The luciferase assays were performed at 24 h post-transfection. Cell lysates were harvested for the western blot analysis. The luciferase assays were performed as A. Expression levels of corresponding proteins were confirmed by western blot. Having established that PB1-F2 could induce complete mitophagy, resulting in the removal of the damaged mitochondria, it was reasonable to speculate that the suppression of type I IFN response by PB1-F2 was dependent on the PB1-F2-induced mitophagy.
In brief, these results indicated that PB1-F2-induced mitophagy was crucial for its attenuated innate immune response. Mitophagy is a type of autophagy that selectively sequesters the damaged mitochondria by autophagosome via the interaction of mitochondrial receptors with LC3B [ 4 ].
Cell lysates were immunoprecipitated with anti-GFP antibody and then were subjected to western blot analysis. Cell lysates were immunoprecipitated with anti-GFP antibody and then analyzed by western blot. Cell lysates were harvested for western blot analysis. Mitophagy, the selective engulfment, and clearance of mitochondria are essential for the homeostasis of a healthy network of functioning mitochondria and prevent excessive production of cytotoxic ROS reactive oxygen species from damaged mitochondria.
Mitochondrial dynamics and mitophagy are closely related [ 67 ]. Many viruses impair mitochondrial networks and induce mitophagy [ 3 , 11 , 14 , 15 , 18 , 44 ]. Several viral proteins can serve as viral components that directly mediate mitophagy induction [ 3 , 10 , 11 ].
Because IAV infection induces incomplete autophagic process [ 80 ], we further determined which type of mitophagy PB1-F2 induced, and showed that PB1-F2 protein triggered complete mitophagy. The mitochondrially targeted PINK1 and the PRKN are well-established synergistic mediators of mitophagy of dysfunctional mitochondria, which relies on the ubiquitination of a number of mitochondrial outer membrane substrates and subsequent docking of autophagy receptors to selectively clear mitochondria [ 71 , 81 ].
However, it cannot exclude the possibilities that other proteins in mitochondria may also facilitate the recruitment of PB1-F2 to mitochondria. Intriguingly, our study provided compelling evidence showing that TUFM, a mitochondrial protein, acted as an adaptor protein that bound to PB1-F2 to bridge the interaction of PB1-F2 with LC3B on the mitochondria, thereby recruiting mitochondria to the autophagosome, thus resulting in the induction of mitophagy.
Then, they mediate mitochondrial sequestration by autophagosome via their interaction with LC3B, leading to mitophagy [ 10 , 12 ]. It has been well-established that the late stage of autophagy, the process of autolysosome formation, is involved in the removal of the aggregated proteins such as MAVS aggregates by the lysosome enzyme [ 70 ]. It promotes the activation of NLRP3 inflammasomes [ 42 ]. However, the detailed mechanisms remain unclear.
Mitophagy promotes viral replication by inhibiting the innate antiviral immunity [ 44 ]. PB1-F2 protein is a virulent gene of IAV which influences viral pathogenicity, promotes lung inflammation, and increases the mortality and morbidity by modulating the host innate immune response [ 35 , 83 ]. The C-terminal of PB1-F2 is critical for its mitochondria translocation, in which the amino acids 61—87 contributes to IAV pathogenesis and severe inflammation [ 35 ].
Moreover, the N66S substitution in PB1-F2 protein is associated with enhanced virulence in mice, as shown previously [ 38 ]. In this study, we provided evidence for the first time that the PB1-F2 protein acted as a mitophagy receptor that was recognized specifically by LC3B. Third, PB1-F2-induced mitophagy appeared to depend on its mitochondrial localization, as TUFM knockout not only decreased the localization of PB1-F2 to mitochondria but also abolished its capacity to induce autophagic and subsequent mitophagy signaling.
Thus, we tried to assure that the LIR motif was responsible for PB1-F2-induced mitophagy and attenuated innate immunity in infection. Thus, improved understanding of mitophagy induction during IAV infection will facilitate the development of antiviral therapeutics strategies to combat influenza viral infection and pathogenesis.
The supernatants were collected, and equal amounts of proteins were separated by SDS-PAGE, and then were transferred to a nitrocellulose membrane GE Healthcare Life sciences, 10,, and 10,, All bands of western blots were detected within the linear range. The A Renilla control plasmid pGL4. The medium was replaced 12 h later. Viral particles were collected 60 h post-transfection and filtered with low-protein-binding filters Millex-HV, 0.
After 48 hpi, the lentivirus-infected cells were selected in 1. Finally, the monoclonal cells were acquired by using the limiting dilution method in well plates. Cells were then washed with PBS and incubated with 0.
