Influenza A virus PB1-F2 gene in recent Taiwanese isolates.
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ABSTRACT:
SUBMITTER: Chen GW
PROVIDER: S-EPMC3323094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:RIG-I plays an essential role in the duck innate immune response to influenza infection. RIG-I engages the critical adaptor protein mitochondrial antiviral signaling (MAVS) to activate the downstream signaling pathway. The influenza A virus non-structural protein PB1-F2 interacts with MAVS in human cells to inhibit interferon production. As duck and human MAVS share only 28% amino acid similarity, it is not known whether the influenza virus can similarly inhibit MAVS signaling in avian cells. Using confocal microscopy we show that MAVS and the constitutively active N-terminal end of duck RIG-I (2CARD) co-localize in DF-1 cells, and duck MAVS is pulled down with GST-2CARD. We establish that either GST-2CARD, or duck MAVS can initiate innate signaling in chicken cells and their co-transfection augments interferon-beta promoter activity. Demonstrating the limits of cross-species interactions, duck RIG-I 2CARD initiates MAVS signaling in chicken cells, but works poorly in human cells. The D122A mutation of human 2CARD abrogates signaling by affecting MAVS engagement, and the reciprocal A120D mutation in duck 2CARD improves signaling in human cells. We show mitochondrial localization of PB1-F2 from influenza A virus strain A/Puerto Rico/8/1934 (H1N1; PR8), and its co-localization and co-immunoprecipitation with duck MAVS. PB1-F2 inhibits interferon-beta promoter activity induced by overexpression of either duck RIG-I 2CARD, full-length duck RIG-I, or duck MAVS. Finally, we show that the effect of PB1-F2 on mitochondria abrogates TRIM25-mediated ubiquitination of RIG-I CARD in both human and avian cells, while an NS1 variant from the PR8 influenza virus strain does not.
| S-EPMC7232376 | biostudies-literature
Project description:Influenza A virus (IAV) infection induces mitophagy, which is essential for the clearance of damaged mitochondria. Dysfunctional mitochondria can be selectively targeted by PINK1, which recruits PRKN/PARK2 and leads to subsequent mitochondrial sequestration within autophagosomes. The IAV PB1-F2 protein translocates to mitochondria, accelerates the mitochondrial fragmentation and impairs the innate immunity. However, whether PB1-F2 mediates IAV-induced mitophagy and the relation between mitophagy and PB1-F2-attenuated innate immunity remain obscure. Here, we showed that PB1-F2 translocated to mitochondria by interacting and colocalizing with TUFM (Tu translation elongation factor, mitochondrial). Further studies revealed that PB1-F2 induced complete mitophagy, which required the interactions of PB1-F2 with both TUFM and MAP1LC3B/LC3B that mediated the autophagosome formation. PB1-F2-induced mitophagy was critical for the MAVS (mitochondrial antiviral signaling protein) degradation and led to its suppression of the type I IFN production. Importantly, the C-terminal LIR motif of PB1-F2 protein was demonstrated to be essential for its mitophagy induction and attenuated innate immunity. In conclusion, PB1-F2-induced mitophagy strongly correlates with impaired cellular innate immunity, revealing it is a potential therapeutic target.Abbreviations: BCL2L13: BCL2 like 13; BECN1: beclin 1; BNIP3L/Nix: BCL2 interacting protein 3 like; CQ: chloroquine; DDX58: DExD/H-box helicase 58; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOI, multiplicity of infection; mRFP: monomeric red fluorescent protein; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NLRP3: NLR family pyrin domain containing 3; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RLR: RIG-I-like-receptor; ROS: reactive oxygen species; SEV: sendai virus; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TM: transmembrane; TOMM20/40: translocase of outer mitochondrial membrane 20/40; TUFM: Tu translation elongation factor, mitochondrial.
| S-EPMC8007153 | biostudies-literature
Project description:The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.
| S-EPMC1563817 | biostudies-literature
Project description:BACKGROUND:PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. METHODOLOGY/PRINCIPAL FINDINGS:Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAV(PR8)), from A/Brevig Mission/1/1918(H1N1) (IAV(SF2)) or the H5N1 consensus sequence (IAV(BF2)) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. CONCLUSION/SIGNIFICANCE:The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.
