Project Summary Double-stranded RNA (dsRNA) is a potent pathogen-associated molecular pattern (PAMP) that activates cell intrinsic antiviral defenses that inhibit protein synthesis and viral replication. Viral RNAs were previously regarded as the primary ligands for cellular dsRNA sensors, however, cellular RNAs have recently been revealed to be major regulators of nucleic acid PAMP responses capable of inhibiting and promoting their activation. Though HSV1 is restricted by dsRNA-dependent antiviral effectors PKR and RNase L, what RNA activates these enzymes and the contribution of newly characterized cellular calibrators of these responses is completely unknown. Our overall objective is to understand how cellular RNAs contribute to antiviral immunity in HSV1 infection. DsRNA-dependent responses are counteracted by several HSV1-encoded proteins, underlining the importance of the task. These include vhs, an endoribonuclease which accelerates mRNA decay minimizing dsRNA accumulation and Us11, which binds dsRNA preventing activation of the eIF2α kinase PKR and RNase L. Our preliminary results indicate two opposing mechanisms exist by which cellular RNAs also regulate dsRNA-dependent cell intrinsic defenses in HSV1 infected cells. First, we hypothesize that cellular circular (circ) RNAs, protected from RNase L activation by Us11 and selectively preserved from vhs cleavage, inhibit PKR activation. Second, we hypothesize that HSV1-induced dsRNA is not exclusively virus-encoded and instead contains a cellular RNA component dependent on cytosolic DNA-sensing by cGAS-STING, the sensing pathway that induces type I interferon (IFN) in response to HSV1. This would constitute a novel mode of signal amplification and cross-talk between pathogenic DNA and RNA cytosolic detection systems. We will test these hypotheses in two specific aims that (i) determine the role of circRNAs in HSV1 infection biology; and (ii) define how the cGAS-STING dsDNA sensing pathway influences dsRNA abundance and/or antiviral dsRNA-dependent innate immune responses. Completion of these specific aims will reveal how cell intrinsic innate immune responses are regulated by host RNAs and parallel innate immune signaling pathways during infection with HSV1, a medically important human pathogen. The results of this exploratory study are potentially applicable to a wide variety of pathogenic human viruses and could reveal new strategies for treating virus infections. In addition, they will provide important insights into cross-talk between innate immune signaling pathways involving dsDNA and dsRNA that are important therapeutic targets for cancer and autoimmune disease.

Project Narrative Sensing of the pathogen-associated molecular pattern double-stranded RNA (dsRNA) activates potent antiviral defense pathways that inhibit protein synthesis and viral replication, but the extent to which cellular RNAs participate in and regulate these pathways is unclear. Here, we investigate the hypotheses that previously uncharacterized host RNAs (circRNAs and cellular RNAs that are dependent on pathogenic dsDNA-sensing by cGAS-STING) regulate dsRNA-dependent anti-viral responses to herpes simplex virus-1, a medically important virus responsible for a variety of conditions ranging from self-limiting skin sores to life threatening encephalitis. Understanding how host RNAs regulate dsRNA signaling and anti-viral immunity could reveal new strategies for treating a wide variety of viral infections and has significant implications for understanding autoimmune disease, anti-tumor immunity and infection biology.