UC San Diego

Mammalian cells can detect the presence of viral double- stranded RNA (dsRNA) through extracellular and intracellular pathogen recognition receptors (PRRs). Toll- like receptor 3 (TLR3) can be localized on the endosomal membrane of cells and detects endocytosed dsRNA (extracellular). Conversely, RIG-I-like receptors (RLRs), which include retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (Mda-5) receptors detect dsRNA in the cytoplasm (intracellular). Most IEC types that have been used to study rotavirus infection lack robust TLR3 signaling. Here, via analysis of type I interferon (IFN) induction and apoptosis, the HCT-116 cell line was identified to harbor both TLR3 and RIG/Mda-5 dsRNA signaling pathways. This cell type may be useful for future in vitro studies of the role of these extracellular and intracellular signaling pathways in rotavirus infections. Through a series of experiments using the SA11-4F and SA11-5S strains of rotavirus, it was determined that SA11-4F exerts a suppressive effect on IFN response in HCT-116 cells, likely through an inhibitory effect on IFN regulatory factor 3 (IRF-3) caused by nonstructural protein 1 (NSP1), as has been shown by others. It was also observed that MAVS is required for maximal IFN-[Beta] induction in HCT-116 cells following rotavirus infection. Surprisingly, it was determined that TLR3 stimulation exhibits a negative regulatory effect on mitochondrial antiviral signaling protein (MAVS, also known as IPS-1 or Cardif), a mitochondrial surface protein that serves as a common adaptor to RIG-I-like receptors (RLRs). IFN[Beta] induction increased in the absence of TLR3, and this strengthened innate immune response exhibits a biologically relevant reduction in viral replication. These results observed in HCT-116 cells suggest that TLR3 may up-regulate apoptosis following viral infection, and down-regulate MAVS levels and IFN- [Beta] production