34Pattern recognition receptors (PRRs) protect against host invasion by detecting specific 35 molecular patterns found in pathogens and initiating an immune response. While 36 microbial-derived PRR ligands have been extensively characterized, the contribution and 37 relevance of endogenous ligands to PRR activation during viral infection remain 38 overlooked. In this work, we characterize the landscape of endogenous ligands that 39 engage RIG-I-like receptors (RLRs) upon infection by a positive-sense RNA virus, a 40 negative-sense RNA virus or a retrovirus. We found that several endogenous RNAs 41 transcribed by RNA polymerase 3 (Pol3) specifically engage RLRs, and in particular the 42 family of small non-coding repeats Y-RNAs, which presents the highest affinity as RIG-I 43 ligands. We show that this recognition is dependent on Y-RNA mimicking viral secondary 44 structure and its 5'-triphosphate extremity. Further, we found that HIV-1 infection triggers 45 a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, 46 leading to an increase of Y-RNA 5'-triphosphorylation that enables their immunogenicity. 47Pattern Recognition Receptors (PRRs) were initially described as innate immune sensors 53 of molecular patterns commonly found in pathogens but rarely, if ever, found in their hosts. 54In recent years, this view has been challenged by evidence that ligands originating from 55 self can engage these same PRRs. Notably, sensing of self-RNA by innate receptors has 56 been observed in various settings such as autoimmune disorders (1-3), tumorigenesis 57 and cancer therapies (4-9) or infection by DNA viruses (10, 11). While the importance of 58 endogenous ligands in priming immune responses is progressively uncovered, little is 59 known about the breadth of biological processes in which they happen, nor about their 60 functional and evolutionary interplay with immune sensors. Furthermore, we lack 61 understanding of what features confers self-RNAs the ability to activate sensors and 62 whether this is a general response to aberrant transcription or is dominated by specific 63
RNA species. 64Further confounding matters, we previously determined that conventional RNA 65 sequencing approaches fail to capture the full spectrum of RNA expression in tumors (12). 66In particular, repetitive RNA, which can harbor immunostimulatory features (13), require 67 further computational analysis for unbiased screening of their transcription. Here, we 68 apply these approaches to identify novel RNA agonists of RIG-I-like receptors (RLRs). 69RLRs are a family of cytosolic RNA sensors composed of three members: RIG-I, LGP2 70 and MDA5 (14). Their intracellular localization and proximity with host RNA species 71 implies a delicate balance between a need to develop high affinity for microbial features 72 and the possibility to encounter self-RNAs that display similar structures. However, 73 despite a growing knowledge of the role of RLRs during RNA virus infection and the 74 microbial-derived ligands they recognize...