The flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. In vitro production of fragments of the DENV genome confirmed the NGS data and revealed that the 5' end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells.

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.

The interaction between LGP2 and a component of the RNA-silencing machinery enables coordination of innate immune responses.

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. IMPORTANCE 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 worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/ ). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, “panhandle” or copy-back (cb) and “hairpin” or snap-back (sb) DI genomes are generated during RNA virus replication. 5′ cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed DI-tector, a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. DI-tector confirmed the presence of 5′ cb genomes in cells infected with measles virus (MV). DI-tector also identified a novel 5′ cb genome, as well as a variety of 3′ cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of DI-tector to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of DI-tector. We propose DI-tector as a universal tool for the unbiased detection of DI viral genomes, including 5′ cb/sb DI genomes, in NGS data.