Assessment of immune sensor responses to a viral small non-coding RNA

Provisionally accepted

Gammaherpesviruses are widespread pathogens that cause persistent infections that are associated with the development of numerous types of lymphomas in humans. During latency, most viral protein-coding genes are repressed, allowing adaptive immune recognition of protein antigens to be evaded. In contrast, many noncoding RNA (ncRNA) molecules are expressed in infected cells and can regulate important cellular signaling pathways while evading adaptive immune recognition. To counteract this, many cells express internal pattern recognition receptors that can intrinsically sense ongoing infections and initiate cellular defenses. Murine gammaherpesvirus 68 (MHV68) is a valuable model for studying aspects of gammaherpesvirus pathogenesis in vivo. The MHV68 ncRNA TMER4 (tRNA-miRNA-encoding RNA 4) promotes lymph node egress of infected B cells: In the absence of TMER4, MHV68-infected B cells accumulate in the lymph node in a manner similar to B cells activated by a specific antigen encounter. Based on this finding, we hypothesized that TMER4 might alter intrinsic immune activation. In the research described here, we aimed to investigate the immunomodulatory functions of TMER4 by examining its influence on signaling through the critical immune sensors Toll-like receptor 4 (TLR4), TLR3, TLR7, and retinoic acid-inducible gene I (RIG-I). To this end, we developed a system for testing noncoding RNAs using commercially available reporter cell lines. We optimized the experimental procedure to ensure ncRNA expression and to quantify the induction or inhibition of immune sensory molecules by the expressed ncRNA. Although TMER4 did not alter TLR or RIG-I signaling, this study provides a clear experimental framework that can be applied to test other small ncRNAs for their effects on various innate immune sensor proteins.