ABSTRACT
Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.
Subject(s)
Autophagy-Related Proteins , Vacuolar Proton-Translocating ATPases , Vacuolar Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Humans , Autophagy-Related Proteins/metabolism , Autophagy-Related Proteins/genetics , Animals , Mice , Protein Binding , Neurons/metabolism , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Protein 8 Family/genetics , Autophagy , HEK293 Cells , Induced Pluripotent Stem Cells/metabolism , Influenza, Human/virology , Influenza, Human/metabolism , Influenza, Human/genetics , Mice, Inbred C57BL , Signal Transduction , Carrier Proteins/metabolism , Carrier Proteins/genetics , Mice, KnockoutABSTRACT
The influenza virus (IFV) imposes a considerable health and economic burden globally, requiring a comprehensive understanding of its pathogenic mechanisms. Ferroptosis, an iron-dependent lipid peroxidation cell death pathway, holds unique implications for the antioxidant defense system, with possible contributions to inflammation. This exploration focuses on the dynamic interplay between ferroptosis and the host defense against viruses, emphasizing the influence of IFV infections on the activation of the ferroptosis pathway. IFV causes different types of cell death, including apoptosis, necrosis, and ferroptosis. IFV-induced ferroptotic cell death is mediated by alterations in iron homeostasis, intensifying the accumulation of reactive oxygen species and promoting lipid peroxidation. A comprehensive investigation into the mechanism of ferroptosis in viral infections, specifically IFV, has great potential to identify therapeutic strategies. This understanding may pave the way for the development of drugs using ferroptosis inhibitors, presenting an effective approach to suppress viral infections.
Subject(s)
Ferroptosis , Influenza, Human , Iron , Lipid Peroxidation , Orthomyxoviridae , Reactive Oxygen Species , Humans , Influenza, Human/virology , Influenza, Human/metabolism , Animals , Orthomyxoviridae/physiology , Orthomyxoviridae/pathogenicity , Reactive Oxygen Species/metabolism , Iron/metabolism , ApoptosisABSTRACT
Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.
Subject(s)
Active Transport, Cell Nucleus , Protein Biosynthesis , RNA, Messenger , RNA, Viral , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Animals , Influenza A virus/genetics , Influenza A virus/physiology , Influenza A virus/metabolism , Virus Replication , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H1N1 Subtype/physiology , Cell Nucleus/metabolism , Cell Nucleus/virology , 5' Untranslated Regions/genetics , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/genetics , Madin Darby Canine Kidney Cells , HEK293 Cells , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Dogs , Influenza, Human/virology , Influenza, Human/metabolism , Influenza, Human/genetics , Mutation , Host-Pathogen Interactions/genetics , Viral Core Proteins/metabolism , Viral Core Proteins/geneticsABSTRACT
Upregulation of ADAMTS-4 has been reported to have an important role in lung injury, and ADAMTS-4 expression is regulated by miR-126a-5p in abdominal aortic aneurysms. The aim of this study was to investigate whether miR-126a-5p/ADAMTS-4 plays a role in influenza-virus-induced lung injury. Lung fibroblasts were infected with H1N1 influenza virus to detect changes in miR-126a-5p and ADAMTS-4 expression, and cell viability was measured by CCK-8 assay. Inflammatory factors and matrix protease levels were examined using ELISA kits, and cell apoptosis was assessed by measuring the levels of apoptosis-related proteins. A dual luciferase assay was used to verify the regulatory relationship between miR-126a-5p and ADAMTS-4. H1N1 influenza virus reduced fibroblast viability, inhibited miR-126a-5p expression, and promoted ADAMTS-4 expression. Overexpression of miR-126a-5p attenuated the cellular inflammatory response, apoptosis, matrix protease secretion, and virus replication. Luciferase reporter assays revealed that miR-126a-5p inhibited ADAMTS-4 expression by targeting ADAMTS-4 mRNA. Further experiments showed that overexpression of ADAMTS-4 significantly reversed the inhibitory effects of miR-126a-5p on fibroblast inflammation, apoptosis, matrix protease secretion, and virus replication. Upregulation of miR-126a-5p inhibits H1N1-induced apoptosis, inflammatory factors, and matrix protease secretion, as well as virus replication in lung fibroblasts.
