Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation.

Pathogens and cellular danger signals activate sensors such as RIG-I and NLRP3 to produce robust immune and inflammatory responses through respective adaptor proteins MAVS and ASC, which harbor essential N-terminal CARD and PYRIN domains, respectively. Here, we show that CARD and PYRIN function as bona fide prions in yeast and that their prion forms are inducible by their respective upstream activators. Likewise, a yeast prion domain can functionally replace CARD and PYRIN in mammalian cell signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, a conserved fungal NOD-like receptor and prion pair can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion-like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.

PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation.

The NLRP3 inflammasome, which has been linked to human inflammatory diseases, is activated by diverse stimuli. How these stimuli activate NLRP3 is unknown. Here we show that different NLRP3 stimuli lead to disassembly of the trans-Golgi network (TGN). NLRP3 is recruited to the dispersed TGN (dTGN) through ionic bonding between its conserved polybasic region and negatively charged phosphatidylinositol-4-phosphate (PtdIns4P) on the dTGN. The dTGN then serves as a scaffold for NLRP3 aggregation into multiple puncta, leading to polymerization of the adaptor protein ASC, thereby activating the downstream signalling cascade. Disruption of the interaction between NLRP3 and PtdIns4P on the dTGN blocked NLRP3 aggregation and downstream signalling. These results indicate that recruitment of NLRP3 to dTGN is an early and common cellular event that leads to NLRP3 aggregation and activation in response to diverse stimuli.

Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids.

Microbial infections are recognized by the innate immune system through germline-encoded pattern recognition receptors (PRRs). As most microbial pathogens contain DNA and/or RNA during their life cycle, nucleic acid sensing has evolved as an essential strategy for host innate immune defense. Pathogen-derived nucleic acids with distinct features are recognized by specific host PRRs localized in endolysosomes and the cytosol. Activation of these PRRs triggers signaling cascades that culminate in the production of type I interferons and proinflammatory cytokines, leading to induction of an antimicrobial state, activation of adaptive immunity, and eventual clearance of the infection. Here, we review recent progress in innate immune recognition of nucleic acids upon microbial infection, including pathways involving endosomal Toll-like receptors, cytosolic RNA sensors, and cytosolic DNA sensors. We also discuss the mechanisms by which infectious microbes counteract host nucleic acid sensing to evade immune surveillance.

NLRP9 in innate immunity and inflammation.

The nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) are a family of evolutionarily conserved proteins. Several members of NLRs, notably NLRP1, NLRP3 and NLRC4, are able to form cytosolic oligomeric signalling platforms termed inflammasomes to mediate immune response towards pathogens, damage and stress. However, the functions of many NLRs still remain elusive. In the past few years, a couple of less-characterized NLR members are emerging as important signalling molecules with fundamental functions in host defence and inflammation. Among them, NLRP9 is an NLR originally proposed to be expressed and function solely in the reproductive system. Recent evidence has suggested that NLRP9 is also capable of initiating inflammasome formation in the intestine to restrict replication and damage brought by rotavirus infection. Here, we highlight the latest progress in characterization of the role of NLRP9 in infectious and inflammatory diseases, as well as the newest crystallographic and biochemical studies on NLRP9. Finally, we discuss some important questions remained to be answered regarding the molecular and cellular mechanisms governing NLRP9's function in innate immunity and inflammation.

Direct, noncatalytic mechanism of IKK inhibition by A20.

A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20's seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.

SARS-CoV-2 ORF3a drives dynamic dense body formation for optimal viral infectivity.

SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the trans-Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV. 3DBs recruit the viral structural proteins spike (S) and membrane (M) and undergo dynamic fusion/fission to facilitate efficient virion assembly. A recombinant SARS-CoV-2 virus with an ORF3a mutant specifically defective in 3DB formation showed dramatically reduced infectivity for both extracellular and cell-associated virions. Our study uncovers the crucial role of 3DB in optimal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.

Comparison of ureteral stone measurements for predicting the efficacy of a single session of extracorporeal shockwave lithotripsy: one-, two-, and three-dimensional computed tomography measurements.

