ABSTRACT
PURPOSE: A patient with X-linked agammaglobulinemia (XLA) and severe tick-borne encephalitis (TBE) was treated with TBE virus (TBEV) IgG positive plasma. The patient's clinical response, humoral and cellular immune responses were characterized pre- and post-infection. METHODS: ELISA and neutralisation assays were performed on sera and TBEV PCR assay on sera and cerebrospinal fluid. T cell assays were conducted on peripheral blood the patient and five healthy vaccinated controls. RESULTS: The patient was admitted to the hospital with headache and fever. He was not vaccinated against TBE but receiving subcutaneous IgG-replacement therapy (IGRT). TBEV IgG antibodies were low-level positive (due to scIGRT), but the TBEV IgM and TBEV neutralisation tests were negative. During hospitalisation his clinical condition deteriorated (Glasgow coma scale 3/15) and he was treated in the ICU with corticosteroids and external ventricular drainage. He was then treated with plasma containing TBEV IgG without apparent side effects. His symptoms improved within a few days and the TBEV neutralisation test converted to positive. Robust CD8+ T cell responses were observed at three and 18-months post-infection, in the absence of B cells. This was confirmed by tetramers specific for TBEV. CONCLUSION: TBEV IgG-positive plasma given to an XLA patient with TBE without evident adverse reactions may have contributed to a positive clinical outcome. Similar approaches could offer a promising foundation for researching therapeutic options for patients with humoral immunodeficiencies. Importantly, a robust CD8+ T cell response was observed after infection despite the lack of B cells and indicates that these patients can clear acute viral infections and could benefit from future vaccination programs.
Subject(s)
Agammaglobulinemia , Antibodies, Viral , Encephalitis Viruses, Tick-Borne , Encephalitis, Tick-Borne , Genetic Diseases, X-Linked , Immunoglobulin G , T-Lymphocytes , Humans , Encephalitis, Tick-Borne/immunology , Encephalitis, Tick-Borne/diagnosis , Encephalitis, Tick-Borne/therapy , Male , Agammaglobulinemia/immunology , Agammaglobulinemia/therapy , Encephalitis Viruses, Tick-Borne/immunology , Genetic Diseases, X-Linked/immunology , Genetic Diseases, X-Linked/therapy , Immunoglobulin G/blood , Immunoglobulin G/immunology , Antibodies, Viral/blood , T-Lymphocytes/immunology , Treatment Outcome , Adult , Immunization, Passive/methodsABSTRACT
Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Copper/metabolism , Animals , Bacterial Outer Membrane Proteins/chemistry , Binding Sites , Ions , Male , Methionine/metabolism , Mice , Models, Molecular , Protein Conformation , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/metabolismABSTRACT
Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.
Subject(s)
Anti-Bacterial Agents/metabolism , Cell Membrane/physiology , Nutrients/metabolism , Porins/metabolism , Pseudomonas aeruginosa/physiology , Bacterial Outer Membrane Proteins/metabolism , Biological Transport/physiology , Cell Membrane PermeabilityABSTRACT
Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.
Subject(s)
Drug Resistance, Bacterial , Metabolome , Phenotype , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Aconitate Hydratase/metabolism , Adenosine Triphosphate/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cells, Cultured , Humans , Mice , Mice, Inbred C57BL , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Staphylococcal Infections/drug therapy , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicityABSTRACT
We performed 2 surveys during 2022 to estimate point prevalences of SARS-CoV-2 infection compared with overall seroprevalence in Sweden. Point prevalence was 1.4% in March and 1.5% in September. Estimated seroprevalence was >80%, including among unvaccinated children. Continued SARS-CoV-2 surveillance is necessary for detecting emerging, possibly more pathogenic variants.
