Siyuan Ding

Dynamic expression profiling of type I and type III interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression

Despite activating similar signaling cascades, the type I and type III interferons (IFNs) differ in their ability to antagonize virus replication. However, it is not clear whether these cytokines induce unique antiviral states, particularly in the liver, where the clinically important hepatitis B and C viruses cause persistent infection. Here, clustering and promoter analyses of microarray‐based gene expression profiling were combined with mechanistic studies of signaling pathways to dynamically characterize the transcriptional responses induced by these cytokines in Huh7 hepatoma cells and primary human hepatocytes. Type I and III IFNs differed greatly in their level of interferon‐stimulated gene (ISG) induction with a clearly detectable hierarchy (IFN‐β > IFN‐α > IFN‐λ3 > IFN‐λ1 > IFN‐λ2). Notably, although the hierarchy identified varying numbers of differentially expressed genes when quantified using common statistical thresholds, further analysis of gene expression over multiple timepoints indicated that the individual IFNs do not in fact regulate unique sets of genes. The kinetic profiles of IFN‐induced gene expression were also qualitatively similar with the important exception of IFN‐α. While stimulation with either IFN‐β or IFN‐λs resulted in a similar long‐lasting ISG induction, IFN‐α signaling peaked early after stimulation then declined due to a negative feedback mechanism. The quantitative expression hierarchy and unique kinetics of IFN‐α reveal potential specific roles for individual IFNs in the immune response, and elucidate the mechanism behind previously observed differences in IFN antiviral activity. While current clinical trials are focused on IFN‐λ1 as a potential antiviral therapy, the finding that IFN‐λ3 invariably possesses the highest activity among type III IFNs suggests that this cytokine may have superior clinical activity. (Hepatology 2014;59:1262‐1272)

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, 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.

Long distance interferon signaling within the brain blocks virus spread

ABSTRACT Serious permanent neurological or psychiatric dysfunction may result from virus infections in the central nervous system (CNS). Olfactory sensory neurons are in direct contact with the external environment, making them susceptible to infection by viruses that can enter the brain via the olfactory nerve. The rarity of full brain viral infections raises the important question of whether unique immune defense mechanisms protect the brain. Here we show that both RNA (vesicular stomatitis virus [VSV]) and DNA (cytomegalovirus [CMV]) virus inoculations of the nasal mucosa leading to olfactory bulb (OB) infection activate long-distance signaling that upregulates antiviral interferon (IFN)-stimulated gene (ISG) expression in uninfected remote regions of the brain. This signaling mechanism is dependent on IFN-α/β receptors deep within the brain, leading to the activation of a distant antiviral state that prevents infection of the caudal brain. In normal mice, VSV replication is limited to the OB, and these animals typically survive the infection. In contrast, mice lacking the IFN-α/β receptor succumbed to the infection, with VSV spreading throughout the brain. Chemical destruction of the olfactory sensory neurons blocked both virus trafficking into the OB and the IFN response in the caudal brain, indicating a direct signaling within the brain after intranasal infection. Most signaling within the brain occurs across the 20-nm synaptic cleft. The unique long-distance IFN signaling described here occurs across many millimeters within the brain and is critical for survival and normal brain function. IMPORTANCE The olfactory mucosa can serve as a conduit for a number of viruses to enter the brain. Yet infections in the CNS rarely occur. The mechanism responsible for protecting the brain from viruses that successfully invade the OB, the first site of infection subsequent to infection of the nasal mucosa, remains elusive. Here we demonstrate that the protection is mediated by a long-distance interferon signaling, particularly IFN-β released by infected neurons in the OB. Strikingly, in the absence of neurotropic virus infection, ISGs are induced in the posterior regions of the brain, activating an antiviral state and preventing further virus invasion.

Epigenetic reprogramming of the type III interferon response potentiates antiviral activity and suppresses tumor growth.

The tissue-specific IFN-λ receptor expression program can be epigenetically remodeled via HDAC inhibition to strengthen anti-viral and anti-tumor activities in the central nervous system.

Peroxisomal MAVS activates IRF1-mediated IFN-λ production

Infection with an RNA virus induces the interferons IFN-β and IFN-λ via the adaptor MAVS located in mitochondria, while peroxisomal MAVS selectively activates an IFN-λ response.

Comparative Proteomics Reveals Strain-Specific β-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.

VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans

Individual human VP4- and VP7-binding monoclonal antibodies mediate serotype cross-reactive neutralizing immunity to rotaviruses. Investigating heterotypic immunity to rotavirus There are many serotypes of rotavirus, and currently available vaccines include only one or a handful of different strains. However, these vaccines are generally able to induce cross-reactive immunity that prevents diarrheal disease in young children. To get a better understanding of heterotypic immunity, Nair et al. isolated rotavirus-specific B cells from human intestinal tissue and cloned the antibodies for functional analyses. They discovered that heterotypic immunity was often derived from antibodies targeting the VP5* stalk epitope. Although only a small number of antibodies were studied, these results suggest that vaccines focused on the VP5* region may be better able to induce broadly protective immunity to rotavirus. Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell–sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.

New mitochondrial DNA synthesis enables NLRP3 inflammasome activation

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.

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

Significance Many clinically significant human viral and bacterial pathogens use dynamin-dependent endocytosis to initiate infection or deliver toxin into host cells. Owing to the complex nature of this cellular process, the molecular mechanisms that regulate this pathway remain to be fully elucidated. Here, we use rotavirus (RV) as a model and identify drebrin as a regulatory protein that restricts the cell entry of multiple viruses. We demonstrate that genetic depletion or chemical inhibition of drebrin leads to enhanced RV infection in vitro and increased diarrhea incidence and virus shedding in vivo. Our current study provides insights into endocytosis regulation in general and highlights the potential broad application of blocking drebrin to augment the uptake of viruses and other dynamin-mediated cargo. Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Cytidine deamination and cccDNA degradation: A new approach for curing HBV?

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-a treatment can clear HBV but is limited by systemic side effects. We describe how interferon-a can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-b receptor activation as a therapeutic alternative. Interferon-a and lymphotoxin-b receptor activation upregulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-b receptor activation— allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.