Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

Abstract

Whether host DNA receptors have any capacity to distinguish self from nonself at the molecular level is an outstanding question in the innate immunity of mammals. Here, by using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve as an integral mechanism by which human IFN-inducible protein-16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders, such as Sjögren syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16·dsDNA oligomers are clearly different from the conventional noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA-binding pyrin domain of IFI16 that drives the cooperative filament assembly. The surface residues on the pyrin domain that mediate the cooperative DNA binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling foci in a switch-like manner and that it is capable of using the size of naked dsDNA as a molecular ruler to distinguish self from nonself.cooperative filament formation | inflammasome R ecognition of foreign intracellular DNA is a widely conserved defense mechanism by which the innate immune system of mammals detects and responds to invading pathogens (1, 2). Using such a universal molecule as a major danger signal for detecting pathogens must be regulated in a stringent yet efficient manner. However, only a few deciding factors are known for the host innate immune system to selectively engage foreign DNA (i.e., nonself-DNA) while minimizing interactions with self-DNA, which include the compartmentalization of mammalian cells and the size of foreign DNA. These features, however, only raise more questions than provide answers. For example, the footprints of intracellular DNA receptors usually fall below 20 bp, and yet a long foreign DNA fragment [e.g., poly(dA:dT); ≥1,000 bp] is required to induce a robust innate immune response even in a normally DNA-free environment like the cytoplasm (1, 2). On the other hand, foreign DNA-sensing pathways also exist in the host nucleus in which DNA receptors must not respond to abundant self-DNA to prevent spurious activities (1, 2). Indeed, one of the major unresolved questions in understanding the DNA-sensing pathways of mammals is whether the host intracellular DNA receptors have any capacity to distinguish self-from nonself-DNA at the molecular level (2).Human IFN inducible protein-16 (IFI16) is an intracellular DNA receptor of innate immunity that belongs to the family of absent-in-melanoma-2 (AIM2)-like receptors (ALRs) (1-4). IFI16 senses DNA from invading pathogens in both the nucleus and cytoplasm [e.g., vaccinia virus...

cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection

“…S2A). These filaments, which have been reported to be associated with IFI16 oligomerization along foreign DNA (25), were directly adjacent to or colocalized with cGAS foci and plasmid DNA. Furthermore, intranuclear cGAS foci were also observed in the nuclei of a portion of transfected cells.…”

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…This allows IFI16 to detect dsDNA from many sources including Kaposi's sarcoma-related herpesvirus (KSHV) [11], Epstein-Barr virus (EBV) [44], herpes simplex virus 1 (HSV-1) [45] and human immunodeficiency virus (HIV-1) [46]. On the contrary, dsDNA sensing by IFI16 is length dependent [2], with dsDNA fragments of 150 base pairs reportedly being favourable for the formation of an optimal binding cluster of approximately ten IFI16 protomers [47]. In order to bind stimulatory dsDNA, the HIN domains of IFI16 associate with both DNA strands across both the major and minor strands, in keeping with the requirement of dsDNA rather than ssDNA for the initiation of signalling from IFI16 [2,41].…”

“…A recent paper examining IFI16 oligomerisation on DNA has provided insights into how this distinction might occur. Firstly, Morrone et al found that following DNA binding, IFI16 continues to assemble on the dsDNA strand in a cooperative manner via PYD:PYD associations, forming IFI16 foci from which downstream signal activation can be initiated [47]. For optimal polymerisation to occur a region of approximately 150 bp of DNA is required.…”

“…However, as no difference in caspase-1 cleavage was noted, the importance of IFI16 oligomerisation for inflammasome activation remains unclear. Interestingly, amino acids in the IFI16PYD which are critical for IFI16 oligomerisation are not well conserved in AIM2 [47], raising the possibility that AIM2PYD either oligomerises differently or not at all, although overexpression of the AIM2PYD does result in filament formation [29]. Since AIM2 can nucleate ASCPYD, leading to the polymerisation of ASC in the AIM2 inflammasome [43,58], it remains a possibility that AIM2PYDs which are in very close proximity to each other rather than polymerised per se may be sufficient to nucleate ASC for inflammasome formation.…”

“…It is becoming apparent that oligomerisation is a unifying mechanism critical for downstream signal activation [29,43,47,58]. DsDNA binding to IFI16 and AIM2 induces the polymerisation of IFI16 PYD domains, or the polymerisation of AIM2PYD and ASCPYD respectively.…”