OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

RIPK3–ZBP1–MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2′-5′ oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3–ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein–protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3–ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

Necroptosis is a regulated cell death process that contributes to pathogen-mediated host immune defence by inducing inflammation upon cell death.This intricate balance between induction of inflammatory responses and clearance of pathogens via 'suicidal death' is a noteworthy contribution to host-pathogen standoff, as it determines the degree of pathogenic invasion and pathogenicity.Necroptosis signalling is mediated through the formation of a multiprotein complexthe so-called necrosome-in which receptor interacting protein serine/ threonine kinase 3 (RIPK3) plays a major effector role.In the absence of caspase-8-mediated apoptosis, tumour necrosis factor (TNF) induces the assembly of the canonical necrosome complex composed of two RIPKs: RIPK1 and the effector RIPK3 (refs.1-4).More recently, necroptosis has been recognized as an antiviral mechanism after virus infections, such as murine cytomegalovirus (MCMV) 5,6 , herpes simplex virus-1 (HSV-1) 7,8 , vaccinia virus (VACV) 2 and influenza A virus (IAV) 9 , by forming a non-canonical necrosome composed of RIPK3 and an interferon-stimulated gene (ISG), Z-DNA-binding protein 1 (ZBP1) 10,11 .RIPK3-mediated phosphorylation of its substrate, mixed lineage kinase Article https://doi.org/10.1038/s41556-022-01039-ybarriers, many proteins, such as FUS, α-synuclein, and STING, undergo phase transition over time to a more rigid hydrogel or amyloid-like fibril structure [35][36][37][38][39] .Thus, phase separation facilitates efficient formation of signalling complexes by inducing concentrated homotypic and heterotypic interactions within the partitioned core.
In this study, we identify OASL as a regulator of virus-induced necroptosis that promotes antiviral activity to restrict viral replication and dissemination.OASL undergoes LLPS and chaperones the assembly of RIPK3-ZBP1 necrosome by recruiting RIPK3 and ZBP1 into its phase-separated droplets via protein-protein interactions.These phase condensates serve as a platform for RIPK3 to nucleate, ultimately inducing RIPK3 amyloid-like fibre formation and enzymatic activation.Subsequent activation of the non-canonical necroptosis pathway elicits antiviral activity by restricting virus replication and dissemination in vivo.Herein, we elucidate the molecular action of the IFN-inducible OASL as a key signalling adaptor for the RIPK3-ZBP1 necroptotic pathway after virus invasion.

Identification of OASL as a binding partner of RIPK3
MCMV-encoded M45 is a viral inhibitor of RIP activation that blocks necroptosis through the disassembly of the RIPK3-ZBP1 non-canonical necrosome.Thus, we utilized a necroptosis-sensitive mutant MCMV carrying the RHIM mutation of M45 (MCMV-M45mutRHIM) to identify key regulators of the necrosome complex in the context of virus infection 5,6 .We infected mouse primary tail fibroblasts with the necroptosis-sensitive MCMV-M45mutRHIM, and at 6 h post-infection (h.p.i.), endogenous mouse RIPK3 was immune-purified and subjected to mass spectrometry analysis.In agreement with previous reports 14 , a number of TNF signalling proteins and phosphoinositide-related enzymes were specifically detected in the RIPK3 complex after virus infection (Fig. 1a and Supplementary Table 1).Notably, an IFN-inducible protein, OASL1, was detected from the RIPK3 complex following MCMV-M45mutRHIM infection.Mouse OASL1 shares high sequence similarity (74%) with human OASL and is predicted to contain an amino-terminal enzymatically inactive OAS domain (N-OAS) and two carboxy-terminal ubiquitin-like (UBL) domains (C-UBL) like human OASL.By contrast, mouse OASL2 preserves the 2-5As synthesis activity within its N-terminal OAS domain and carries a C-terminal single UBL domain.Co-immunoprecipitation assays showed that RIPK3 specifically interacted with OASL1 but not with OASL2 (Fig. 1b).
RIPK3 autophosphorylation is the primary prerequisite for its self-activation to relay downstream signals for inducing necroptotic cell death; however, this fundamental mechanism remains hypothetical.It has been proposed that intermolecular interactions mediated by the RIP homotypic interaction motif (RHIM) result in high-order hetero-amyloid structures, which may serve as a platform for RIPK3 autophosphorylation [18][19][20][21] .Recent findings have also revealed that artificially induced RIPK3 homo-oligomerization is sufficient to induce necroptosis [22][23][24] , which suggests that the kinase activity of RIPK3 could be activated by proximity within RIPK3 oligomers.In addition, RIPK3 assembles into discrete functional amyloid-like foci in the cytosol, but it remains unclear how RIPK3 amyloid formation contributes to necroptosis signalling 18,25,26 .
The indispensable role of ZBP1 in virus-induced necroptosis, along with other growing evidence, suggests that robust activation of necroptosis during virus infection requires synergistic interplay between type I interferon (IFN) and TNF signalling to intensify the activation of RIPK3 (refs.27,28).During virus infection, rapid activation of type I IFN signalling gives rise to strong expression of ISGs, among which include the oligoadenylate synthetase (OAS) gene family.OAS family proteins belong to the nucleotidyltransferase superfamily and confer protection against viruses through their 2′-5′-phosphodiester-linked oligoadenylates (2-5As) synthetase activity that is triggered after binding to viral double-stranded RNA (dsRNA).Unlike OAS proteins, the protein OAS-like (OASL) lacks enzymatic activity, yet still has regulatory functions in innate immunity, suggesting that OASL has a role in a RNase L-independent antiviral mechanism.Previous studies have shown that OASL has potent target-specific antiviral activity against a panel of RNA and DNA viruses, enhancing antiviral activity in concert with other ISGs 29,30 .By contrast, other studies 31,32 have reported that binding of OASL to RIG-I enhances IFN production during RNA virus infection, whereas binding to cGAS suppresses IFN production during DNA virus infection.Although the definitive antiviral role of OASL remains unclear, the subcellular localization of OASL appeared as distinct foci in the cytoplasm and the nucleus, which suggests that OASL has a behaviour-specific function.These contentious findings prompt further investigation of the precise role of OASL during virus infections.
Liquid-liquid phase separation (LLPS) is an emerging paradigm in the formation of membraneless biomolecular condensates that enables spatiotemporal regulation of biochemical signalling [33][34][35][36] .Liquid droplets serve as a molecular platform to nucleate biomolecules, including proteins and nucleic acids, which discretely tune condition-specific reactions within the complex.Although these liquid condensates are initially highly mobile and dynamic owing to the lack of physical Fig. 1 | The IFN-stimulated protein OASL is required for efficient virusinduced necroptosis.a, Mouse primary fibroblasts were mock-infected or infected with MCMV-M45mutRHIM virus (multiplicity of infection (m.o.i.) = 5) for 6 h.RIPK3 protein complexes were enriched and immunoprecipitated (IP) using RIPK3 antibody-conjugated agarose beads and analysed by mass spectrometry.Functional-related or biological-related proteins are grouped in boxes.b, HEK 293T cells were transfected with the indicated constructs, and cell lysates were immunoprecipitated with V5-specific antibody.Immunoprecipitates and whole cell extracts (input) were analysed by immunoblotting with the indicated antibodies.c, Cell death kinetics of Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with MCMV-WT or MCMV-M45mutRHIM (m.o.i.= 5).Necrotic cell death was measured on the basis of the uptake of Sytox Green and quantified in real-time from 4 to 12 h.p.i.(n = 4 biological replicates).d, Left: microscopy analysis of cell death in Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with MCMV-M45mutRHIM at 16 h.p.i.Arrows indicate cells with necrotic features after infection.Scale bar, 20 μm.Right: quantification of necrotic cell death by measuring the release of LDH from Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with MCMV-M45mutRHIM (m.o.i.= 5) for 8 h.e, Quantification of necrotic cell death by measuring intracellular ATP levels (n = 4) and LDH release (n = 3) in supernatants of Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with HSV-1 (m.o.i.= 5).f, Immunoblot analysis of RIPK3 and MLKL phosphorylation (P-RIPK3 and P-MLKL, respectively) in Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with MCMV-M45mutRHIM (left) or HSV-1 (right) at m.o.i.= 5 for the indicated hours.Infected cells were collected and subjected to immunoblotting with the indicated antibodies.g, Viral replication of MCMV-M45mutRHIM and HSV-1 in Oasl1 +/+ (n = 4 for MCMV-M45mutRHIM, n = 3 for HSV-1) and Oasl1 -/- (n = 2) primary fibroblasts were determined by plaque assays by titrating culture supernatants at the indicating time points.Data are representative of two (b,f,g) or three (c-e) independent experiments.For c-e,g, data are presented as the mean ± s.e.m.Statistical analyses were performed using two-tailed unpaired t-test (d) or two-way analysis of variance (ANOVA) (c-e,g).NS, not significant.

