Probing the viral replication of HCV and XMRV : biochemical characterization, inhibition kinetics and role of host proteins in viral replication

The studies described in this thesis focus on RNA viruses XMRV and HCV. In Chapter II we focused on Xenotropic Murine leukemia-Related Virus (XMRV). We characterized the biochemical activity and kinetics of the XMRV reverse transcriptase and discovered key mechanistic differences between XMRV, Moloney murine leukemia virus (MoMLV), and human immunodeficiency virus (HIV-1) reverse transcriptase (RT) enzymes. In Chapter III and IV we focused on Hepatitis C Virus (HCV), the causative agent of hepatitis C infection. We studied the role of an antiviral host factor Mov10 in the viral replication of HCV. We demonstrate that Mov10 overexpression in human hepatoma cells restricts HCV RNA production in a fully infectious virus cell culture system, leading to decreased virus production over time. Additionally, overexpression of Mov10 in producer cells decreases the infectivity of the produced virus. Confocal microscopy shows HCV infection results in redistribution of endogenous Mov10 to circular structures surrounding lipid droplets, proximal to viral proteins core and NS5A. Finally, we demonstrate that the RNA-binding function of Mov10 is responsible for its antiviral effect. Decreasing Mov10 protein expression levels decreased HCV replication and infection levels. Our data reveal a complex balance between Mov10 and HCV. In Chapter IV we studied several aspects of HCV. (i) We discovered two novel small molecule inhibitors of the HCV helicase that have antiviral function. (ii) We discovered that Dcp2 is a novel HCV host restriction factor that can block HCV replication, and (iii) we provide insights into the mechanism(s) of action of approved and clinically advanced direct-acting antiviral agents (DAAs)

Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action