Subject(s)
ADAMTS4 Protein , Apoptosis , Fibroblasts , Inflammation , Influenza A Virus, H1N1 Subtype , Lung , MicroRNAs , MicroRNAs/genetics , MicroRNAs/metabolism , Fibroblasts/virology , Fibroblasts/metabolism , Humans , Lung/virology , Lung/pathology , ADAMTS4 Protein/genetics , ADAMTS4 Protein/metabolism , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Inflammation/genetics , Cell Survival , Virus Replication , Influenza, Human/virology , Influenza, Human/genetics , Influenza, Human/metabolism , Cell LineABSTRACT
Although rapid progression and a poor prognosis in influenza A virus (IAV) infection-induced acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are frequently associated with metabolic energy disorders, the underlying mechanisms and rescue strategies remain unknown. We herein demonstrated that the level of resting energy expenditure increased significantly in IAV-induced AECOPD patients and that cellular energy exhaustion emerged earlier and more significantly in IAV-infected primary COPD bronchial epithelial (pDHBE) cells. The differentially expressed genes were enriched in the oxidative phosphorylation (OXPHOS) pathway; additionally, we consistently uncovered much earlier ATP exhaustion, more severe mitochondrial structural destruction and dysfunction, and OXPHOS impairment in IAV-inoculated pDHBE cells, and these changes were rescued by melatonin. The level of OMA1-dependent cleavage of OPA1 in the mitochondrial inner membrane and the shift in energy metabolism from OXPHOS to glycolysis were significantly increased in IAV-infected pDHBE cells; however, these changes were rescued by OMA1-siRNA or melatonin further treatment. Collectively, our data revealed that melatonin rescued IAV-induced cellular energy exhaustion via OMA1-OPA1-S to improve the clinical prognosis in COPD. This treatment may serve as a potential therapeutic agent for patients in which AECOPD is induced by IAV.
Subject(s)
Energy Metabolism , GTP Phosphohydrolases , Influenza A virus , Melatonin , Pulmonary Disease, Chronic Obstructive , Humans , Energy Metabolism/drug effects , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Influenza A virus/drug effects , Influenza, Human/metabolism , Influenza, Human/drug therapy , Melatonin/pharmacology , Metalloendopeptidases , Oxidative Phosphorylation/drug effects , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/drug therapyABSTRACT
Recently, substantial evidence has demonstrated that pseudogene-derived long noncoding RNAs (lncRNAs) as regulatory RNAs have been implicated in basic physiological processes and disease development through multiple modes of functional interaction with DNA, RNA, and proteins. Here, we report an important role for GBP1P1, the pseudogene of guanylate-binding protein 1, in regulating influenza A virus (IAV) replication in A549 cells. GBP1P1 was dramatically upregulated after IAV infection, which is controlled by JAK/STAT signaling. Functionally, ectopic expression of GBP1P1 in A549 cells resulted in significant suppression of IAV replication. Conversely, silencing GBP1P1 facilitated IAV replication and virus production, suggesting that GBP1P1 is one of the interferon-inducible antiviral effectors. Mechanistically, GBP1P1 is localized in the cytoplasm and functions as a sponge to trap DHX9 (DExH-box helicase 9), which subsequently restricts IAV replication. Together, these studies demonstrate that GBP1P1 plays an important role in antagonizing IAV replication.IMPORTANCELong noncoding RNAs (lncRNAs) are extensively expressed in mammalian cells and play a crucial role as regulators in various biological processes. A growing body of evidence suggests that host-encoded lncRNAs are important regulators involved in host-virus interactions. Here, we define a novel function of GBP1P1 as a decoy to compete with viral mRNAs for DHX9 binding. We demonstrate that GBP1P1 induction by IAV is mediated by JAK/STAT activation. In addition, GBP1P1 has the ability to inhibit IAV replication. Importantly, we reveal that GBP1P1 acts as a decoy to bind and titrate DHX9 away from viral mRNAs, thereby attenuating virus production. This study provides new insight into the role of a previously uncharacterized GBP1P1, a pseudogene-derived lncRNA, in the host antiviral process and a further understanding of the complex GBP network.
Subject(s)
DEAD-box RNA Helicases , Influenza A virus , Pseudogenes , Virus Replication , Humans , A549 Cells , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Influenza A virus/genetics , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Signal Transduction , Influenza, Human/virology , Influenza, Human/genetics , Influenza, Human/metabolism , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , HEK293 Cells , Host-Pathogen Interactions , Dogs , Neoplasm ProteinsABSTRACT
Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.