The objective of this study was to compare the value of one-, two- and three-dimensional computed tomography (CT) measurements for predicting the efficacy of a single session of extracorporeal shock wave lithotripsy (ESWL) in patients with a single ureteral stone. A total of 165 patients were included based on the inclusion and exclusion criteria. Different models were constructed using a combination of patients' clinical data and measurements obtained by manual sketching and automated extraction software. Multivariate logistic regression was used to develop the models. Receiver operating characteristic curves were used to assess the performance of the models. There was good interobserver agreement for all measurements in different dimensions (P < 0.001). We also found that hydronephrosis, the largest diameter, the largest area, volume, and mean CT value were significantly greater in the failure group than in the success group (P < 0.01). Furthermore, all sizes and CT measurement values were found to be independent predictors for predicting efficacy after one session of ESWL (P < 0.05). In addition, the multivariate logistic analysis showed that the area under the curve (AUC) for two-dimensional and three-dimensional measurements was superior to that of one-dimensional measurement (P < 0.01). However, when size alone was included as a measurable predictor, there was no significant difference in the AUC among the one-, two-, and three-dimensional measurements (P > 0.05). In summary, after adjusting for clinical data, two- and three-dimensional measurements combining ureteral stone size and CT values were found to be the best predictors of ESWL efficacy, and software-based three-dimensional measurements should be considered to avoid interobserver variability in clinical practice.

Selection of global Metarhizium isolates for the control of the rice pest Nilaparvata lugens (Homoptera: Delphacidae).

BACKGROUND: This study was initiated to search for fungal candidates for microbial control of brown planthopper (BPH) Nilaparvata lugens Stål, to which little attention has been paid in the past two decades. RESULTS: Thirty-five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and M. flavoviride Gams & Rozsypal from different host insects worldwide were bioassayed for their lethal effects against third-instar BPH nymphs at 25 degrees C and a 14:10 h light:dark photoperiod at ca 1000 conidia mm(-2). On day 9 post-treatment, mortality attributable to mycosis ranged from 6.5 to 64.2% and differed significantly among the tested isolates with no apparent relationship to their host origin. Only two BPH-derived M. anisopliae isolates from the Philippines (ARSEF456) and Indonesia (ARSEF576) killed >50% of the nymphs. Both isolates were further bioassayed for time-concentration-mortality responses of the nymphs to the sprays of 19-29, 118-164 and 978-1088 conidia mm(-2) in repeated bioassays. The resultant data fitted a time-concentration-mortality model very well. Their LC(50) values were estimated as 731 and 1124 conidia mm(-2) on day 7 and fell to 284 and 306 conidia mm(-2), respectively, on day 10. CONCLUSION: The two M. anisopliae isolates are potential biocontrol agents of BPH for further research. This is the first report of the lethal effects of global Metarhizium isolates on the rice pest.

Regulation of NF-κB by ubiquitination.

The nuclear factor κ enhancer binding protein (NF-κB) family of transcription factors regulates the expression of a large array of genes involved in diverse cellular processes including inflammation, immunity and cell survival. Activation of NF-κB requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Studies in the past few years have provided new insights into the mechanisms underlying regulation of NF-κB by ubiquitination, including the involvement of multiple linkages of ubiquitin, the essential role of ubiquitin binding, and the function of unanchored polyubiquitin chains. In this review, we will focus on recent advances in understanding the role of ubiquitination in NF-κB regulation in various pathways.

MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades.

RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI:http://dx.doi.org/10.7554/eLife.00785.001.

Evaluation of alternative rice planthopper control by the combined action of oil-formulated Metarhizium anisopliae and low-rate buprofezin.

BACKGROUND: High resistance of brown planthopper (BPH) Nilaparvata lugens Stål to common insecticides is a challenge for control of the pest. An alternative control strategy based on the combined application of fungal and chemical agents has been evaluated. RESULTS: Three gradient spore concentrations of oil-formulated Metarhizium anisopliae (Metschnikoff) Sorokin (Ma456) were sprayed onto third-instar nymphs in five bioassays comprising the low buprofezin rates of 0, 10, 20, 30 and 40 µg mL(-1) respectively. Fungal LC(50) after 1 week at 25 °C and 14:10 h light:dark photoperiod decreased from 386 conidia mm(-2) in the buprofezin-free bioassay to 40 at the highest chemical rate. Buprofezin (LC(50): 1647, 486 and 233 µg mL(-1) on days 2 to 4) had no significant effect on the fungal outgrowths of mycosis-killed cadavers at the low application rates. The fungal infection was found to cause 81% reduction in reproductive potential of BPH adults. In two 40 day field trials, significant planthopper (mainly BPH) control (54-60%) was achieved by biweekly sprays of two fungal candidates (Ma456 and Ma576) at 1.5 × 10(13) conidia ha(-1) and elevated to 80-83% by incorporating 30.8 g buprofezin ha(-1) into the fungal sprays. CONCLUSION: The combined application of the fungal and chemical agents is a promising alternative strategy for BPH control.