Subject(s)
COVID-19 , Child , Humans , COVID-19/epidemiology , Prevalence , SARS-CoV-2 , Sweden/epidemiology , Seroepidemiologic StudiesABSTRACT
BACKGROUND: In order to estimate the prevalence and understand the spread of SARS-CoV-2 in Sweden, the Public Health Agency of Sweden, with support from the Swedish Armed Forces, conducted a series of point prevalence surveys between March and December 2020. METHODS: Sampling material and instructions on how to perform self-sampling of the upper respiratory tract were delivered to the homes of the participants. Samples were analysed by real-time PCR, and the participants completed questionnaires regarding symptoms. FINDINGS: The first survey in the Stockholm region in March 2020 included 707 participants and showed a SARS-CoV-2 prevalence of 2.5%. The following five surveys, performed on a national level, with between 2461 and 2983 participants, showed SARS-CoV-2 prevalences of 0.9% (April), 0.3% (May), 0.0% (August), 0.0% (September), and 0.7% (December). All positive cases who responded to questionnaires reported experiencing symptoms that occurred from 2 weeks before the date of sampling up to and including the date of sampling. INTERPRETATION: None of the individuals shown to be PCR-positive were asymptomatic at the time of sampling or in the 14 days prior to sampling. This is in contrast to many other surveys in which a substantial proportion of positive cases have been reported to be asymptomatic. Our surveys demonstrate a decreasing ratio between notified cases and the observed prevalence throughout the year, in line with increasing testing capacity and the consecutive inclusion of all symptomatic individuals in the case definition for testing.
Subject(s)
COVID-19 , Humans , COVID-19/epidemiology , Prevalence , SARS-CoV-2 , Sweden/epidemiology , Public HealthABSTRACT
We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.
Subject(s)
Chikungunya Fever/epidemiology , Chikungunya virus/isolation & purification , Communicable Diseases, Emerging/prevention & control , Disease Outbreaks/prevention & control , Laboratories/standards , Chikungunya Fever/diagnosis , Disease Notification , Humans , Laboratories/organization & administration , Phylogeny , Thailand/epidemiology , TravelABSTRACT
Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.
Subject(s)
Influenza A virus/pathogenicity , Influenza, Human/metabolism , Macrophages/virology , Phosphoproteins/analysis , Proteomics/methods , Animals , Computational Biology/methods , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Macrophages/metabolism , Mice , Signal TransductionABSTRACT
Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.
Subject(s)
DNA/metabolism , Transcription, Genetic/physiology , Viral Nonstructural Proteins/metabolism , Animals , Cell Line , Chromatin/metabolism , Humans , Influenza A virus/physiology , Protein Binding , Viral Nonstructural Proteins/physiology , Virus ReplicationABSTRACT
Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses.
Subject(s)
14-3-3 Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Keratinocytes/metabolism , Proteomics/methods , RNA, Double-Stranded/metabolism , Repressor Proteins/metabolism , Sirtuin 1/metabolism , Cardiovirus Infections/immunology , Cardiovirus Infections/metabolism , Cell Line , Cytosol/metabolism , Encephalomyocarditis virus/genetics , Encephalomyocarditis virus/immunology , Humans , Immunity, Innate , Keratinocytes/cytology , Keratinocytes/immunology , Keratinocytes/virology , Phosphorylation , RNA, Double-Stranded/immunology , RNA, Viral/immunology , RNA, Viral/metabolism , Signal TransductionABSTRACT
Sendai virus (SeV) is a common respiratory pathogen in mice, rats, and hamsters. Host cell recognition of SeV is mediated by pathogen recognition receptors, which recognize viral components and induce intracellular signal transduction pathways that activate the antiviral innate immune response. Viruses use host proteins to control the activities of signaling proteins and their downstream targets, and one of the most important host protein modifications regulated by viral infection is phosphorylation. In this study, we used phosphoproteomics combined with bioinformatics to get a global view of the signaling pathways activated during SeV infection in human lung epithelial cells. We identified altogether 1347 phosphoproteins, and our data shows that SeV infection induces major changes in protein phosphorylation affecting the phosphorylation of almost one thousand host proteins. Bioinformatics analysis showed that SeV infection activates known pathways including MAPK signaling, as well as signaling pathways previously not linked to SeV infection including Rho family of GTPases, HIPPO signaling, and mammalian target of rapamycin (mTOR)-signaling pathway. Further, we performed functional studies with mTOR inhibitors and siRNA approach, which revealed that mTOR signaling is needed for both the host IFN response as well as viral protein synthesis in SeV-infected human lung epithelial cells.
