A protein-interaction network of interferon-stimulated genes extends the innate immune system landscape.

Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.

Oxeiptosis, a ROS-induced caspase-independent apoptosis-like cell-death pathway.

Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 -/- mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 -/- mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

Viperin Targets Flavivirus Virulence by Inducing Assembly of Noninfectious Capsid Particles.

Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. We describe here a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly and involves release of malfunctioning membrane-associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad-spectrum antiviral interferon-stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) as the cellular protein targeted by viperin. Viperin-induced antiviral activity, as well as C-particle release, was stimulated by GBF1 inhibition and knockdown and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive noninfectious virus particles via a GBF1-dependent mechanism. This as-yet-undescribed antiviral mechanism allows potential therapeutic intervention.IMPORTANCE The interferon response can target viral infection on almost every level; however, very little is known about the interference of flavivirus assembly. We show here that interferon, through the action of viperin, can disturb the assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appears to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study, we show that viperin induces capsid particle release by interacting and inhibiting the function of the cellular protein Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and is essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

UNLABELLED: The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE: Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Sequestration by IFIT1 impairs translation of 2'O-unmethylated capped RNA.

Viruses that generate capped RNA lacking 2'O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2'O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2'O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2'O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2'O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.

Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp.

Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.

mRNA 3'UTR lengthening by alternative polyadenylation attenuates inflammatory responses and correlates with virulence of Influenza A virus.

Changes of mRNA 3'UTRs by alternative polyadenylation (APA) have been associated to numerous pathologies, but the mechanisms and consequences often remain enigmatic. By combining transcriptomics, proteomics and recombinant viruses we show that all tested strains of IAV, including A/PR/8/34(H1N1) (PR8) and A/Cal/07/2009 (H1N1) (Cal09), cause APA. We mapped the effect to the highly conserved glycine residue at position 184 (G184) of the viral non-structural protein 1 (NS1). Unbiased mass spectrometry-based analyses indicate that NS1 causes APA by perturbing the function of CPSF4 and that this function is unrelated to virus-induced transcriptional shutoff. Accordingly, IAV strain PR8, expressing an NS1 variant with weak CPSF binding, does not induce host shutoff but only APA. However, recombinant IAV (PR8) expressing NS1(G184R) lacks binding to CPSF4 and thereby also the ability to cause APA. Functionally, the impaired ability to induce APA leads to an increased inflammatory cytokine production and an attenuated phenotype in a mouse infection model. Investigating diverse viral infection models showed that APA induction is a frequent ability of many pathogens. Collectively, we propose that targeting of the CPSF complex, leading to widespread alternative polyadenylation of host transcripts, constitutes a general immunevasion mechanism employed by a variety of pathogenic viruses.

Surfactin Shows Relatively Low Antimicrobial Activity against Bacillus subtilis and Other Bacterial Model Organisms in the Absence of Synergistic Metabolites.

Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E. coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin.

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153.

Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked ß-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

Comprehensive proteome analysis of bread deciphering the allergenic potential of bread wheat, spelt and rye.

BACKGROUND/OBJECTIVES: Cereal products like flour and bread are known to trigger diseases such as wheat allergy, celiac disease and non-celiac wheat sensitivity (NCWS). Some of these diseases are caused by allergenic proteins, the expression of which might vary depending on the grain type and manufacturing processes. Therefore, we examined the protein composition and abundance of potentially allergenic proteins in flours from bread wheat, spelt and rye, and corresponding breads. MATERIALS AND METHODS: Using Nano-LC-ESI-MS/MS and label free quantification (LFQ) we analyzed the proteome of six different bread flours (wholegrain and superfine flours from rye, spelt and bread wheat) and 14 bread types (yeast and sourdough fermented breads from all flours and wheat breads plus/minus bread improver). Potentially allergenic proteins in flours and breads were functionally categorized using the Pfam database and relatively quantified by LFQ. RESULTS: We could show that almost equal numbers of proteins can be identified in rye- and spelt samples compared to wheat samples using the Uniprot bread wheat protein database, indicating high sequence conservation between cereals. In total, 4424 proteins were identified in the 20 flour and bread samples. The average number of identified proteins in flour (2719 ± 243) was slightly higher than in bread (2283 ± 232; P < 0.001). In wheat- and spelt wholegrain flour higher protein numbers (wheat: 2891 ± 90; spelt: 2743 ± 140) were identified on average than in superfine flour (wheat: 2562 ± 79; P = 0.009; spelt: 2431 ± 140; P = 0.004). Neither the absolute number nor the abundance distribution of potentially allergenic proteins were dependent on the flour type or the fermentation process, but known allergenic proteins like gliadins showed higher relative abundance in spelt- and wheat samples, compared to rye samples. CONCLUSION: We provide comprehensive proteome data for six flour types and related breads showing that the grain species have greater influence on proteome composition than milling and fermentation processes. Our data indicate that allergenic proteins are not selectively degraded during bread production and are more abundant in bread wheat and spelt compared to rye. SIGNIFICANCE: Our proteomics study revealed that bread contains a number of potentially and proven allergenic proteins. Most likely allergenicity is not dependent on milling or conventional fermentation processes, but on the grain type. Relative abundance of allergenic proteins was higher in spelt- and wheat samples than in rye samples. Considering rye bread as better suited to atopic individuals predisposed to react to cereal allergens, clinical trials are warranted to verify this assumption.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

HIV-1 Vpu affects the anterograde transport and the glycosylation pattern of NTB-A.

HIV-1 Vpu induces downregulation of cell surface NTB-A to evade lysis of HIV-1 infected cells by NK cells. Here we show that Vpu affects the anterograde transport and the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, these results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules.