The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.DOI: http://dx.doi.org/10.7554/eLife.11275.001

Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5' copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5' copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six." We correlated the presence of DI-RNAs with a larger stimulation of the IFN-β pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling. Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.

The retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I– and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I–dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.

Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = −0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine.

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5' copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.

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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...

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