HEK T cells grown in well plates were co-transfected with 0. Data were presented as relative firefly luciferase activities normalized to Renilla luciferase activities and were representative of 3 independent experiments. Cells were washed with ice-cold PBS twice and then detached with a cell scraper. The supernatants were considered as the cytosolic fraction. P value equal or lower to 0. We thank Ms. Supplemental data for this article can be accessed here.
National Center for Biotechnology Information , U. Journal List Autophagy v. Published online Feb Author information Copyright and License information Disclaimer. This article has been cited by other articles in PMC.
Introduction Mitophagy is a process of the selective engulfment of mitochondria by autophagosomes and their subsequent degradation by lysosomes, which functions as selective removal of damaged mitochondria [ 1 , 2 ]. Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. PB1-F2 acts as a potential receptor of mitophagy to interact with LC3B via the LIR motif Mitophagy is a type of autophagy that selectively sequesters the damaged mitochondria by autophagosome via the interaction of mitochondrial receptors with LC3B [ 4 ].
Figure 6. Discussion Mitophagy, the selective engulfment, and clearance of mitochondria are essential for the homeostasis of a healthy network of functioning mitochondria and prevent excessive production of cytotoxic ROS reactive oxygen species from damaged mitochondria. Luciferase reporter assay HEK T cells grown in well plates were co-transfected with 0. Supplementary Material Supplemental Material: Click here for additional data file.
Acknowledgments We thank Ms. Disclosure statement No potential conflict of interest was reported by the authors. Supplementary material Supplemental data for this article can be accessed here. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys. PINK1-and Parkin-mediated mitophagy at a glance. J Cell Sci. Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis.
PLoS Pathog. Mechanisms of mitophagy. There is no indication of a persistent poration effect, however, which could be responsible for the measured channel currents. This observation requires further investigation in future work considering oligomers of the PB1-F2 peptide. Early studies described PB1-F2 as a proapoptotic protein, which was assumed to remove host immune cells responding to IAV infection [4] , [11].
The molecular mechanisms of PB1-F2 induced apoptosis is still not totally understood, though recent results confirmed that its proapoptotic function is cell specific and related to PKC activation after infection of primary human monocytes [17].
However, since the protein is localized predominantly in the mitochondria of transfected or infected cells and found to alter the mitochondrial morphology [4] it was hypothesized that apoptosis induction occurs due to direct interaction with subunits of the mitochondrial PTPC or destabilisation of the mitochondrial membrane by pore formation [10].
In previous experiments it has been demonstrated that PB1-F2 causes an elevation of membrane leakage [14] and increases the electrical conductance of membranes [12]. However, the currents elicited by the PB1-F2 protein were not showing defined conductance levels and channel like fluctuations. The results of these experiments therefore fostered the hypothesis that the PB1-F2 protein is not forming canonical protein mediated transmembrane pores.
It was speculated that the protein may destabilize the lipid bilayer due to the formation of lipidic pores [12]. The question of how this protein destabilizes the mitochondrial membrane potential, by channel like or lipidic pore formation, is still under investigation.
Thus, it can be concluded that PB1-F2 can short circuit the mitochondria by inserting channel forming proteins into the organelle membrane.
This finding might explain the property of PB1-F2 to disrupt the reticulotubular mitochondrial organization and to dissipate the inner membrane potential [4] , [10]. A similar strategy for inducing apoptosis is known from a number of other viruses [19]. This together with the results from MD simulations, which show that the truncated fragment is stable in the membrane, suggests that the c-terminus is part of the channel forming domain in the protein.
But since the truncated and the full-length protein evoke different unitary conductances we must nonetheless assume that also the n-terminal part of the protein is involved in channel formation.
Indeed the simulations show that the monomer alone leads to membrane denting, but is apparently incapable of inducing ion permeation; we did not find evidence for sufficient destabilization of the membrane to allow for passive ion translocation.
Although we made no attempt to model a multi-protein complex, it is very likely that such a complex is necessary to facilitate ion transport. This hypothesis is supported by the finding that PB1-F2 has indeed the tendency to form oligomers. The latter involves distinct domains not only in the the C- but also in the N terminus [13] in line with the hypothesis, that both termini are important for the complete channel activity.
The data however do not yet explain how a peptide with that many charges is able to insert into the membrane. The unusual behavior of these proteins can be explained by an interaction of the membrane with the distinct peptide structure; the charged amino acids in these membrane-inserting cationic peptides have to be evaluated in the overall context and their specific position in the entire peptide [20].
