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The most dangerous man of this century has employed the most dangerous weapon yet. Criminal mastermind Harlan DeVane has developed -- and spread -- a deadly, genetically engineered \"superbug\" resistant to all known cures. A microscopic time bomb, it will lie dormant inside its human hosts until detonated by specific biochemical triggers.

Dysregulated NLRP3 inflammasome activity results in uncontrolled inflammation, which underlies many chronic diseases. Although mitochondrial damage is needed for the assembly and activation of the NLRP3 inflammasome, it is unclear how macrophages are able to respond to structurally diverse inflammasome-activating stimuli. Here we show that the synthesis of mitochondrial DNA (mtDNA), induced after the engagement of Toll-like receptors, is crucial for NLRP3 signalling. Toll-like receptors signal via the MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, a rate-limiting enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments after exposure to NLRP3 activators. Cytosolic oxidized mtDNA associates with the NLRP3 inflammasome complex and is required for its activation. The dependence on CMPK2 catalytic activity provides opportunities for more effective control of NLRP3 inflammasome-associated diseases. New mitochondrial DNA synthesis links the priming and activation of the NLRP3 inflammasome.

Type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.

Functional expression of the calcium‐sensing receptor (CaSR) in calcitropic tissues, for example, parathyroid glands and kidneys, is important for maintaining Ca2+ homeostasis. It is also established that the CaSR is present in tissues previously thought to be noncalcitropic and this review discusses the role of the CaSR in vascular function, focusing mainly on contractility but also outlining its role in cell proliferation and calcification. Stimulation of the CaSR by extracellular Ca2+ concentration ([Ca2+]o) on perivascular sensory nerves and vascular endothelial cells is associated with vasodilatation through the release of vasoactive substances and stimulation of IKCa channels and nitric oxide synthesis, respectively, which mediate endothelium‐derived hyperpolarizations and activation of BKCa channels and KATP channels in vascular smooth muscle cells (VSMCs). CaSR‐induced vasoconstrictions are mediated by the CaSR expressed in VSMCs, which are coupled to the Gq/11 protein‐coupled pathway. In addition, the CaSR expressed on VSMCs also regulates proliferation and calcification. Consequently, the CaSR has been implicated in regulating systemic and pulmonary blood pressure and calcimimetics and calcilytics are potential therapeutic targets for cardiovascular diseases, such as hypertension, pulmonary artery hypertension, and atherosclerosis. Representation of the role of the CaSR in the vasculature shows that a rise in extracellular Ca2+ concentration ([Ca2+]o) stimulates the CaSR expressed at the plasma membrane of perivascular sensory nerves, VECs, and VSMCs, which leads to the release of vasodilator substances (e.g., calcitonin‐related gene‐related peptide (CGRP), substance P (Sub P), and lipid substances), activation of vasodilatation via nitric oxide (NO)‐ and endothelium‐derived hyperpolarization (EDH)‐mediated pathways and vasoconstriction, and proliferation and calcification of VSMCs, respectively.

We previously demonstrated that in Ifnar1 -/- Ifngr1 -/- or Stat1 -/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

The inflammasome receptor Nlrp9b defends against enteric viruses by interacting with double-stranded viral RNA-bound helicase Dhx9, triggering gasdermin-D-dependent pyroptotic cell death of infected cells and secretion of Il-18. Inflammasome defends against gastric bug Rotaviruses cause severe and often fatal gastroenterological illnesses in young children. The mechanism by which such enteric viruses are detected and restricted in vivo is largely unknown. Richard Flavell and colleagues report how the inflammasome receptor Nlrp9 helps to defend against enteric viruses, via RNA helicase Dhx9, by interacting with double-stranded viral RNA. This interaction triggers gasdermin-D-dependent pyroptotic cell death of infected cells and secretion of IL-18. This innate immune signalling functions uniquely in intestinal epithelial cells and could reveal useful targets in the modulation of viral defences. Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide 1 . Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling 2 , 3 , 4 , 5 , raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo , especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.

In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5' phosphate groups. RNAs lacking 2'-O-methylated 5' cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5' capping or 2'-O-methylation of the 5' cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.

In vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs), phosphatidylinositol 4,5‐bisphosphate (PIP2) acts as a substrate for phospholipase C (PLC)‐ and phosphoinositol 3‐kinase (PI3K)‐mediated signaling pathways and an unmodified ligand at ion channels and other macromolecules, which are key processes in the regulation of cell physiological and pathological phenotypes. It is envisaged that these distinct roles of PIP2 are achieved by PIP2‐binding proteins, which act as PIP2 buffers to produce discrete pools of PIP2 that permits targeted release within the cell. This review discusses evidence for the expression, cell distribution, and role of myristoylated alanine‐rich C‐kinase substrate (MARCKS), a PIP2‐binding protein, in cellular signaling and function of VSMCs. The review indicates the possibilities for MARCKS as a therapeutic target for vascular disease involving dysfunctional cell proliferation and migration, endothelial barrier permeability, and vascular contractility such as atherosclerosis, systemic and pulmonary hypertension, and sepsis. Representation of the role of MARCKS in VSMCs and VECs, showing that unstimulated MARCKS is expressed at the plasma membrane where it forms electrostatic interactions with PIP2. Cell stimulation by G‐protein‐coupled and tyrosine kinase receptors induces to phosphorylation MARCKS by various mechanisms including protein kinase C (PKC), Ca2+‐calmodulin (CaM), and H2O2 production, leading to the translocation of MARCKS from the plasma membrane into the cytoplasm and release of PIP2 into the local environment where it interacts with transmembrane proteins. Both phosphorylation and translocation of MARCKS and PIP2–protein interactions were associated with changes in cell function such as proliferation and migration, endothelial barrier permeability, and smooth muscle contractility.

Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. Rotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro , it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1 day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development. IMPORTANCE Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate, for the first time in a pathologically valid homologous small animal model, that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon or inflammasome signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo , making it an attractive target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.