Abstract
Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short- hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify an shRNA that potently activates the IFN pathway in human cells in a sequence- and 50-triphosphate- dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation.
Hepatitis C virus (HCV) is a leading cause of liver disease and much about its life cycle is still poorly understood. We have isolated a mutant virus, named SAV III, though serially passaging JFH-1 through a cell line expressing siRNA against SRBI, a known HCV receptor. We have shown that this virus spreads faster in cell culture and produces more infectious viral particles as compared to wild type HCV. In addition, we have also shown that the HCV core protein is present in higher concentrations despite other viral proteins having similar levels of expression between WT and SAV III. SAV III also has 10 fold more intracellular and extracellular infectious virus as compared to WT, suggesting enhanced viral assembly. We propose that more SAV III core is assembled into viral particles, resulting in the core protein being stabilized. Several mutations have been identified and we are working to determine what mutation, or groups of mutations are responsible for the mutant phenotype.
|