OASL chaperones the assembly of the RIPK3-ZBP1 necrosome
The RHIM-mediated interaction between ZBP1 and RIPK3 forms a non-canonical necrosome during virus infection 6,41,42 .More recently, the Zα domains of ZBP1 were reported to be essential for ZBP1-mediated necroptosis, but the precise role of the Zα domains in RIPK3-ZBP1 interactions during virus infection remains elusive 43,44 .To test whether OASL might play a role in the assembly of the RIPK3-ZBP1 necrosome, MCMV-M45mutRHIM-infected Oasl1 +/+ and Oasl1 -/-primary fibroblasts were collected at 8 h.p.i. for immunoprecipitation with an anti-RIPK3 antibody.RIPK3-ZBP1 interaction was readily detected in Oasl1 +/+ fibroblasts, whereas the interaction was barely detected in Oasl1 -/- fibroblasts (Fig. 2a).In addition, the RIPK3-OASL1 interaction was evidently detected in Oasl1 +/+ fibroblasts (Fig. 2a).Immunoblotting with an anti-PKR antibody was included as a negative control.The RIPK3-ZBP1 interaction was further probed in Oasl1 +/+ and Oasl1 -/- primary fibroblasts by in situ proximity ligation assay (PLA).Oasl1 +/+ primary fibroblasts displayed numerous PLA puncta as early as 4 h.p.i. and gradually increased to a peak at 6 h.p.i., whereas Oasl1 -/-primary fibroblasts exhibited minimal numbers of PLA puncta during the course of infection (Fig. 2b,c and Extended Data Fig. 2a,b).These data suggest that OASL is a crucial mediator in the formation of the RIPK3-ZBP1 necrosome during virus-induced necroptosis.
To further characterize the interaction of OASL and RIPK3, we truncated OASL into the N-OAS domain and the C-UBL domain and identified that RIPK3 bound to N-OAS (Fig. 2d).The serine/ threonine kinase RIPK3 has an N-terminal kinase domain and a C-terminal proline-rich region adjacent to the single RHIM (Fig. 2e).Co-immunoprecipitation revealed that the C-terminal proline-rich/ RHIM-containing domain, but not the N-terminal kinase domain, of RIPK3 interacted with OASL1 as efficiently as full-length RIPK3 (Fig. 2e).Notably, human OASL also effectively interacted with human ZBP1 in a transient expression assay (Extended Data Fig. 2c).ZBP1 carries two Z-DNA-binding domains (Zα and Zβ) at the N terminus and a pair of adjacent RHIM motifs at the C terminus (Fig. 2f).Owing to the small size of the Zα and Zβ domains, we utilized a yeast two-hybrid system to dissect the protein-protein interaction and revealed that the N-terminal Zα domain of ZBP1 bound to OASL1 as effectively as full-length ZBP1 (Fig. 2f).Furthermore, ZBP1 specifically interacted with OASL1, but not OASL2, and the C-UBL domain of OASL1 was crucial for the interaction with ZBP1 (Extended Data Fig. 2d).To test the functional consequence of these interactions, Oasl1 -/-primary fibroblasts were complemented with full-length OASL or with the N-OAS or C-UBL domain of OASL1, followed by MCMV-M45mutRHIM or HSV-1 infection to induce necroptosis.Compared with expression of the control vector, expression of full-length OASL1 readily enhanced RIPK3-mediated MLKL Ser345 phosphorylation in Oasl1 -/-fibroblasts infected with MCMV-M45mutRHIM or HSV-1 (Fig. 2g).Subsequently, full-length OASL1 expression in Oasl1 -/-primary fibroblasts resulted in significantly higher RIPK3-mediated cell death in HSV-1-infected Oasl1 -/-fibroblasts (Fig. 2g,h).However, neither N-OAS nor C-UBL expression alone was able to reach the level of MLKL phosphorylation and necrotic cell death as robustly as full-length OASL1 expression.This result highlights the requirement of full-length OASL to induce maximal level of necroptosis upon virus infection.Overall, these data indicate that the interactions of OASL with RIPK3 and ZBP1 through its N-OAS and C-UBL domains, respectively, are crucial for RIPK3-ZBP1 assembly and virus-induced necroptosis, thereby establishing OASL as a chaperone for the formation of the non-canonical necrosome (Extended Data Fig. 2e).
Fig. 2 | OASL chaperones the assembly of the RIPK3-ZBP1 necrosome complex.a, Oasl1 +/+ and Oasl1 -/-primary fibroblasts were infected with MCMV-M45mutRHIM (m.o.i.= 5) for 0 or 8 h.Whole-cell lysates were immunoprecipitated using an anti-RIPK3 antibody, followed by immunoblot analysis using the indicated anti-ZBP1, anti-OASL1, anti-RIPK3 or anti-PKR antibody.b,c, In situ PLA using anti-RIPK3 and anti-ZBP1 antibodies in Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with MCMV-M45mutRHIM (m.o.i.= 5) for 0 or 6 h.Fluorescence microscopy was used to detect the discrete fluorescent PLA signals (red dots).Representative images of dots indicate the interaction of endogenous RIPK3 and ZBP1.Scale bar, 10 μm.c, Quantification of the dot signals of b per cell (n = 32 cells).Each dot indicates a single cell.d,e, HEK 293T cells were transfected with the indicated Flag, HA, V5 or untagged constructs, and cell lysates were immunoprecipitated with anti-RIPK3 (d) or anti-HA antibodies (e).Immunoprecipitates and whole cell extracts (input) were analysed by immunoblotting with the indicated antibodies.f, AH109 yeasts were transformed with pGBKT7-OASL1 or pACT2-ZBP1 constructs.Interactions were determined by triple dropout (Trp-Leu-His-) synthetic medium or secreted α-galactosidase activity assay using X-Gal.g, OASL1-mediated phosphorylation of the necroptosis effector MLKL.Oasl1 -/-primary fibroblasts were complemented with empty vector (Vector), HA-tagged full-length OASL1, N-OAS domain or C-UBL domain and infected with MCMV-M45mutRHIM or HSV-1 (m.o.i.= 5) for the indicated times.Total cell lysates were subjected to immunoblot analysis with the indicated antibodies.h, Measurement of necrotic cell death on the basis of released LDH in Oasl1 -/-primary fibroblasts reconstituted with empty vector or the indicated OASL1 constructs infected with HSV-1 (m.o.i.= 5) at various time points.Data are representative of two (b-f) or three (a,g,h) independent experiments.For c,h, data are presented as the mean ± s.e.m. and statistical analysis was performed using two-way ANOVA.

