Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)–like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo–electron microscopy (cryo-EM) structure of the human NLRP3 PYD filament and identify a pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends designated as A- and B-ends. Coupling a titration polymerization assay to cryo-EM, we demonstrate that ASC adaptor protein elongation on NLRP3 PYD nucleation seeds is unidirectional, associating exclusively to the B-end of the filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 and ASC. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3, ASC, and Caspase-1 proteins.

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1β release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNTblocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNTpartitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand-foot-mouth disease to life-threatening conditions such as pancreatitis, myocarditis and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc result in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3M) and one with partial (CVB3K) resistance. DNA sequence alignment of the resistant virus variant CVB3M with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus's interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3N2139S to sCAR-Fc. Interestingly, development of resistance by CVB3-H3N2139S and the exemplarily investigated CVB3M-clone 2 (CVB3M2) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and the viral replication in comparison to the parental strains. These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single amino acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness. IMPORTANCE The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, thus representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules.