Abstract 1383 The circular RNA circ_ATP8B regulates ROS production and antiviral immunity

Circular RNAs (circRNAs), which are products of "head-to-tail" back-splicing events, have been discovered in diverse eukaryotes, and constitute a new addition to the non-coding and regulatory RNA collection. CircRNAs have been implicated in a wide variety of biological processes, including antibacterial innate immunity, neurodevelopment, and gene control. To investigate the role of circRNAs in antiviral defense in Drosophila, we performed RNA sequencing and identified and validated of a collection of circRNAs that display significant changes in expression levels upon viral infection. We found that depletion of the pro-viral circular RNA circ_ATP8B, but not its linear sibling, led to a reduction in levels of viral RNA, protein and titer in cultured Drosophila SL2 cells upon infection by a panel of RNA viruses, including Flock House virus (FHV), Drosophila C virus (DCV) and Cricket paralysis virus (CrPV). Similarly, ubiquitous depletion of circ_ATP8B in vivo reduced viral load and enhanced host survival upon viral infection. Importantly, restoring circ_ATP8B expression in circ_ATP8B-depleted cells or flies suppressed the viral replication and/or host survival phenotypes. In addition, our analyses revealed that circ_ATP8B is a gut-enriched circRNA that is induced specifically by oral ingestion of viruses but not bacteria, and that gut-specific depletion of circ_ATP8B compromises viral replication in an oral infection model. Furthermore, we found that defects in viral RNA replication in circ_ATP8B-depleted flies correlated with elevated levels of ROS and enhanced expression of Dual oxidase (Duox), which encodes a ROS-producing enzyme. Notably, circ_ATP8B and Duox associate with each other, and expression of various versions of circ_ATP8B that are competent in binding Duox, but not a mutant circ_ATP8B that is incapable of binding Duox, restored physiological levels of ROS in circ_ATP8B-depleted cells. Lastly, we show that feeding flies with N-acetyl-L-cysteine (NAC), a ROS inhibitor, depletion of Duox or Gaq, a G protein required for optimal Duox activity, or overexpression of immune-regulated catalase (IRC), an enzyme that removes ROS, led to a reduction in ROS levels and suppressed the viral replication defects elicited by circ_ATP8B depletion. Thus, our study demonstrates that ROS acts as an antiviral agent against infection via the oral route and that circ_ATP8B regulates antiviral immunity, at least in part, by modulating Duox-dependent ROS production. This work was supported by the American Heart Association: (14GRNT18970089 and 16GRNT31360017 to R.Z.); NIH (1R01AI140049 to R.Z.; 1R01NS124668 to R.J.P.); Florida Department of Health Bankhead-Coley Cancer Research Program (5BC08 to R.J.P.); and Johns Hopkins University (R.Z. and R.J.P.). J.L. is supported by the Intramural Research Program of the NIH.