OASL undergoes LLPS
LLPS drives the formation of membraneless biomolecular condensates, which depends on the presence of intrinsically disordered regions within the proteins 45 .Notably, a computational algorithm-based predictor, PONDR 46 , revealed multiple intrinsically disordered regions distributed throughout the sequences of human OASL, RIPK3 and ZBP1 (Extended Data Fig. 3a).To assess the potential phase separation capability of these proteins, full-length and fragments of OASL fused with enhanced GFP (eGFP), RIPK3-mCherry and ZBP1 tagged with blue fluorescent protein (ZBP1-BFP) were purified from bacteria and subjected to phase separation in vitro.Neither RIPK3 (full-length and RIPK3 295-518 ) nor ZBP1 was able to undergo phase separation (Extended Data Fig. 3b,c).By contrast, OASL formed liquid droplets under physiological pH, salt concentration and temperature at a minimum Article https://doi.org/10.1038/s41556-022-01039-yconcentration of 0.05 μM (Fig. 3a).The frequency and size of the OASL droplets correlatively increased with the increasing concentrations of OASL (Extended Data Fig. 3d).Notably, N-OAS was sufficient to undergo LLPS as efficiently as full-length OASL, whereas C-UBL did not form any liquid droplets (Fig. 3a and Extended Data Fig. 3d).The dynamic activity of OASL droplets was demonstrated by the increase in droplet size over time (Fig. 3b), whereby liquid droplets coalesced after coming into contact with one another to form larger droplets (Fig. 3c).Furthermore, a time-course spectrometric assay that measures protein turbidity showed a considerable increase in OASL or N-OAS phase separation, but not for C-UBL (Fig. 3d).In accordance with previous studies 47,48 , increasing the salt concentration or incubation with the aliphatic chemical 1,6-hexanediol considerably disrupted OASL droplet formation (Extended Data Fig. 3e,f).Conversely, as divalent cations control phase separation 49,50 , increasing the concentration of Mg 2+ ions increased the size of OASL droplets (Extended Data Fig. 3g).
Refractive index (RI), a measure of intrinsic optical property, enables the characterization of intracellular structures in a label-free and noninvasive manner 51,52 .A combination of three-dimensional (3D) holotomographic and fluorescence imaging of purified OASL-GFP liquid droplets defined OASL droplets at specific RI values, which enabled quantitative measurement of the morphological and biochemical properties of OASL droplets (Fig. 3e,f).3D RI tomograms quantitatively demonstrated the increases in volume and surface area of the OASL droplets, as well as the mass and concentration of OASL within the droplets after phase-separation induction.The results correlated with the increased mean RI values (Fig. 3f), which indicated that the droplets fuse over time.In addition, the average sphericity index of the OASL droplets was 0.8368 ± 0.005769 after phase-separation induction, which indicated that the droplets are a near-perfect spherical shape (Fig. 3f).These results suggest that OASL, but not RIPK3 or ZBP1, forms liquid droplets that are fused over time and increase in size and mass.
N-OAS that sufficiently undergoes phase separation contains a dsRNA-binding groove 53 .Notably, OASL droplet formation increased in frequency following treatment with poly(I:C) low molecular weight (LMW), and a large increase in frequency and size was observed after longer treatment with poly(I:C) high molecular weight (HMW) in vitro (Fig. 3g).As seen with OASL-GFP, fluorescence-labelled untagged human OASL, as well as mouse OASL1-GFP, formed phase-separated droplets that were further enhanced after poly(I:C) HMW treatment (Extended Data Fig. 3h-i).Accordingly, RNase A treatment disrupted in vitro OASL phase separation (Fig. 3g).Furthermore, a dsRNA-binding-deficient OASL mutant (OASL-RK; R45E, K66E, R196E and K201E) failed to undergo LLPS in vitro (Fig. 3h).These results indicate that binding of dsRNA contributes to the in vitro phase separation of OASL.

