While novel direct-acting antivirals (DAA) against hepatitis C virus (HCV), such as anti-polymerase and anti- proteinase compounds are effective in many patients, these treatments are still very expensive and not available to most of the 170 million people world-wide infected with HCV. In addition, it is not known whether every patient population will respond to the current DAA treatments. Thus, it is significant to continue to search for new compounds that target both HCV and viral host susceptibility factors to combat virus infection. An astonishing recent discovery made by our laboratory revealed that the HCV genomic RNA is processed during viral infection to yield hundreds of different virus-derived circular RNAs (circRNAs). These circRNAs are generated from all parts of the 10,000-nucleotide viral RNA genome, including circRNAs that contain the viral internal ribosome entry site (IRES). It is hypothesized that these circRNAs present a novel class of viral RNA species that likely display novel functions in infected and uninfected bystander cells. Because these viral circRNAs are predicted to be long-lived they could also have important roles as biomarkers. Thus, exploring the pro- and anti-viral effects of the HCV-derived circular RNAs is a significant venue of research and will point to new Achilles’ heels in RNA viruses. The long-term goals of this application are to explore the roles for the identified viral circRNAs in pro- and anti-viral responses. Two specific aims are proposed to accomplish these goals. First, the mechanism by which the viral circRNAs are generated is being investigated. The hypothesis is that HCV subverts a cellular splicing mechanism that modulates the unfolded protein response in the endoplasmic reticulum. Secondly, it will be examined whether highly abundant circRNAs and IRES-containing circRNAs have functional roles on HCV gene expression in infected cells. The functions of IRES-circRNA translation products in the viral infectious cycle will be examined both in cultured liver cells and human liver organoids that can be infected with autologous HCV. The expected outcomes of this application will address fundamental aspects about the functions of novel circular HCV-derived RNAs in the viral infectious cycle. This proposed research is innovative because it will examine whether novel virus-derived circRNAs can be targeted in antiviral approaches in RNA virus-infected cells.