results obtained indicated that SNPs in OAS-1 are associated with HCV recurrence within 12 months post-orthotopic liver transplantation (OLT) and OAS-1 SNP genotypes were significantly associated with the development of fibrosis within the first year. Additionally we observed an association between the SNP genotypes of OAS-1 and ISG-15 and CMV infection post-OLT. A significant distribution of ISG-15 genotypes was also found to correlate with acute rejection. These findings might help identify patients at high risk of developing complications within the first year.

2'-5'-oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding to viral double-stranded RNAs, OAS enzymes produce 2'-5'-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to West Nile virus (WNV) infection, highlighting the importance of the OAS1 enzyme. Here I report that the 5'-terminal region (5'-TR) of the WNV genome, comprising both the 5'-untranslated region (5'-UTR) and initial coding region, is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I (SLI) is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. Solution conformations of both the 5'-TR RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. I also report that the 3' terminal region (3'-TR) is able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-TR that is sufficient for activation of the enzyme. The solution confirmation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-TR in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-TRs, a required step for replication, is not sufficient to protect WNV from OAS1 recognition. The purity, monodispersity and homogeneity of all samples subjected to SAXS analysis were evaluated using dynamic light scattering and/or analytical ultra-centrifuge. These data provide a framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme. / October 2015