OASL phase condensates after virus infection
To assess the biological role of OASL phase separation in the context of virus infection, Oasl1 -/-primary fibroblasts expressing WT OASL1-mCherry or the dsRNA-binding mutant OASL1-RK-mCherry were infected with MCMV-M45mutRHIM.WT OASL1-mCherry was observed as discrete cytosolic foci after MCMV-M45mutRHIM infection that specifically colocalized with dsRNA-containing granules stained by the dsRNA-specific antibody J2 (ref.54) (Fig. 3i).By contrast, the OASL1-RK mutant showed little or no cytoplasmic foci formation upon MCMV-M45mutRHIM infection (Fig. 3j).We next examined the dynamics of OASL1 foci in virus-infected live cells using fluorescence recovery after photobleaching (FRAP) assay.Cytoplasmic foci stained with SYTO Blue, a cell-permeable double-stranded nucleic acid dye, were extensively colocalized with OASL1-mCherry foci in MCMV-M45mutRHIM-infected Oasl1 -/-primary fibroblasts (Fig. 3k).After partially photobleaching a section of the SYTO-stained OASL1-mCherry foci, the fluorescent signal underwent rapid and near-complete recovery by 120 s after bleaching (Fig. 3k, bottom, and Supplementary Video 1).These OASL droplets featured dynamic liquid-like phase condensation and continuous exchange between the foci and the surrounding environment during virus-induced necroptosis (Supplementary Video 1).To further assess whether the OASL droplets display propensity for liquid-to-solid-like transition, FRAP curves were acquired during early and late time points of MCMV-M45mutRHIM infection.Notably, the fluorescence recovery of the droplets was faster and higher at 4 and 6 h.p.i.than at 8 h.p.i.(Fig. 3l, Extended Data Fig. 3j,k and Supplementary Videos 1-3).As liquid droplets exhibit changes in viscoelasticity during droplet maturation 37 , our data suggest that OASL1 foci may manifest a transition from a liquid-like state to a gel-like state over time during virus infection.Overall, these results demonstrate that OASL dynamically phase condensates into dsRNA-containing liquid droplets during virus-induced necroptosis.

OASL phase condensate promotes RIPK3 amyloid-like fibril formation
Notably, staining with thioflavin T (ThT), an aromatic cross β-sheet-specific dye for amyloid-like structures, detected endogenous RIPK3 foci in MCMV-M45mutRHIM-infected Oasl1 +/+ primary fibroblasts that resembled previously reported RIPK3 amyloid fibrils (Fig. 5a) (ref.18).By contrast, MCMV-M45mutRHIM-infected Oasl1 -/- or mock-infected Oasl11 +/+ and Oasl1 -/-primary fibroblasts showed diffuse cytoplasmic localization of endogenous RIPK3 without any distinct ThT-positive staining (Fig. 5a and Extended Data Fig. 5a).Furthermore, RIPK3 295-518 -mCherry fibril formation substantially increased after in vitro incubation with OASL in a dose-dependent manner (Extended Data Fig. 5b,c).In particular, KCl was more efficient in inducing OASL-mediated RIPK3 295-518 -mCherry fibril formation than NaCl (Extended Data Fig. 5c).Finally, ThT-positive RIPK3 295-518 -mCherry fibrils were readily observed after in vitro incubation with OASL or N-OAS, but not with C-UBL (Fig. 5b and Extended Data Fig. 5d).These data collectively indicate that the N-OAS domain of OASL is vital for efficient RIPK3 fibril formation.Phase-separated structures provide a platform for protein nucleation, which often results in a transition from a highly mobile liquid-state to a gel-like state owing to the increase in viscoelasticity 37,38,56,57 .High-resolution transmission electron microscopy (TEM) showed that OASL formed dense, fibril-like structures inside the liquid droplets (Fig. 5c).Incubation of OASL with RIPK3 295-518 induced amyloid-like fibril formation within the OASL liquid droplets (Fig. 5c).These RIPK3 295-518 amyloid fibrils, detected by anti-RIPK3 immunogold labeling, within the OASL droplets were dense and extensively branched following incubation with ZBP1 (Fig. 5c and Extended Data Fig. 5e).3D electron tomograms reconstructed from consecutive virtual sections of TEM images with incrementally tilted angles revealed that whereas RIPK3 295-518 alone formed irregular and short fibril intermediates 18 , incubation with ZBP1 or OASL led to elongated amyloid fibril formation (Fig. 5d and Extended Data Fig. 5f,g).In particular, incubation with OASL led to highly ordered and homomorphic amyloid formation, whereas incubation with the RHIM-containing ZBP1 resulted in irregular and polymorphic amyloid structures (Fig. 5d and Extended Data Fig. 5f,g).Finally, RIPK3 295-518 amyloids were substantially ordered and highly divaricated after incubation with both OASL and ZBP1 (Fig. 5d and Extended Data Fig. 5f,g).Time-dependent and interaction-dependent amyloidogenesis of RIPK3 295-518 was noted by the growth of RIPK3 295-518    5h).Holotomography imaging also revealed that RIPK3 increasingly nucleated over time as OASL droplets grew in size and concentration in vitro.This RIPK3 nucleation was not observed after incubation with ZBP1 alone (Fig. 5e and Extended Data Fig. 5i-k which highlights the role of OASL phase condensation in nucleating RIPK3 and promoting RIPK3 amyloid fibril formation.The growth and maturation of RIPK3 amyloid-like fibrils within the OASL droplets were further evidenced in MCMV-M45mutRHIM-infected primary fibroblasts by the colocalization of ThT-positive OASL1 and RIPK3 foci and the increasing ThT signal over time (Fig. 5f).
To further demonstrate that fibrils of and activated RIPK3 directly transduce downstream signalling to its substrate MLKL, an in vitro kinase reaction was performed after induction of phase separation of OASL, RIPK3 and ZBP1 followed by the addition of MLKL and ATP to induce the kinase reaction.OASL phase separation regulated RIPK3 autophosphorylation, which subsequently induced MLKL phosphorylation (Fig. 5g).However, the RHIM-mediated interaction between RIPK3 and ZBP1 was not sufficient for inducing RIPK3 amyloid fibril formation and MLKL phosphorylation without OASL (Fig. 5d,e,g and Extended Data Fig. 5f-k).These data demonstrate that the recruitment of RIPK3 and ZBP1 into OASL droplets results in pervasive amyloid formation of RIPK3 during virus infection, which indicates that OASL phase condensation is a spatial hub for nucleating RIPK3 to accelerate amyloid fibril formation.
As a consequence of necroptosis, levels of released interleukin-1α (IL-1α) in the serum were considerably higher in Oasl1 +/+ mice infected with either MCMV-WT or MCMV-M45mutRHIM compared with those in Oasl1 -/-mice (Fig. 6d and Extended Data Fig. 6f).Consistent with the reduced footpad swelling and serum IL-1α levels, MCMV-WT-infected or M45mutRHIM-infected Oasl1 -/-mice showed a marked reduction in immune cell infiltration and epidermal hyperplasia (Fig. 6e and Extended Data Fig. 6g,h).Indeed, immunohistochemistry of virus-infected mouse footpads showed intense phospho-RIPK3 and phospho-MLKL signals in the dermis region of the footpads from MCMV-WT-infected or MCMV-M45mutRHIMinfected Oasl1 +/+ mice, whereas the signals were substantially reduced in Oasl1 -/-mice (Fig. 6f,g), which suggests that OASL1 positively regulates RIPK3 and MLKL activation.Collectively, the markedly reduced activation of the key necroptotic kinase RIPK3 and the effector MLKL upon virus infection in Oasl1 -/-mice suggests that OASL1 has a pivotal role in the execution of necroptosis in vivo to restrict MCMV replication and elicit antiviral inflammatory responses.

