Filament-like Assemblies of Intracellular Nucleic Acid Sensors: Commonalities and Differences

Abstract

Self versus non-self discrimination by innate immune sensors is critical for mounting effective immune responses against pathogens while avoiding harmful auto-inflammatory reactions against the host. Foreign DNA and RNA sensors must discriminate between self versus non-self nucleic acids, despite their shared building blocks and similar physicochemical properties. Recent structural and biochemical studies suggest that multiple steps of filament-like assembly are required for the functions of several nucleic acid sensors. Here, we discuss ligand discrimination and oligomerization of RIG-I-like receptors, AIM2-like receptors, and cGAS. We discuss how filament-like assembly allows for robust and accurate discrimination of self versus non-self nucleic acids and how these assemblies enable sensing of multiple distinct features in foreign nucleic acids, including structure, length, and modifications. We also discuss how individual receptors differ in their assembly and disassembly mechanisms and how these differences contribute to the diversity in nucleic acid specificity and pathogen detection strategies.

Figure 1.

Signaling pathways of RIG-I-like receptors (RLRs), cGAS, and AIM2-like receptors (ALRs). RLRs recognize dsRNA and activate MAVS, while cGAS recognizes dsDNA and activates STING. Activated MAVS and STING commonly stimulate TBK1 and IKK for production of type I/III interferons (IFNs) and other pro-inflammatory cytokines. As with cGAS, ALRs also recognize dsDNA, but the ligand engagement leads to assembly of inflammasomes, which include receptors, adaptor ASC and effector caspase-1. Within the assembled inflammasome, caspase-1 cleaves IL-1β, IL-18, and gasdermin D, which results maturation of the inflammatory cytokines and induction of pyroptosis. IFI16 was also reported to activate the interferon pathway through STING, albeit through a poorly understood mechanism.

Figure 2.

Filament formation and higher order oligomerization of RIG-I-like receptors. A) Both RIG-I and MDA5 share the same domain architecture, consisting of CARDs, a helicase domain and a C-terminal domain (CTD). CARDs are responsible for signal activation, while helicase and CTD are for RNA binding. The downstream adaptor MAVS contains the N-terminal CARD, followed by ^~400 residue-long linker containing TRAF binding sites and a C-terminal transmembrane domain (TM). MAVS CARD interacts with RLR CARDs, while TM anchors MAVS to mitochondria. B) RIG-I and MDA5 both form filaments on dsRNA, but their assembly mechanisms differ. RIG-I first binds the 5’ppp at the end of the dsRNA, then uses ATP hydrolysis to translocate to the interior. MDA5 binds the interior of the dsRNA then cooperatively forms a filament in a ATP-independent manner towards the end of the dsRNA. C) RIPLET uses bivalency to selectively bind RIG-I filaments (intra-filament binding). However, on long RIG-I filaments, RIPLET can induce higher order oligomerization by cross-bridging RIG-I filaments (inter-filament binding). This results in clustering of RIG-I filaments and further amplification of antiviral signaling. D) A model of how filament formation and higher order oligomerization of RIG-I and MAVS enable self vs. non-self discrimination and antiviral signaling. RIG-I can bind short dsRNA as a monomer, but RNA-binding is insufficient to activate downstream signaling. Filament formation along long dsRNA allows recruitment of RIPLET, which in turn bridges RIG-I filaments and conjugates K63-linked Ub chains. The high local receptor concentration and K63-polyUb together promote RIG-I CARD tetramer formation, which then acts as a nucleus to induce MAVS filament formation. MAVS filament then functions as a signaling platform to recruit and activate further downstream signaling molecules, such as TRAFs. The multiple steps of receptor oligomerization serve as independent check-points to filter out self RNAs and allow only non-self RNAs to activate downstream signaling. Once MAVS filament is nucleated, signal amplifies through polymerization of MAVS. Thus, the combination of multi-step oligomerization, both by the receptor and the signaling adaptor, ensures the accuracy and robustness of antiviral signaling in response to infection.

Figure 3.

cGAS forms ladder-like structures on long dsDNA. A) cGAS has an N-terminal basic region and an NTase-like domain. The latter is responsible for DNA binding and DNA-dependent cGAMP synthesis. B) on short DNA, the cGAS:DNA complex a 2:2 stoichiometry, with each cGAS monomer bridging two DNA strands. C) On longer DNA, a ‘founding’ cGAS dimer can promote binding of other cGAS dimers, allowing cooperative multimer assembly. These cGAS dimers arrange into ‘ladder-like’ structure. A direct dimer-to-dimer contact is unnecessary for the cooperative assembly.

Figure 4.

Model of AIM-2-like receptor inflammasome assembly. A) AIM2 contains single PYD and HIN domains, while IFI16 contains one PYD but two HIN domains. B) domain architecture of the signaling adaptor ASC and effector caspase-1. Together with receptors in (A), ASC and caspase-1 constitute inflammasomes. C) The model of AIM2 inflammasome assembly. Upon dsDNA binding, the HIN domain of AIM2 coats the dsDNA, while the PYD forms an oligomer that seeds the filament formation of ASC PYD. ASC PYD filaments recruit the effector caspase-1 through CARD-CARD interactions and activates the caspase activity. The assembled inflammasome cleaves precursors of IL-1β, IL-18, and gasdermin D, leading to the maturation of the inflammatory cytokines and formation of the gasdermin D pore. IFI16 can also activate the inflammasome, likely though a similar mechanism as AIM2.