Non-self RNA sensing in virus infected cells and activation of antiviral immunity

Colloque organisé par Jean-Luc Imler (Université de Strasbourg) et Raul Andino (UCSF, USA) du 27 août au 1er septembre 2012


Shizuo Akira, Raul Andino, Bruce Beutler, Stephen Cusack, Ding Shou-Wei, Takashi Fujita, Akira Tajima-Goto, Otto Haller, Jules Hoffmann, Veit Hornung, Jean-Luc Imler, Karim Majzoub, Carine Meignin, Eric Miska, Jan Rehwinkel, Carla Saleh, Rotem Sorek, Volker Thiel, Joao Trindade Marques, Ronald van Rij, Olivier Voinnet, Friedemann Weber


Innate immunity is the first line host-defense that operates to contain infections in all organisms. Activation of this response relies on the sensing of molecular patterns present in the infecting microorganism but absent from the host. Among pathogens, viruses pose a particular challenge to the innate immune system. Indeed, these intracellular parasites are intimately associated with their host cell, and use many of its enzymatic machineries for replication and expression of their genomes. Nevertheless, it is now clearly established that all cells are equipped with sensors detecting the presence of viral ribonucleic acids (RNAs) in infected cells, and triggering immunity. This immunity can be either based on the production of small RNA species targeting the virus, or on the induction of proteins, such as the interferons (IFN) and the products of IFN stimulated genes. A group of scientists working in a range of different models (bacteria, plants, invertebrate and vertebrate animals) gathered in Les Treilles the last week of August 2012 to discuss how viral RNAs are sensed by the innate immune system.


RNA vs protein based antiviral defenses

In plants and invertebrates, antiviral defenses are dominated by an RNA-based system of defense, which relies on the production of small interfering RNAs (siRNAs) by enzymes of the Dicer family to trigger sequence specific degradation or inhibition of translation of RNAs with complementary sequences. Plant and insect viruses adapt to this response and express viral suppressors of RNAi. For example, Ronald van Rij described how the protein VP1 from Nora virus interacts with AGO2 in flies and affects the antiviral function of the preassembled RISC complex. In mosquitoes, a second class of small RNAs, piRNAs, are also produced in response to viral infection and it will be interesting to find out how these small RNAs are generated. In plants, Olivier Voinnet described how RNA interference can control genomic invaders like retrotransposons, in addition to viruses. The Dicer-like genes from plants act in hierarchical manner, and are saturable. In the case of retrotransposons, saturation of DCL2 leads to activation of DCL3, which then triggers DNA methylation and transcriptional gene silencing. Besides invertebrates and plants, bacteria use a conceptually similar RNA based immune system as described by Rotem Sorek. This adaptive system to counter genomic invaders rely on small RNAs produced from transcripts of clustered regularly interspaced short palindromic repeats (CRISPR), which are used to direct a DNA nuclease towards foreign DNA. This system, present in about 40% of bacteria, and which involves insertion of foreign DNA in CRISPR loci, can be used in metagenomic studies to identify the bacterial species that can be targeted by a particular phage.

In vertebrates and mammals in particular, detection of viral RNAs triggers the production of interferons, which induce in stimulated cells the production of antiviral molecules. Otto Haller described one of these molecules, the dynamin-like Mx protein, which target viral nucleocapsids. The pleckstrin homology domain found in dynamin is replaced by a loop known as L4 in Mx proteins. The sequence of this loop is extremely variable between species and is thought to interact with viral target structures. The current model is that activation of the GTPase activity of Mx proteins assembled in rings around the viral nucleocapsids could twist and squeeze them, leading to their destruction. Shizuo Akira described another type of antiviral strategy used by neutrophils to neutralize HIV. Engagement of Toll-like receptor (TLR) 7 and 8 by viral nucleic acids triggers the production of neutrophil extracellular traps (NETs), which include the antiviral factors myeloperoxydase and D-defensin, leading to elimination of HIV. An induced antiviral response also participates in the control of viral infection in drosophila, but remains poorly characterized. Akira Goto and Jules Hoffmann reported that the fly orthologue of the IKKE molecule, a core component of NF-NB signaling, plays an important role in the control of infection by the picornalike virus DCV, in vivo but also in tissue culture cells, opening the way to the characterization of both the signals activating this pathway, and the genes it regulates in the context of viral infections.