Subject(s)
Endocytosis , Mice, Knockout , Receptors, Metabotropic Glutamate , Animals , Receptors, Metabotropic Glutamate/metabolism , Receptors, Metabotropic Glutamate/genetics , Mice , Humans , Virus Internalization , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Clathrin/metabolism , Orthomyxoviridae Infections/virology , Orthomyxoviridae Infections/metabolism , HEK293 Cells , Actins/metabolism , Dogs , Madin Darby Canine Kidney Cells , Receptors, Virus/metabolism , Receptors, Virus/genetics , Influenza, Human/virology , Influenza, Human/metabolism , Orthomyxoviridae/physiology , Orthomyxoviridae/genetics , Orthomyxoviridae/metabolismABSTRACT
The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch [P-Patch]), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation. P-Patch phosphorylation occurs during the G1 phase of the cell cycle, recruiting CMTR1 to RNA polymerase II during a period of rapid transcription and RNA cap formation. CMTR1 phosphorylation is required for the expression of specific RNAs, including ribosomal protein gene transcripts, and promotes cell proliferation. CMTR1 phosphorylation is also required for interferon-stimulated gene expression. The cap-snatching virus, influenza A, utilizes host CMTR1 phosphorylation to produce the caps required for virus production and infection. We present an RNA cap methylation control mechanism whereby CK2 controls CMTR1, enhancing co-transcriptional capping.
Subject(s)
Casein Kinase II , Methyltransferases , RNA Caps , Animals , Humans , Casein Kinase II/metabolism , Casein Kinase II/genetics , HEK293 Cells , Influenza A virus , Influenza, Human/virology , Influenza, Human/metabolism , Influenza, Human/genetics , Methylation , Methyltransferases/metabolism , Phosphorylation , RNA Caps/metabolism , RNA Polymerase II/metabolismABSTRACT
Influenza virus infection poses a great threat to human health globally each year. Non-coding RNAs (ncRNAs) in the human genome have been reported to participate in the replication process of the influenza virus, among which there are still many unknowns about Long Intergenic Non-Coding RNAs (LincRNAs) in the cell cycle of viral infections. Here, we observed an increased expression of Linc01615 in A549 cells upon influenza virus PR8 infection, accompanied by the successful activation of the intracellular immune system. The knockdown of Linc01615 using the shRNAs promoted the proliferation of the influenza A virus, and the intracellular immune system was inhibited, in which the expressions of IFN-ß, IL-28A, IL-29, ISG-15, MX1, and MX2 were decreased. Predictions from the catRAPID website suggested a potential interaction between Linc01615 and DHX9. Also, knocking down Linc01615 promoted influenza virus proliferation. The subsequent transcriptome sequencing results indicated a decrease in Linc01615 expression after influenza virus infection when DHX9 was knocked down. Further analysis through cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) in HEK293 cells stably expressing DHX9 confirmed the interaction between DHX9 and Linc01615. We speculate that DHX9 may interact with Linc01615 to partake in influenza virus replication and that Linc01615 helps to activate the intracellular immune system. These findings suggest a deeper connection between DHX9 and Linc01615, which highlights the significant role of Linc01615 in the influenza virus replication process. This research provides valuable insights into understanding influenza virus replication and offers new targets for preventing influenza virus infections.
Subject(s)
DEAD-box RNA Helicases , Influenza, Human , RNA, Long Noncoding , Virus Replication , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , A549 Cells , HEK293 Cells , Influenza, Human/virology , Influenza, Human/genetics , Influenza, Human/immunology , Influenza, Human/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Influenza A virus/physiology , Animals , Dogs , Gene Knockdown Techniques , Neoplasm ProteinsABSTRACT
Obesity and metabolic syndrome alter serum lipid profiles. They also increase vulnerability to viral infections and worsen the survival rate and symptoms after infection. How serum lipids affect influenza virus proliferation is unclear. Here, we investigated the effects of lysophosphatidylcholines on influenza A virus (IAV) proliferation. IAV particles in the culture medium were titrated using extraction-free quantitative PCR, and viral RNA and protein levels were assessed using real-time PCR and Western blot, respectively. RNA sequencing data were analyzed using PCA and heatmap analysis, and pathway analysis was performed using the KEGG mapper and PathIN tools. Statistical analysis was conducted using SPSS21.0. LPC treatment of THP-1 cells significantly increased IAV proliferation and IAV RNA and protein levels, and saturated LPC was more active in IAV RNA expression than unsaturated LPC was. The functional analysis of genes affected by LPCs showed that the expression of genes involved in IAV signaling, such as suppressor of cytokine signaling 3 (SOCS3), phosphoinositide-3-kinase regulatory subunit 3 (PI3K) and AKT serine/threonine kinase 3 (AKT3), Toll-like receptor 7 (TKR7), and interferon gamma receptor 1 (IFNGR1), was changed by LPC. Altered influenza A pathways were linked with MAPK and PI3K/AKT signaling. Treatment with inhibitors of MAPK or PI3K attenuated viral gene expression changes induced by LPCs. The present study shows that LPCs stimulated virus reproduction by modifying the cellular environment to one in which viruses proliferated better. This was mediated by the MAPK, JNK, and PI3K/AKT pathways. Further animal studies are needed to confirm the link between LPCs from serum or the respiratory system and IAV proliferation.