Subject(s)
Epithelial Cells/metabolism , Lung Neoplasms/metabolism , Phosphoproteins/metabolism , Proteomics/methods , Respirovirus Infections/metabolism , Sendai virus/physiology , TOR Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , Computational Biology , Cricetinae , Epithelial Cells/cytology , Humans , Interferons/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/virology , Mice , Phosphoproteins/genetics , Phosphorylation , Protein Array Analysis , RNA, Messenger/genetics , Rats , Real-Time Polymerase Chain Reaction , Respirovirus Infections/virology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , TOR Serine-Threonine Kinases/genetics , Tumor Cells, CulturedABSTRACT
Non-structural protein NS1 of influenza A viruses interacts with cellular factors through its N-terminal RNA-binding, middle effector and C-terminal non-structured domains. NS1 attenuates antiviral responses in infected cells and thereby secures efficient virus replication. Some influenza strains express C-terminally truncated NS1 proteins due to nonsense mutations in the NS1 gene. To understand the role of the NS1 C-terminal region in regulation of antiviral responses, we engineered influenza viruses expressing C-terminally truncated NS1 proteins using A/WSN/33(H1N1) reverse genetics and tested them in human macrophages and in mice. We showed that a WSN virus expressing NS1 with a 28 aa deletion from its C terminus is a more powerful inducer of antiviral responses than the virus expressing full-length NS1, or one with a 10 aa truncation of NS1 in vitro. Thus, our findings suggest that the C-terminal region of NS1 is essential for regulation of antiviral responses. Moreover, viruses expressing truncated NS1 proteins could be good vaccine candidates.
Subject(s)
Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/immunology , Macrophages/immunology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/immunology , Amino Acid Motifs , Animals , Female , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/virology , Macrophages/virology , Mice , Mice, Inbred BALB C , Viral Nonstructural Proteins/genetics , Virus ReplicationABSTRACT
The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro. In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus.
Subject(s)
Heterocyclic Compounds, 3-Ring/pharmacology , Influenza A Virus, H1N1 Subtype , Influenza, Human/prevention & control , Oncogene Protein v-akt/antagonists & inhibitors , Protease Inhibitors/pharmacology , Cell Line , Cytokines/metabolism , Host-Pathogen Interactions/drug effects , Humans , In Vitro Techniques , Influenza, Human/virology , Molecular Sequence Data , Phosphoproteins/metabolism , RNA, Small Interfering/genetics , Transfection , Viral Plaque AssayABSTRACT
BACKGROUND: It is possible to identify thousands of phosphopeptides and -proteins in a single experiment with mass spectrometry-based phosphoproteomics. However, a current bottleneck is the downstream data analysis which is often laborious and requires a number of manual steps. RESULTS: Toward automating the analysis steps, we have developed and implemented a software, PhosFox, which enables peptide-level processing of phosphoproteomic data generated by multiple protein identification search algorithms, including Mascot, Sequest, and Paragon, as well as cross-comparison of their identification results. The software supports both qualitative and quantitative phosphoproteomics studies, as well as multiple between-group comparisons. Importantly, PhosFox detects uniquely phosphorylated peptides and proteins in one sample compared to another. It also distinguishes differences in phosphorylation sites between phosphorylated proteins in different samples. Using two case study examples, a qualitative phosphoproteome dataset from human keratinocytes and a quantitative phosphoproteome dataset from rat kidney inner medulla, we demonstrate here how PhosFox facilitates an efficient and in-depth phosphoproteome data analysis. PhosFox was implemented in the Perl programming language and it can be run on most common operating systems. Due to its flexible interface and open source distribution, the users can easily incorporate the program into their MS data analysis workflows and extend the program with new features. PhosFox source code, implementation and user instructions are freely available from https://bitbucket.org/phintsan/phosfox. CONCLUSIONS: PhosFox facilitates efficient and more in-depth comparisons between phosphoproteins in case-control settings. The open source implementation is easily extendable to accommodate additional features for widespread application use cases.