Discussion
Robust activation of necroptosis during virus infection requires contemporaneous engagement of intact TNF and type I IFN signalling, which suggests that there is a node of pathway crosstalk between the two signalling pathways.Here we demonstrated that the IFN-inducible protein OASL is a previously undescribed component of the non-canonical necrosome complex that governs a sequential event during virus-induced necroptosis.Our study revealed that OASL undergoes dsRNA-dependent LLPS, which serves as a scaffold to facilitate the assembly of the RIPK3-ZBP1 necrosome during virus infection.These OASL liquid droplets effectively recruit RIPK3 and ZBP1, providing a spatially partitioned platform for the amyloidogenic protein RIPK3 to nucleate and induce amyloid fibril formation and prompt activation during immediate challenges (Fig. 7h).Subsequently, activated RIPK3 phosphorylates MLKL to execute high levels of virus-induced necroptosis and proinflammatory responses.A rigorous structural study is needed to further characterize the formation, as well as its functionality Biomolecular condensates formed by LLPS through multivalent protein-nucleic acid interactions enable spatially segregated molecular platforms to fine-tune intricate biological signalling.The physical properties of droplet-like phase separation of RNA-binding proteins is not solely dependent on the negative charge of its target RNAs 60,61 .We found that OASL formed larger phase-separated condensates following treatment with poly(I:C) HMW than with poly(I:C) LMW in vitro, which implies that the length of dsRNA, and hence the multivalency, Article https://doi.org/10.1038/s41556-022-01039-ycould be an important factor that determines the magnitude of OASL droplet formation 61 .Likewise, the availability of cytosolic viral dsRNA during virus infection may determine the duration and frequency of OASL LLPS.The identification of specific target dsRNA in the future can provide insights into our understanding of not only how the assembly of OASL is spatially patterned with viral dsRNA but also what secondary structure of target RNAs may trigger the necroptotic activity of OASL 60 .
Although RIPK3 is not able to undergo phase separation on its own, it has been reported that RIPK3 can heterocomplex with its RHIM-containing adaptor molecules, RIPK1 or ZBP1, to form amyloid-like structures in vitro, ultimately forming an energetically favourable conformation 18 .These RHIM-RHIM hetero-amyloids display high energetic preference over RIPK3 homo-amyloids, thereby implicating RIPK3 hetero-amyloids for a broader range of signalling activities before the formation of homo-amyloids 19 .These findings collectively suggest that an efficient assembly of higher-order signalling platforms, such as amyloid-like fibrous structures, often requires scaffolding molecules that facilitate the polymerization of subunits to become energetically favourable and consequently execute condition-specific signals.Here we showcased OASL liquid droplets that serve as a molecular platform to provide a spatial segregation region for RIPK3 to induce polymerization and amyloid-like fibril formation.Our model suggests that the OASL-RIPK3 interaction enhances the formation of the RIPK3 amyloid-like fibril signalling complex, which prolongs RIPK3 activation and drives high levels of MLKL phosphorylation, ultimately inducing robust necroptosis during virus infection.As OASL nucleates RIPK3, together with ZBP1 via RHIM-mediated interactions, within its liquid droplet, stronger intermolecular and intramolecular interactions within the droplets may allow RIPK3 to promptly achieve the minimal binding proximity and concentration for homo-oligomerization, triggering the formation of amyloid fibrils.These polymeric platforms may then exert augmented forces to trigger efficient signal transduction activity.Indeed, higher-order assemblies like amyloid fibrils have gained attention as a platform to induce signal transduction, such as apoptosis-associated spec-like protein (ASC)-dependent inflammasome assembly or FAS-associated death domain protein (FADD) and caspase-8 in apoptosis 62,63 .In-depth study of the structural perspectives of OASL liquid phase condensates is needed to elucidate how this RIPK3-ZBP1-OASL necrosome regulates condition-specific signal transduction activity.
In summary, this study elucidated a previously uncharacterized mechanism of RIPK3 activation during virus-induced necroptosis and identified a vital role of the IFN-inducible protein OASL in exploiting phase separation to activate executioner signals by scaffolding the assembly of RIPK3 and ZBP1.OASL-mediated necroptosis is an effector pathway of the IFN-mediated antiviral response that reduces virus burden by initiating proinflammatory cell death: necroptosis.This pathway is functionally distinct from the previously described role of the OAS protein family in the 2-5A synthetase-directed RNase L pathway as well as the role of OASL in IFN production.Although OASL has been implicated in differential aspects of antiviral immunity, herein we described an antiviral mechanism of OASL by defining the biochemical characteristics of OASL.This crucial role of OASL in the crosstalk between the OAS family proteins and necroptosis brings insights into our understanding of its function as a regulator of necroptosis-mediated inflammatory response to combat pathogenic challenges.
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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.© The Author(s) 2023 https://doi.org/10.1038/s41556-022-01039-y in an 8-well chamber coverslip.After overnight incubation at 37 °C, cells were infected with MCMV-M45mutRHIM for the indicated times, washed with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized for 10 min at room temperature.The PLA pair of monoclonal rabbit RIPK3 (Cell Signaling Technology) and monoclonal mouse ZBP1 (Adipogen) antibodies were incubated overnight at 4 °C.Images were captured using a fluorescence microscope (Keyence, BZ-X710 series) and quantified using ImageJ (Fiji) software.