The structural and charge density features of the PB1-F2 protein are apparently similar to those of cationic peptides, which are able to insert spontaneously into membranes. Due to the large net charge it is very likely that the voltage dependency of the PB1-F2 conductance is related to a voltage-induced change of the positioning and orientation within the membrane. This issue needs further scrutiny by MD simulations employing an external transmembrane potential.
Both experimental and theoretical data support the view that channel activity is achieved by protein pores, which are formed by complex formation of the PB1-F2 protein. The importance of the c-terminus for channel activity is consistent with the finding that similar channel fluctuations are observed here with all three PB1-F2 analogs. The three proteins are variable in length with an overall identity of ca. Interestingly, all human H1N1 isolates circulating since were reported to code for a truncated PB1-F2 of 57 amino acids [5] lacking the c-terminal domain.
Moreover, the current H1N1 swine flu isolates are not able to express any form of PB1-F2 since multiple Stop-codon mutations only allow the expression of the first 11 amino acids. This is consistent with initial findings that expression of PB1-F2 is species dependent and often interrupted in swine isolates [4]. The fact that the PB1-F2 peptides derived from different isolates were all able to generate similar channel like activity in planar lipid biliayers implies that the different PB1-F2 variants have the same potential of ion channel conductance within the mitochondria of infected cells.
We can only speculate about the reasons why this channel type activity was recorded here but not in a previous study [12]. It is possible that the fusion efficiency of the peptides was in the present study lower than in a previous study.
A lower number of proteins in the bilayer however can be beneficial for the resolution of single channel activity because individual events are not masked by the simultaneous activity of many channels.
Also in the case of other pore forming peptides step like channel fluctuations can only be observed when the number of for proteins inserted into the bilayer is low [20] , [23].
The analysis of PB1-F2 evoked unitary channel currents in buffers with different ion compositions reveals that the channel has two main conductance levels and both are non-selective; they transport cations as well as anions with a marginal preference for anions. The low selectivity of the channel and in particular the permeability to gluconate implies a very simple and wide channel pore.
The tendency of PB1-F2 to form oligomers [13] presumably results in a self-assembly of multiple monomers in membranes with a central water filled pore. Similar channel pores with low selectivity can also be formed by aggregation of small membrane proteins from other viruses [24] — [26] or by the aforementioned alamethicin [22].
One particular feature of the PB1-F2 induced channel activity is the occurrence of at least two defined conductance levels, which can be achieved directly from the closed state.
This is not unique to PB1-F2 but can also be observed with other small channel forming membrane proteins such phospholamban [27]. Also the antibiotic peptide alamethicin generates multiple unitary current levels whose conductances obey a geometrical progression; each transition is thought to result from the uptake or release of an individual monomer within the conducting bundle [28].
This explanation however does not hold true for the PB1-F2 generated conductances because inspections of the current traces also revealed intermediate closures from the high conductance level to lower levels see Fig. Such behaviour cannot be explained by dynamic uptake or release of individual monomers from a conducting bundle. We must hence conclude that the low conductance level is a real sub-state of a channel protein build with a fixed number of monomers.
A single amino acid exchange in the c-terminal region of the protein from Asn66 to Ser66 is sufficient to convert a virus of moderate pathogenicity into a highly pathogenic one [7]. This finding is important because most IAV related deaths are due to bacterial super-infection [6]. The present data show that the three PB1-F2 peptides from IAV of different pahtogenic potential have in spite of their sequence differences the same ability to generate ion channel activity.
Hence the difference in biological function between the three PB1-F2 proteins must be due to other properties such as mitochondrial targeting or association with other proteins. Experiments with planar lipid bilayers were performed as described by Schrempf et al. Before adding the protein to the trans chamber the bilayer conductance was recorded for some time in order to exclude artefacts from contaminations.
Only perfectly silent bilayers were used for reconstitution of PB1-F2 proteins. During thawing periods the liposomes were kept in an ultrasonic bath for 5 minutes. This procedure resulted in the loading of liposomes with dye. The fluorescence signals Fig.
In the microplate experiments Fluo3 was excited at nm and lucigenin at nm. All titratable residues were kept at their standard protonation states. The simulation has been performed with a modified version of the program NAMD 2. Starting with the experimental NMR structure [13] , the protein was inserted into a pre-equilibrated POPC membrane by removing overlapping lipid molecules, leading to 26 and 28 POPC molecules in each layer.
