RNA-DNA hybrids and ssDNA differ in intracellular half-life and Toll-like receptor 9 activation

Abstract The innate immune system senses viral and bacterial RNA or DNA via different cytoplasmic or endosomal localized pattern recognition receptors. In general, the preference of these receptors for single-stranded (ss), double-stranded (ds) RNA or DNA has been thoroughly characterized. Recently, RNA-DNA hybrids have also been identified as ligands for pattern recognition receptors such as Toll-like receptor 9 (TLR9). However, a comparison of RNA-DNA hybrids and ssDNA in terms of TLR9 stimulation potential and intracellular stability has not been addressed. RNA-DNA hybrids are formed transiently during normal cellular processes (e.g. replication), consist as part of some viral genomes (e.g. cytomegalovirus; CMV or hepatitis B virus; HBV) and exist during retroviral infection. Here we report that virus-derived synthetic RNA-DNA hybrids stimulate human peripheral blood mononuclear cells (PBMCs) as well as murine FMS-like tyrosine kinase 3 ligand (FLT3L) induced dendritic cells to secrete interferon alpha (IFN-α) in a TLR9-dependent manner. Furthermore, we could show that RNA-DNA hybrids exhibit increased intracellular stability, which correlates with enhanced activation of TLR9 in comparison to corresponding ssDNA. Overall, these data suggest a prominent role for TLR9 in the immune recognition of RNA-DNA hybrids in retroviral or CMV infection.