Protein expression and purification
Expression constructs were generated in a pGEX-6p-1 vector to contain a PreScission-cleavable site.eGFP C-terminal fused full-length human OASL, N-OAS and C-UBL; mCherry C-terminal fused full-length human RIPK3 and C-terminal unstructured region (RIPK3 295-518 ); and BFP C-terminal fused full-length human ZBP1 were expressed and purified from Escherichia coli strain BL21 DE3 (pLys).E. coli harbouring a glutathione S-transferase (GST)-tagged plasmid encoding fluorescence-fused human OASL or RIPK3 was induced by adding 0.4 mM IPTG at 16 °C for less than 16 h.Bacteria pellets were collected and lysed in lysis buffer containing 10 mM HEPES (pH 7.4), 450 mM KCl, 1 mg ml -1 zymolyase, 1 mM DTT, 0.1% Triton X-100 and protease inhibitor cocktail without EDTA (Roche) at room temperature for 1 h and at 4 °C for 4 h, followed by centrifugation at 16,000g, 4 °C for 30 min.RNase A (1 mg ml -1 ) was supplemented in the lysis buffer for human ZBP1-BFP protein purification.Supernatant was collected for overnight purification using glutathione sepharose 4B beads (Cytiva).The GST tag was removed using PreScission protease (GE Healthcare), incubating at 4 °C for 18 h.The protein concentration of the fractions containing fluorescence-tagged proteins were measured by SDS-PAGE with BSA standards.

In vitro phase-separation assay
Purified fluorescence-tagged protein in 450 mM salt buffer was diluted to 125 mM KCl in 10 mM HEPES, pH 7.4, in 96-well plates.The plates were incubated at 37 °C and images were captured at indicated time points after incubation.All phase separation of purified protein was performed in phase-separation buffer (10 mM HEPES, pH 7.4, 125 mM KCl) without adding any crowding reagents.For RIPK3 295-518 -mCherry and ZBP1-BFP recruitment to OASL-GFP droplets, variant GFP-tagged OASL proteins, mCherry-tagged RIPK3 295-518 and BFP-tagged ZBP1 were mixed and incubated in 96-well plates at 37 °C for 1 h at the indicated concentrations in the phase-separation buffer.Image acquisition of phase separation was performed using a fluorescence microscope (Keyence, BZ-X710 series) under ×20, ×40 and ×60 1.49-NA oil-immersion objectives.At least four independent imaging areas were analysed for each condition of each replicate.Data shown are representative of at least three independent experiments across five protein preparations.

In vitro fibril formation assay with ThT measurement
In vitro fibril formation assays were performed as previously described 65 .In brief, 30 μM ThT (Abcam) was added to the mixtures of 0.5 μM RIPK3 295-318 -mCherry with different amounts of purified OASL and incubated at 37 °C for 16 h.ThT binding was excited using a wavelength of 430 nm and evaluated by monitoring the emission fluorescence spectrum at wavelengths from 480 nm to 550 nm.

Electron microscopy and immunogold labelling
A total of 5 μl of individual or mixtures of OASL-GFP, RIPK3 295-518 -mCherry and ZBP1-BFP proteins were spotted onto Parafilm, and a carbon-formvar-coated copper grid (200 mesh, Ted Pella) was placed on the surface of the droplet.After 1 min of absorption at room temperature, the sample was wicked off the grid by touching the filter paper.The grids were washed with deionized water three times and stained by floating the grids on a droplet of 2% uranyl acetate.For immunogold labelling, the grids were blocked in 1% BSA in PBS for 10 min, followed by staining with rabbit monoclonal anti-RIPK3 antibody (Cell Signaling Technology) at 1:100 dilution for 2 h.Next, the grids were washed with 1% BSA in PBS for six times, followed by incubation with secondary antibodies (anti-rabbit conjugated with 6 nm and 10 nm gold particles) for 30 min.The grids were washed with PBS six times, followed by deionized water washing and stained with 2% uranyl acetate.Grids were examined and imaged using a transmission electron microscope (FEI, Tecnai G2 Spirit) at 120 kV operated by the Imaging Core of the Lerner Research Institute at the Cleveland Clinic.

Transmission electron tomography
Carbon-formvar-coated copper grids (200 mesh, Ted Pella) were coated with 0.1% (w/v) poly-l-lysine (Sigma) for 10 min and blotted to remove residual liquid.After drying, the grids were incubated with a droplet of sample for 5 min and subsequently washed with distilled water for 5 s, followed by 2% uranyl acetate staining.Afterwards, the grids were treated with 10 nm gold particles (741957, Sigma Aldrich) in water (1:1) and blotted.Single-axis tilt electron tomography was recorded from −60° to 60° using tilt increments of 2° using a transmission electron microscope (FEI, Tecnai F20) at 200 kV equipped with a 4,096 × 4,096 pixel CMOS camera (TemCam-F416, TVIPS).The image stack was generated from tilt images and subjected to tomographic reconstructions using the weighted back-projection algorithm in IMOD (v.4.11.2).The 3D model of amyloids was manually traced from the virtual sections of the tomogram.

In vitro kinase assay
In vitro kinase assays were performed as previously described 2,25 .Human RIPK3, kinase-dead RIPK3-K50A and phosphorylation-dead RIPK3-S227A mutant proteins were purified from E. coli using glutathione sepharose beads.After removing the GST tag, 1 ng of RIPK3 protein was mixed with different amounts of purified human OASL protein (5-50 ng) in kinase reaction buffer (20 mM HEPES, pH 7.4, 1 mM DTT, 20 mM MnCl 2 , 20 mM MgCl 2 , 1 mM EDTA and 100 μM ATP) supplemented with phosphatase inhibitor cocktail (Sigma).For RIPK3 and MLKL in vitro kinase assays, OASL-GFP and ZBP1-BFP were purified from E. coli, and GST-RIPK3 (Sigma) and MLKL (Cusabio) were commercially purchased.GST-RIPK3 (10 ng), ZBP1 (50 ng) and OASL (50 ng) were subjected to phase separation at 37 °C before kinase reaction with MLKL and the kinase reaction buffer (50 ng).The kinase reaction was performed at 30 °C for 45 min in a total volume of 20 μl, stopped with 20 μl of 2× Laemmli sample buffer, boiled for 5 min and subjected to immunoblotting analysis with the indicated anti-phospho antibodies.

Immunostaining and confocal microscopy
Primary tail fibroblasts were seeded onto an 8-well chamber slide (Nunc Lab-Tek II Chamber Slide system) for overnight culture at 37 °C and infected with a m.o.i.= 5 of MCMV-M45mutRHIM mutant virus for the indicated times.ThT (25 μM) was added to cells 1 h before fixation 18 .Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked with 0.5% BSA in PBS containing 0.1% saponin at room temperature.Cells were then incubated with the indicated antibodies overnight at 4 °C, followed by washing with PBS-T three times at room temperature and incubation with anti-mouse IgG Cascade Blue, anti-mouse IgG Alexa Fluor 568, anti-rabbit IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 568 (Thermo Fisher) at 1:1,000 dilution.The coverslips were mounted and counterstained using Pro-Long Gold Antifade mountant with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher).All images were captured using identical settings on a Nikon laser scanning confocal microscope or a Leica SP8 confocal microscope with a ×60 oil-objective, and processed using Nikon's NIS elements, Leica's LAS X and ImageJ software.