The system was solvated by ca. The total system contained atoms. The simulation time step was 2 fs, electrostatics were treated by the particle mesh Ewald method [38] on a 64x64x grid. Bond distances to hydrogen atoms were kept fixed.
Production simulations were performed in the isothermal-isobaric NpT ensemble using the Langevin piston algorithm at 1 atm [39] , [40] with an oscillation period of fs and damping constant of fs, and the Langevin thermostat with a coupling constant of 5 ps After minimization over steps we successively turned on the barostat and the thermostat over 0. Data for the fully free system were collected over ns. Competing Interests: The authors have declared that no competing interests exist.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. PLoS One. Published online Jun DiFrancesco , 5 Leonhard M. Henkes , 1 Michael Kreim , 1 Stefan M. Mattia L. Leonhard M. Stefan M. Ralph Tripp, Editor. Author information Article notes Copyright and License information Disclaimer.
Received Mar 10; Accepted May Copyright Henkel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. This article has been cited by other articles in PMC.
Abstract 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. Introduction Influenza A Virus IAV is a permanent threat to humans and animals with the potential to cause disastrous pandemics which appeared periodically in the last century causing millions of fatal casualities [1] , [2].
Open in a separate window. Figure 1. Alignment of predicted amino acid sequences of PB1-F2 proteins. Figure 2. Figure 3. Unitary channel fluctuations and current voltage relation of sPB1-F2 pr8 generated conductance in planar lipid bilayer. Figure 4. Figure 5. Figure 6. The truncated peptide s PB1-F2 pr8 50—87 generates an elevated membrane conductance.
Figure 7. MD simulation The electrical data imply that PB1-F2 is able to form with its c-terminus ion channels in membranes. Figure 8. Dependence of various measures for protein stability over the simulation time after removal of the center-of-mass c. Figure 9. Snapshots of the simulation system after removal of the center-of-mass constraint set to 0 ns. In untreated cells, p65 was detected both in the cytoplasm and in the nucleus Fig.
As expected, stimulation of cells with LPS plus soluble CD14 [39] resulted in an obvious nuclear accumulation of p A shift of the probe was seen when extracts from cells transfected with the empty plasmid were used Fig. A recent study mapped the pro-inflammatory domain of PB1-F2 peptides delivered intranasally and identified residues L62, R75, R79 and L82 as essential for the induced lung immunopathology [48].
Additionally, these residues have been associated with enhanced pathogenicity in the context of bacterial secondary infection [49]. We found no evidence for increased ubiquitination of p65 in cells expressing PB1-F2 data not shown. However, we found no evidence for direct interaction between p65 and PB1-F2 by immunoprecipitation data not shown. The results of deletions of PB1-F2 in the virus genome are conflicting. In contrast, similar experiments carried out by Dudek et al. Conenello et al.
It is remarkable that a virus has evolved two different proteins to interact with the same host kinase. Do they synergize with each other?
The answers to these questions are beyond the scope of this report. However, the mutation of the PB1-F2 start codon has been shown to up-regulate N40 levels [47]. Alignment of the PB1-F2 protein sequences tested in this study. PB1-F2 does not have a general impact on cell signaling.
No statistically significant differences from control empty vector were observed one-way ANOVA followed by Dunnett's test. Expression of PB1-F2 constructs in transfected Vero cells.
Vero cells in a 24 well plate, were transfected with ng of each of the different PB1-F2 constructs and PB1-F2 expression was detected by immunofluorescence using a monoclonal mouse anti-myc antibody Sigma, M We thank Steve Goodbourn, Steve Ley, Katrin Rittinger, for plasmids and discussion; we thank Haixia Xiao, Nicole Friedrich and Saira Hussain for discussion and assistance with reverse genetics and we thank Rod Daniels for discussion and critically reviewing the manuscript.
This work was funded by the Medical Research Council through programme U The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U.
PLoS One. Published online May John W. Paul Digard, Editor. Author information Article notes Copyright and License information Disclaimer. Competing Interests: The authors have declared that no competing interests exists. Received Jan 20; Accepted Apr 7. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
This article has been cited by other articles in PMC. Figure S2: PB1-F2 does not have a general impact on cell signaling. Abstract 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.
Introduction Influenza A viruses have a significant impact on the human population as demonstrated not only by the mortality associated with infection, but also by their economic impact on health care systems around the world. Immunoprecipitation Vero cells 2. Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. TIF Click here for additional data file. Figure S2 PB1-F2 does not have a general impact on cell signaling. References 1.
Nat Rev Microbiol 9 : — Immunol Rev : 68— J Virol 79 : —
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