ABSTRACT
In this review, published applications of microwave-assisted derivatization procedures for gas chromatography/mass spectrometry (GC/MS) are summarized. Among the broad range of analytical techniques available, GC/MS is still the method of choice for most high-throughput screening procedures in forensic/clinical toxicology, doping control and food and environmental analysis. Despite the many advantages of the GC/MS method, time-consuming derivatization steps are often required in order to obtain desirable chromatographic characteristics or to improve the stability and detectability of the target analytes. These derivatization processes typically require reaction times from 30 min up to several hours at elevated temperature. In contrast, microwave protocols have demonstrated to be able to reduce the time required for derivatization to a few minutes, and can thus very effectively shorten the overall analysis time, in particular when carried out in a high-throughput format. Herein, the literature in this field is summarized and recent experimental techniques for performing parallel GC/MS derivatization protocols are discussed.
Subject(s)
Analytic Sample Preparation Methods/methods , Gas Chromatography-Mass Spectrometry/methods , Microwaves , Drug Stability , Gas Chromatography-Mass Spectrometry/trends , High-Throughput Screening Assays/methods , Inorganic Chemicals/chemistry , Inorganic Chemicals/pharmacology , Models, Biological , Organic Chemicals/analysis , Organic Chemicals/chemistry , Organic Chemicals/pharmacologyABSTRACT
Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels and use such compounds as scaffolds for the design of efficiently permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel during infection in rodents. The X-ray crystal structure of DcaP reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and all-atom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically relevant ß-lactamase inhibitor.
Subject(s)
Acinetobacter Infections/metabolism , Acinetobacter baumannii/metabolism , Porins/chemistry , Porins/metabolism , Sulbactam/pharmacology , beta-Lactamase Inhibitors/pharmacology , Acinetobacter baumannii/drug effects , Animals , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Crystallography, X-Ray , Dicarboxylic Acids/metabolism , Mice , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Protein Multimerization , Proteomics , RatsABSTRACT
Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.
Subject(s)
Antiviral Agents/pharmacology , Immunologic Factors/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Kynurenine/antagonists & inhibitors , Metabolic Networks and Pathways/drug effects , Orthomyxoviridae Infections/drug therapy , Animals , Female , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Indoles , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/metabolism , Interferons/genetics , Interferons/immunology , Kynurenine/biosynthesis , Lung/drug effects , Lung/immunology , Lung/virology , Macrophages/drug effects , Macrophages/virology , Metabolic Networks and Pathways/genetics , Metabolic Networks and Pathways/immunology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Oxazoles/pharmacology , Oximes/pharmacology , Primary Cell Culture , Pyrroles/pharmacology , Sulfonamides/pharmacology , Thiazoles/pharmacology , Transcriptome , Tryptophan/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus ReplicationABSTRACT
JNJ-63623872 (formally known as VX-787; referred to here as JNJ872) is an orally bioavailable compound, which is in phase II clinical trials for the treatment of influenza A virus (IAV) infections. Here we show that JNJ872 inhibits at nanomolar concentrations the transcription of viral RNA in IAV-infected human macrophages by targeting a highly conserved site on the cap-snatching domain of influenza polymerase basic 2 protein (PB2). Furthermore, even lower concentrations of JNJ872 protected macrophages from IAV-mediated death when given in combination with 100 nM gemcitabine, which also attenuated transcription and replication of viral RNA. Importantly, treating human macrophages with JNJ872 allowed expression of many immune-related and other genes, involved in antiviral responses, such as indoleamine 2,3-dioxygenase 1 (IDO), and cytosolic 5'-nucleotidase 3A (NT5C3A). Moreover, our targeted metabolomics analysis indicate that treatment with JNJ782 did not interfere with metabolic responses to infection, which further supported our transcriptomics results. Thus, VX-737 alone or in combination with other drugs could be beneficial for treating IAV infected patients, because it would allow the development of antiviral responses and, thereby, protect individuals from current and future infections with closely related IAV strains.
Subject(s)
Antiviral Agents/pharmacology , Host-Pathogen Interactions/drug effects , Influenza A virus/drug effects , Influenza A virus/physiology , Virus Replication/drug effects , Antiviral Agents/chemistry , Binding Sites , Cluster Analysis , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Drug , Gene Expression Profiling , Host-Pathogen Interactions/genetics , Humans , Influenza, Human/genetics , Influenza, Human/metabolism , Influenza, Human/virology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/virology , Metabolome , Metabolomics/methods , Models, Molecular , Molecular Conformation , Protein Binding , Protein Interaction Domains and Motifs , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.