Fluorescent recovery after photobleaching assay
Cellular FRAP experiments were performed using a LSM880 Airyscan microscope at 37 °C in a live-cell imaging chamber.Oasl1 -/-primary https://doi.org/10.1038/s41556-022-01039-yfibroblasts expressing OASL1-mCherry were grown on glass-bottom dishes (MatTek) overnight to reach the desired density.Cells were then inoculated with MCMV-M45mutRHIM for 2 h.OASL1-mCherry condensates were identified by live-staining with SYTO 45 (Thermo Fisher) 30 min before bleaching and partially photobleaching with 50% laser power using a 560 nm laser.Time-lapse images were acquired with 2-s intervals over 2 min after photobleaching.Images were processed using ImageJ, and FRAP data were fit to a single exponential model using GraphPad Prism.The background intensity was subtracted, and values are reported relative to pre-bleaching time points.

Tomography and analysis of parameters
A commercial optical diffraction tomographic system with modifications for 3D fluorescence imaging (HT-2, Tomocube) was utilized to measure the RI tomograms of individual liquid-like droplets.All morphological and biochemical parameters were quantitatively obtained from RI tomograms, as previously described 66 , using the TomoStudio software.

Infection of mice and organ collection
Oasl1 +/+ and Oasl1 -/-mice were anaesthetized with isoflurane, followed by subcutaneous injection of 10 6 p.f.u.MCMV-WT or MCMV-M45mutRHIM virus into the ventral side of the footpads 5 .Infected mice were observed over a period of 14 days for body weight loss and survival.All experiments were performed with sex-matched mice at 6-8 weeks of age.After euthanasia, organs were collected in PBS and stored at -80 °C until thawed for plaque assays.Experiments involving mice were performed under an approved protocol from the Institutional Animal Care and Use committees at the University of Southern California Keck School of Medicine and Cleveland Clinic.

Histological analysis of mouse tissue
Mouse footpads were collected, fixed in 4% paraformaldehyde for 24 h and transferred to 70% ethanol for storage.Tissue sectioning and immunohistochemistry were performed by the University of Southern California Immunohistochemistry Core facility.In brief, footpads were decalcified in formic acid (Immunocal, StatLab), embedded in paraffin and sectioned at 5 μm thickness.Slides were deparaffinized and either stained with haematoxylin and eosin or antigen-retrieved using retrieval buffer for immunohistochemistry staining.Anti-phospho RIPK3 (Cell Signaling Technology) and anti-phospho MLKL (Cell Signaling Technology) were used for staining.Staining was visualized using streptavidin-HRP (Millipore) and DAB substrate (DAKO and Vector Lab).All immunohistochemistry sections were counterstained with haematoxylin.Images were captured using a bright-field microscope (Keyence, BZ-X710 series).Quantification of phosphorylation signals, epidermatitis or inflamed area were calculated into percentage values (positive signal area versus total area of field of view) on individual footpad cross-sections.

ELISA
Cytokines in serum from animal experiments were quantified using ELISA kits for mouse IL-1α (Invitrogen) according to the manufacturer's protocol.

Statistics and reproducibility
Statistical significance was determined using two-tailed unpaired Student's t-test for two component comparisons and one-way analysis of variance (ANOVA) with Tukey's comparison or two-way ANOVA with Bonferroni's comparison for multi-component comparisons in GraphPad Prism v.9.1.Survival curves were generated using the Kaplan-Meier method.Quantification of area was performed using ImageJ (Fiji).All experiments were repeated as indicated in the figure legends with a minimum of two independent replications.The exact value of n, representing the number of mice or samples in the experiments, is indicated in each figure legend.No statistical method was used to predetermine sample sizes.Sample sizes were chosen on the basis of standard practice in the field.Data distribution was assumed to be normal, but was not formally tested.No data were excluded from the analyses.No randomization or blinding was used.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 3 |
Fig. 3 | OASL undergoes LLPS during virus-induced necroptosis.a, Fluorescence images of droplet formation by GFP-tagged full-length OASL, and the N-OAS and C-UBL domains of OASL in vitro 30 min after induction.b, LLPS of GFP-tagged OASL over time in vitro.c, Time-lapse images of the coalescence of OASL liquid droplets over 120 s (indicated by the arrow).d, Turbidity measurement of GFP-tagged full-length OASL, and the N-OAS and C-UBL domains of OASL over time in vitro.e, Representative 3D RI distribution and fluorescence of OASL droplets before and after incubation at physiological conditions (green, GFP fluorescence; purple, RI tomogram; n = 50 condensates per condition).f, Statistical analyses of morphological (top) and biochemical (bottom) parameters of e (n = 7 condensates per condition).ND, not detected.g,Representative images of OASL-GFP (0.05 μM) phase separation mixed with poly(I:C) LMW (50 μg ml -1 ), poly(I:C) HMW (50 μg ml -1 ) or RNase A (300 μg ml -1 ) for 60 min.h, Droplet formation of full-length OASL-GFP and the dsRNAbinding mutant OASL-RK-GFP.i, Representative images of Oasl1 -/-primary fibroblasts reconstituted with OASL1-mCherry (OASL1-mCh) and infected