Subject(s)
Influenza A virus , Lysophosphatidylcholines , MAP Kinase Signaling System , Virus Replication , Humans , Lysophosphatidylcholines/pharmacology , Lysophosphatidylcholines/metabolism , Virus Replication/drug effects , MAP Kinase Signaling System/drug effects , Influenza A virus/physiology , Macrophages/metabolism , Macrophages/virology , Macrophages/drug effects , THP-1 Cells , Cell Differentiation/drug effects , Influenza, Human/virology , Influenza, Human/metabolism , Signal Transduction/drug effects , AnimalsABSTRACT
Influenza A virus (IAV) continues to pose serious threats to the global animal industry and public health security. Identification of critical host factors engaged in the life cycle of IAV and elucidation of the underlying mechanisms of their action are particularly important for the discovery of potential new targets for the development of anti-influenza drugs. Herein, we identified Hydroxyacyl-CoA Dehydratase 3 (HACD3) as a new host factor that supports the replication of IAV. Downregulating the expression of HACD3 reduced the level of viral PB1 protein in IAV-infected cells and in cells that were transiently transfected to express PB1. Silencing HACD3 expression had no effect on the level of PB1 mRNA but could promote the lysosome-mediated autophagic degradation of PB1 protein. Further investigation revealed that HACD3 interacted with PB1 and selective autophagic receptor SQSTM1/p62, and HACD3 competed with SQSTM1/p62 for the interaction with PB1, which prevented PB1 from SQSTM1/p62-mediated autophagic degradation. Collectively, these findings establish that HACD3 plays a positive regulatory role in IAV replication by stabilizing the viral PB1 protein.
Subject(s)
Autophagy , Influenza A virus , Influenza, Human , Viral Proteins , Virus Replication , Animals , Dogs , Humans , A549 Cells , HEK293 Cells , Host-Pathogen Interactions/genetics , Influenza A virus/physiology , Influenza, Human/metabolism , Influenza, Human/virology , Proteolysis , Sequestosome-1 Protein/metabolism , Sequestosome-1 Protein/genetics , Viral Proteins/metabolism , Virus Replication/geneticsABSTRACT
The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.
Subject(s)
Cysteine Endopeptidases , Influenza A virus , Viral Nonstructural Proteins , Virus Replication , Animals , Dogs , Humans , Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/genetics , Deubiquitinating Enzymes/metabolism , HEK293 Cells , Influenza A virus/metabolism , Influenza, Human/metabolism , Influenza, Human/virology , RNA, Viral/metabolism , RNA, Viral/genetics , Ubiquitination , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/genetics , Virus Replication/physiology , Cell Line , Vero Cells , Chlorocebus aethiopsABSTRACT
The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.
Subject(s)
Complement Factor H , Hemagglutinin Glycoproteins, Influenza Virus , Influenza A virus , Influenza, Human , Protein Binding , Humans , Complement Factor H/metabolism , Complement Factor H/immunology , Animals , Influenza, Human/immunology , Influenza, Human/virology , Influenza, Human/metabolism , Influenza A virus/immunology , Influenza A virus/physiology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Binding Sites , Influenza in Birds/virology , Influenza in Birds/immunology , Influenza in Birds/metabolism , Birds/virology , Host-Pathogen Interactions/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza A Virus, H9N2 Subtype/immunologyABSTRACT
Following infection, influenza viruses strive to establish a new host cellular environment optimized for efficient viral replication and propagation. Influenza viruses use or hijack numerous host factors and machinery not only to fulfill their own replication process but also to constantly evade the host's antiviral and immune response. For this purpose, influenza viruses appear to have formulated diverse strategies to manipulate the host proteins or signaling pathways. One of the most effective tactics is to specifically induce the degradation of the cellular proteins that are detrimental to the virus life cycle. Here, we summarize the cellular factors that are deemed to have been purposefully degraded by influenza virus infection. The focus is laid on the mechanisms for the protein ubiquitination and degradation in association with facilitated viral amplification. The fate of influenza viral infection of hosts is heavily reliant on the outcomes of the interplay between the virus and the host antiviral immunity. Understanding the processes of how influenza viruses instigate the protein destruction pathways could provide a foundation for the development of advanced therapeutics to target host proteins and conquer influenza.
