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

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 and CMV infection.