Fig. 2 |
OASL interacts with RIPK3 and ZBP1 through its N-OAS and C-UBL domain, respectively.a, In situ proximity ligation assay (PLA) of RIPK3 and ZBP1 in Oasl1 +/+ and Oasl1 -/-primary fibroblasts infected with mock or MCMV-M45mutRHIM (m.o.i.= 5) for the indicated hours.Fluorescence microscope was used to detect the discrete fluorescent PLA signals (red dots).Representative images of dots indicate the interaction of endogenous RIPK3 and ZBP1.b, Quantification of dot signals of a per cell (n = 30 cells).Each dot indicates single cell.c, HEK 293 T cells were transfected with the indicated FLAG or untagged constructs, and cell lysates were immunoprecipitated with anti-FLAG antibody.Immunoprecipitates and whole cell extracts (input) were analyzed by immunoblotting with the indicated antibodies.d, AH109 yeasts were transformed with the indicated pGBKT7 or pACT2 constructs.Interactions were measured by triple dropout (Trp-Leu-His-) synthetic medium or secreted α-galactosidase activity assays using X-αGal.e, Schematic of the interaction of OASL with RIPK3 and ZBP1 through its N-terminus and C-terminus domains, respectively.a-d, Data are representative of two independent experiments with similar results.b, Data are presented as mean ± SEM and statistical analysis was performed using two-way ANOVA.Extended Data Fig. 3 | Liquid-liquid phase separation properties of OASL, RIPK3, and ZBP1.a, Graph of putative intrinsically disordered regions of human OASL, RIPK3, and ZBP1 as calculated by PONDR (VSL2) algorithm.b, Failure of liquid droplet formation of purified mCherry-tagged full-length RIPK3 or C-terminal RIPK3 295-518 .Scale bar, 10 μm.c, Failure of liquid droplet formation of purified BFP-tagged full-length ZBP1.Scale bar, 10 μm.d, In vitro droplet formation by GFP-tagged full-length OASL, N-OAS, or C-UBL protein in a concentration-dependent manner.Incubation was carried out at physiological temperature and buffer for 1 h.Scale bar, 10 μm.b-d, Histogram represent distribution of quantified droplet amounts and diameters of the liquid droplets.e, Effect of KCl concentration on the formation of OASL liquid droplets.Scale bar, 10 μm.f, OASL-GFP droplets treated with increasing concentration of 1,6-hexanediol.Scale bar, 5 μm.g, Increase of Mg 2+ concentration leads to enlargement of OASL droplets.Scale bar, 10 μm.h, In vitro liquid droplet formation of GFP-tagged mouse OASL (OASL1) treated with mock or poly(I:C) HMW (50 μg/ml) for 1 h.Scale bar, 10 μm.i, (Left) Schematic diagram of Sortase A-mediated labeling of FITC at the C-terminus of OASL with Sortase A-recognition motif (LPETGG).(Right) In vitro phase separation of FITC-labeled OASL treated with mock or poly(I:C) HMW (50 μg/ml).Scale bar, 10 μm.j,k, Representative FRAP images of OASL1 foci observed at (j) 4 h.p.i. and (l) 8 h.p.i.(l).SYTO 45-stained OASL1-Cherry foci were chosen for photobleaching.White rectangle box indicates the photobleached and recovered area within the foci.Scale bar, 10 μm.Data are representative of three (b-i) or two (j,k) independent experiments with similar results.Extended Data Fig. 4 | OASL liquid phase separation during virus-induced necroptosis.a, (Left) Representative images of liquid droplet formation of GFP-tagged full-length OASL, N-terminus OAS-like domain, or C-terminus UBL domain with mCherry-tagged RIPK3 295-518 .Scale bar, 5 μm.(Right) Histogram of the size and formation frequency of RIPK3 295-518 -mCherry droplets with or without the presence of GFP-tagged OASL proteins.b, (Left) Full length RIPK3-mCherry undergoes phase separation in the presence of purified OASL or N-OAS, but not C-UBL.Representative images of liquid droplet formation of mCherry-tagged full-length RIPK3 in the presence of full-length OASL, N-OAS or C-UBL. 2 μM of RIPK3-mCherry was mixed with 0.5 μM of purified OASL proteins.Scale bar, 5 μm.(Right) Histogram of the size and formation frequency of RIPK3 droplets.c, Confocal imaging of mock-infected Oasl1 -/-primary fibroblasts reconstituted with mCherry-tagged vector, OASL1, or OASL1 RK .Scale bar, 10 μm.d, (Top) OASL1-mCherry merged image of Fig. 4c with line across the condensate.Scale bar, 10 μm.(Bottom) Line profile of fluorescence intensity indicates colocalization of OASL1, ZBP1, and RIPK3 signal in OASL1-mCherry expressing Oasl1 -/-primary fibroblasts.e, Quantitative analysis of morphological (top) and biochemical (bottom) parameters of OASL-GFP alone, RIPK3 295-518 -mCherry alone, and OASL-GFP with RIPK3 295-518 -Cherry liquid-like droplets before and after incubation at physiological conditions (n = 13 condensates per group).Morphological parameters include volumes, surface areas, and sphericity.Biochemical parameters include dry mass, concentration, and mean RI.ND, not detected.Data are representative of three (a-c) or two (e) independent experiments with similar results.Extended Data Fig. 5 | OASL phase separation promotes RIPK3 amyloid fibrillation.a, Representative images of mock-infected Oasl1 +/+ and Oasl1 -/- primary fibroblasts immunostained for endogenous RIPK3 and amyloid-like structure by anti-RIPK3 antibody and Thioflavin T (ThT), respectively.Scale bar, 10 μm.b, Fibrillation of RIPK3 295-518 -mCherry in vitro with increasing amounts of OASL at physiological (left) KCl or (right) NaCl concentration for 16 h.c, Dose-response curve of OASL inducing RIPK3 295-518 -mCherry fibrillation assessed by ThT emission.d, Quantification of RIPK3 295-518 -mCherry and ThT signal colocalization upon incubation with OASL, N-OAS, or C-UBL.Box plots show the minimum, first quartile, median, third quartile, and maximum with n = 150 droplets per group.e, TEM imaging of RIPK3 295-518 -mCherry fibrils after phase separation with ZBP1-BFP and OASL-GFP, followed by immunogoldlabeling with anti-RIPK3 antibody upon (top) normal or (bottom) denatured condition.Prominent immunogold labeling was observed in denatured condition.f, Virtual sections of RIPK3 amyloid fibrils through the tomogram and overlays with 3D-model at different z-axis positions.g, Virtual sections of RIPK3 295-518 -mCherry fibrils through tomogram at different z-axis positions.h, TEM images of RIPK3 295-518 -mCherry alone or incubated with OASL or OASL and ZBP1 together after 20 or 120 min of phase separation.Scale bar, 200 nm.Insets: magnified views of the red box regions.e-h, Scale bar, 100 nm.i, Representative 3D refractive index distribution of RIPK3 + ZBP1 or RIPK3 + ZBP1 + OASL at the indicated times.RI tomogram: blue (RIPK3), yellow (ZBP1), purple (OASL).(n = 30 condensates per condition).j,k, 3D tomogram quantitative analysis of ZBP1-BFP ( j) and OASL-GFP (k) upon co-incubation with RIPK3 (n = 15 condensates per group).Data are representative of three (a-d) or two (i-k) independent experiments with similar results.e-h, TEM images are representative of at least 8 fields with four independent experiments.j,k, Data are presented as mean ± SEM and statistical analyses were performed using two-way ANOVA.ND, not detected.Extended Data Fig. 6 | Murine OASL1 is required for MCMV infectioninduced necroptosis to restrict viral dissemination in vivo.a, Measurement of footpad swelling caused by subcutaneous footpad injection of Ripk3 +/+