Subject(s)
Host-Pathogen Interactions , Orthomyxoviridae , Ubiquitination , Virus Replication , Humans , Orthomyxoviridae/metabolism , Orthomyxoviridae/physiology , Influenza, Human/metabolism , Influenza, Human/virology , Proteolysis , AnimalsABSTRACT
Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LCâMS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.
Subject(s)
Mitochondria , Viral Proteins , Virus Replication , Humans , Mitochondria/metabolism , Mitochondria/virology , Viral Proteins/metabolism , Viral Proteins/genetics , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/genetics , Influenza A virus/physiology , Influenza A virus/genetics , Influenza A virus/pathogenicity , Influenza A virus/metabolism , Host-Pathogen Interactions , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/physiology , Influenza A Virus, H3N2 Subtype/metabolism , Autophagy , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H1N1 Subtype/pathogenicity , HEK293 Cells , Influenza, Human/virology , Influenza, Human/metabolism , A549 Cells , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Tandem Mass SpectrometryABSTRACT
Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.
Subject(s)
Influenza A virus , Influenza, Human , RNA, Messenger , RNA, Viral , RNA-Binding Proteins , Animals , Humans , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Cell Nucleus/virology , Influenza A virus/genetics , Influenza, Human/metabolism , Influenza, Human/virology , Influenza, Human/genetics , RNA Transport , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA, Viral/metabolism , RNA, Viral/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Virus ReplicationABSTRACT
The most prevalent glycoprotein on the influenza virus envelope is called hemagglutinin (HA), yet little is known about its involvement in the pathophysiology and etiology of severe influenza pneumonia. Here, after stimulating human bronchial epithelial cells (16-HBE) and mice with HA of H1N1 for 12 h, we investigated the proliferation, migration, inflammatory cytokines expression, and apoptosis in 16-HBE and the pathological damage in mouse lung tissue. The expression of inflammatory cytokines plasminogen activator inhibitor 1(PAI-1), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, and apoptosis were all enhanced by HA, which also prevented the proliferation and migration of bronchial epithelial cells. HA enhanced up-regulated PAI-1, uPA, and tPA protein expression within mouse lung tissue and caused lung injury. In conclusion, HA alone, but not the whole H1N1 virus, induces lung tissue injury by inhibiting cell proliferation and migration, while promoting the expression of inflammatory cytokines and apoptosis.
Subject(s)
Apoptosis , Cell Proliferation , Hemagglutinin Glycoproteins, Influenza Virus , Influenza A Virus, H1N1 Subtype , Animals , Humans , Mice , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Cell Movement , Cytokines/metabolism , Epithelial Cells/metabolism , Epithelial Cells/virology , Lung/metabolism , Lung/virology , Lung/pathology , Cell Line , Pneumonia, Viral/virology , Pneumonia, Viral/metabolism , Pneumonia, Viral/pathology , Influenza, Human/metabolism , Influenza, Human/virology , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolism , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/virology , Plasminogen Activator Inhibitor 1/metabolism , Plasminogen Activator Inhibitor 1/genetics , Pneumonia/metabolism , Pneumonia/virologyABSTRACT
Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.
Subject(s)
CD47 Antigen , Epithelial Cells , Staphylococcal Infections , Staphylococcus aureus , Superinfection , CD47 Antigen/metabolism , CD47 Antigen/genetics , Humans , Animals , Superinfection/microbiology , Mice , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/virology , Staphylococcal Infections/immunology , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Influenza, Human/metabolism , Influenza, Human/immunology , Influenza, Human/virology , Bacterial Adhesion , Respiratory Mucosa/metabolism , Respiratory Mucosa/microbiology , Respiratory Mucosa/virology , Mice, Inbred C57BL , Bronchi/metabolism , Bronchi/cytology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/virology , Mice, Knockout , Influenza A Virus, H1N1 SubtypeABSTRACT
Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.
Subject(s)
Influenza A virus , Viral Nonstructural Proteins , Humans , A549 Cells , Host-Pathogen Interactions , Influenza A virus/genetics , Influenza, Human/virology , Influenza, Human/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/genetics , Virus Replication , Host-Parasite InteractionsABSTRACT
Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-ß, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.