| S-EPMC2886074 | biostudies-literature
Project description:Highly pathogenic avian influenza virus (HPAIV) is a permanent threat due to its capacity to cross species barriers and generate severe infections and high mortality in humans. Recent findings have highlighted the potential role of PB1-F2, a small accessory influenza protein, in the pathogenesis process mediated by HPAIV in mammals. In this study, using a recombinant H5N1 HPAIV (wt) and its PB1-F2-deleted mutant (ΔF2), we studied the effects of PB1-F2 in a chicken model. Unexpectedly, when using low inoculation dose we observed that the wt-infected chickens had a higher survival rate than the ΔF2-infected chickens, a feature that contrasts with what is usually observed in mammals. High inoculation dose had similar mortality rate for both viruses, and comparison of the bio-distribution of the two viruses indicated that the expression of PB1-F2 allows a better spreading of the virus within chicken embryos. Transcriptomic profiles of lungs and blood cells were characterized at two days post-infection in chickens inoculated with the wild type (wt) or the ΔF2 mutant viruses. In lungs, the expression of PB1-F2 during the infection induced pathways related to calcium signaling and repressed a large panel of immunological functions. In blood cells, PB1-F2 was associated with a gene signature specific for mitochondrial dysfunction and down-modulated leucocytes activation. Finally we compared the effect of PB1-F2 in lungs of chickens and mice. We identified that gene signature associated to tissue damages is a PB1-F2 feature shared by the two species; by contrast, the early inhibition of immune response mediated by PB1-F2 observed in chickens is not seen in mice. In summary, our data suggest that PB1-F2 expression deeply affect the immune response in chickens in a way that may attenuate pathogenicity at low infection dose, a feature differing from what was previously observed in mammal species.
| S-EPMC4069075 | biostudies-literature
Project description:PB1-F2, a protein encoded by a second open reading frame of the influenza virus RNA segment 2, has emerged as a modulator of lung inflammatory responses but the molecular mechanisms underlying this are only poorly understood. Here we show that PB1-F2 inhibits the activation of NF-κB dependent signalling pathways in luciferase reporter assays. PB1-F2 proteins from four different viruses interact with IKKβ in yeast two-hybrid assays and by co-immunoprecipitation. PB1-F2 expression did not inhibit IKKβ kinase activity or NF-κB translocation into the nucleus, but NF-κB binding to DNA was severely impaired in PB1-F2 transfected cells as assessed by Electrophoretic Mobility Shift Assay. Neither the N-terminal 57 amino acid truncated forms nor the C-terminus of PB1-F2 were able to inhibit NF-κB dependent signalling, indicating that the full length protein is necessary for the inhibition.
| S-EPMC3660569 | biostudies-literature
Project description:The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.
| S-EPMC3585811 | biostudies-literature
Project description:Influenza A virus (IAV) generally causes caspase-dependent apoptosis based on caspase-3 activation, resulting in nuclear export of newly synthesized viral nucleoprotein (NP) and elevated virus replication. Sulfatide, a sulfated galactosylsphingolipid, enhances IAV replication through promoting newly synthesized viral NP export induced by association of sulfatide with hemagglutinin delivered to the cell surface. Here, we demonstrated that sulfatide is involved in caspase-3-independent apoptosis initiated by the PB1-F2 protein of IAV by using genetically sulfatide-produced cells and PB1-F2-deficient IAVs. Sulfatide-deficient COS7 cells showed no virus-induced apoptosis, whereas SulCOS1 cells, sulfatide-enriched COS7 cells that genetically expressed the two transferases required for sulfatide synthesis from ceramide, showed an increase in IAV replication and were susceptible to caspase-3-independent apoptosis. Additionally, PB1-F2-deficient IAVs, which were generated by using a plasmid-based reverse genetics system from a genetic background of A/WSN/33 (H1N1), demonstrated that PB1-F2 contributed to caspase-3-independent apoptosis in IAV-infected SulCOS1 cells. Our results show that sulfatide plays a critical role in efficient IAV propagation via caspase-3-independent apoptosis initiated by the PB1-F2 protein.
| S-EPMC3617187 | biostudies-literature
Project description:Highly pathogenic Avian influenza (HPAI) is a notifiable viral disease caused by avian influenza type A viruses of the Orthomyxoviridae family. Type A influenza genome consists of eight segments of negative-sense RNA. RNA segment 2 encodes three proteins, PB1, PB1-F2, and N40, which are translated from the same mRNA by ribosomal leaky scanning and reinitiation. Since these proteins are critical for viral replication and pathogenesis, targeting their expression can be one of the approaches to control and resist HPAI. MicroRNAs are short noncoding RNAs that regulate a variety of biological processes such as cell growth, tissue differentiation, apoptosis, and viral infection. In this study, a set of 300 miRNAs expressed in chicken lungs were screened against the HPAI virus (H5N1) segment 2 with different screening parameter like thermodynamic stability of heteroduplex, seed sequence complementarity, conserved target sequence, and target-site accessibility for identifying miRNAs that can potentially target the transcript of segment 2 of H5N1. Chicken miRNAs gga-mir-133c, gga-mir-1710, and gga-mir-146c* are predicted to target the expression of PB1, PB1-F2, and N40 proteins. This indicates that chicken has genetic potential to resist/tolerate H5N1 infection and these can be suitably exploited in designing strategies for control of avian influenza in chicken.
| S-EPMC4069037 | biostudies-literature
Project description:Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon β in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus.
| S-EPMC7302872 | biostudies-literature