Sensors of viral nucleic acids

Unbiased forward genetic screens were used successfully to establish the key role played by TLRs in the sensing of infection. Bruce Beutler used a screen for susceptibility to infection by a sublethal dose of the DNA virus MCMV to identify more components of this pathway. Characterization of mutations in the genes Slc15a4 (feeble) and AP3 (bullet gray), helped establish that a lysosome-related organelle, from which nucleic acid sensing TLRs signal, is critical for antiviral host-defense. Another critical family of receptors are the RIG-I-like receptors RIG-I and MDA5, which sense viral nucleic acids in the cytosol. Stephen Cusack described the structure of RIG-I, and explained how RNA binding, together with ATP binding, triggers a massive conformational change leading to exposition of the CARD domains, and signaling. Conservation of key residues in the helicase domains of RIG-I and Dicer-2 indicate that these two key molecules for sensing viral RNAs in mammals and flies bind dsRNA in the same way, preferentially engaging the extremities of the dsRNA. Eric Miska characterized two strains of Caenorhabditis elegans differing in their sensitivity to nodaviruses infecting worms, and reported that a Dicer-related helicase, bearing strong similarity to mammalian RIG-I like receptors, plays a critical role in resistance to viral infection, acting upstream of Dicer itself. This indicates that the role of RIG-I like receptors in sensing viral RNAs is evolutionarily ancient and predates apparition of the interferons. RIG-I like receptors and Dicer enzymes share an evolutionarily conserved DExD/H box helicase domain, and Carine Meignin presented data on the contribution of this domain to the control of viral infection. She showed that a mutation disabling the ATPase domain of Dicer-2 affected the resistance to some viruses, but not others, pointing to the existence of different modes of viral RNA recognition in drosophila. This may involve coreceptors, as suggested by proteomic data presented by Karim Majzoub, who identified several proteins copurifying with Dicer-2 in virus infected cells.
Besides TLRs and RLRs, the role of two other sensors was addressed. Veit Hornung discussed the role of STING, an ER resident protein containing 4 transmembrane domains, that can function as a sensor for some microbial ligands, such as cyclic dinucleotides. STING is also an essential signaling adaptor involved in the induction of IFN production upon sensing the presence of cytosolic DNA, and several phosphoproteins regulated by this signaling pathway were identified and discussed. Finally, Jan Rehwinkel discussed the role of SAM-HD1, a restriction factor of HIV in myeloid cells, in the regulation of IFN production, and reported on the establishment of a mouse model to study the role of this molecule in the regulation of retroelements.

Features of viral RNAs sensed by the innate immune system

Takeshi Fujita analyzed the interaction of dsRNA with RIG-I (which binds preferentially small dsRNA) and MDA5 (which bind both long and small dsRNA). Using atomic force microscopy, he reported that MDA5 progressively coats long dsRNA molecules, whereas RIG-I forms punctate structures on viral RNA. Friedeman Weber reported that the nucleocapsid, which packages genomic RNA of (-) strand RNA viruses, can trigger activation of RIG-I if the RNA contains a 5’ triphosphate, and this activation does not require viral replication or transcription. In flies, Joao Marques reported that the dsRNA binding protein Loquacious is required for the endogenous siRNA pathway, but not the antiviral siRNA pathway, pointing to differences in the sensing of dsRNA expressed endogenously, or produced in the course of a viral infection. Volker Thiel studied induction of IFN by coronaviruses, which trigger a strong response through TLR7 in plasmacytoid dendritic cells, but not in macrophages or fibroblasts. These viruses express methyl transferases that modify the extremities of viral RNAs and prevent their recognition by MDA5.
Besides viral RNAs, siRNAs produced in infected cells may also be sensed by the innate immune system. Jean-Luc Imler reported that induction of the antiviral gene Vago requires dicing of viral dsRNAs by Dicer-2 and can be blocked in cells expressing the plant VSR p19, which specifically binds to siRNA duplexes. Raul Andino further provided evidence for a novel class of secondary siRNAs, produced in virus infected cells, and exhibiting different features from primary siRNAs. This new class of siRNAs may play a role in the spreading of the antiviral RNA interference to uninfected cells. The origin of these secondary siRNAs is still mysterious, but Carla Saleh reported the presence of viral DNA in persistently infected drosophila cell lines, which may contribute to their formation. Indeed, treatment of infected cells with inhibitors of reverse transcriptase prevents the formation of viral DNA, and leads to increased viral replication, suggestion that the DNA form of the viral genome contributes to host-defense.

RNA based immunity in mammals?

Although synthetic siRNAs can confer protection against viral infection in mammals, there has so far been no evidence for processing of viral RNAs into siRNAs in mammalian cells. Shou-Wei Ding used Nodamura virus, a relative of Flock House Virus, the virus that was instrumental in establishing the essential role of RNAi in the control of viral infection in flies. He showed that he could grow a mutant version of Nodamura virus deleted of its B2 protein (the VSR in FHV) in a mammalian cell line, and that these cells produced virus-derived siRNAs, indicating that under some conditions, the mammalian Dicer could contribute to antiviral defense. Similar results were reported by Olivier Voinnet, who reported the presence of virus-derived siRNAs in embryonic stem cells infected by EMCV. Thus, RNA-based immunity may not be a specific feature of plants and invertebrates, but may also play a role in antiviral defense in critical vertebrate tissues such as the stem cells.

Overall, the meeting was a perfect opportunity to gather a group of expert scientists interested in similar questions, but who did not know each other. In addition to the presentations summarized above, the wonderful setting of Les Treilles enabled many informal discussions that will lead to collaborations and, certainly, more meetings on this fascinating topic.