PB1-F2 Influenza A Virus Protein Adopts a β-Sheet Conformation and Forms Amyloid Fibers in Membrane Environments
2010; Elsevier BV; Volume: 285; Issue: 17 Linguagem: Inglês
10.1074/jbc.m109.067710
ISSN1083-351X
AutoresChristophe Chevalier, Ali Al Bazzal, Jasmina Vidić, Vincent Février, Christiane Bourdieu, Edwige Bouguyon, Ronan Le Goffic, Jean-François Vautherot, Julie Bernard, Mohammed M. Moudjou, Sylvie Noinville, Jean‐François Chich, Bruno Da Costa, Human Rezaei, Bernard Delmas,
Tópico(s)Protein Structure and Dynamics
ResumoThe influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an α-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display β-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a β-sheet structure. Dynamic light scattering revealed that the presence of β-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation. The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an α-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display β-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a β-sheet structure. Dynamic light scattering revealed that the presence of β-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation. IntroductionInfluenza A virus (IAV) 3The abbreviations used are: IAVinfluenza A virusTFEtrifluoroethanolDLSdynamic light scatteringThTthioflavin TThSthioflavin SSPRsurface plasmon resonance. is a major pathogen of humans and animals responsible for three main pandemics of the last century: 1957, 1968, and the most severe in 1918 with 20–40 million deaths reported (1Lamb R.A. Takeda M. Nat. Med. 2001; 7: 1286-1288Crossref PubMed Scopus (48) Google Scholar). Every year, influenza epidemics cause considerable illness and death. Since 2003, one of the greatest worldwide pandemic threats remains the highly pathogenic H5N1 avian viruses that display a high lethality rate (60% of the 438 cases resulted in death) (2Lipatov A.S. Govorkova E.A. Webby R.J. Ozaki H. Peiris M. Guan Y. Poon L. Webster R.G. J. Virol. 2004; 78: 8951-8959Crossref PubMed Scopus (203) Google Scholar, 3Gambotto A. Barratt-Boyes S.M. de Jong M.D. Neumann G. Kawaoka Y. Lancet. 2008; 371: 1464-1475Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Since its identification in April 2009, a novel swine origin influenza A virus H1N1 is currently causing a pandemic as declared by the WHO in June 2009 (around 1% of the 300,000 cases resulted in death) (4Garten R.J. Davis C.T. Russell C.A. Shu B. Lindstrom S. Balish A. Sessions W.M. Xu X. Skepner E. Deyde V. Okomo-Adhiambo M. Gubareva L. Barnes J. Smith C.B. Emery S.L. Hillman M.J. Rivailler P. Smagala J. de Graaf M. Burke D.F. Fouchier R.A. Pappas C. Alpuche-Aranda C.M. López-Gatell H. Olivera H. López I. Myers C.A. Faix D. Blair P.J. Yu C. Keene K.M. Dotson Jr., P.D. Boxrud D. Sambol A.R. Abid S.H. St George K. Bannerman T. Moore A.L. Stringer D.J. Blevins P. Demmler-Harrison G.J. Ginsberg M. Kriner P. Waterman S. Smole S. Guevara H.F. Belongia E.A. Clark P.A. Beatrice S.T. Donis R. Katz J. Finelli L. Bridges C.B. Shaw M. Jernigan D.B. Uyeki T.M. Smith D.J. Klimov A.I. Cox N.J. Science. 2009; 325: 197-201Crossref PubMed Scopus (1986) Google Scholar). IAV belongs to the Orthomyxoviridae viral family, and its genome is composed of eight negative-strand RNA gene segments encoding 11 proteins (5Palese P. Cell. 1977; 10: 1-10Abstract Full Text PDF PubMed Scopus (173) Google Scholar). The virulence of IAV is complex and can be influenced by each of the eight viral segments and notably the HA, NA, and PB1 segments (6Pappas C. Aguilar P.V. Basler C.F. Solórzano A. Zeng H. Perrone L.A. Palese P. García-Sastre A. Katz J.M. Tumpey T.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3064-3069Crossref PubMed Scopus (123) Google Scholar).PB1-F2, the 11th discovered IAV protein, is translated from an alternative reading frame in the PB1 gene (+1 reading frame of PB1, PB1-Frame 2) (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar). PB1-F2 is expressed by many IAV isolates from a wide range of hosts and in nearly all avian and human strains (8Pancuchárová H. Russ G. Acta Virol. 2006; 50: 269-272PubMed Google Scholar, 9Zell R. Krumbholz A. Wutzler P. Emerg. Infect. Dis. 2006; 12 (author reply 1608–1609): 1607-1608Crossref PubMed Scopus (19) Google Scholar, 10Zell R. Krumbholz A. Eitner A. Krieg R. Halbhuber K.J. Wutzler P. J. Gen Virol. 2007; 88: 536-546Crossref PubMed Scopus (124) Google Scholar). Recent studies have described the influence of PB1-F2 on viral pathogenesis in mouse models (11Zamarin D. Ortigoza M.B. Palese P. J. Virol. 2006; 80: 7976-7983Crossref PubMed Scopus (255) Google Scholar, 12Conenello G.M. Zamarin D. Perrone L.A. Tumpey T. Palese P. PLoS Pathog. 2007; 3: 1414-1421Crossref PubMed Scopus (389) Google Scholar, 13Conenello G.M. Palese P. Cell Host Microbe. 2007; 2: 207-209Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14McAuley J.L. Hornung F. Boyd K.L. Smith A.M. McKeon R. Bennink J. Yewdell J.W. McCullers J.A. Cell Host Microbe. 2007; 2: 240-249Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Loss of PB1-F2 expression results in attenuation of the virus and in a more rapid clearance of the virus from the lungs of infected mice. Insertion of a single N66S mutation present in the PB1-F2 of the 1918 pandemic strain is sufficient to increase virulence of a low pathogenic IAV in mice (12Conenello G.M. Zamarin D. Perrone L.A. Tumpey T. Palese P. PLoS Pathog. 2007; 3: 1414-1421Crossref PubMed Scopus (389) Google Scholar). Moreover, a mouse model of secondary Streptococcus pneumoniae pneumonia following A/PR/834 virus infection shows greater weight loss of infected mice, more severe pneumonia, and a higher lethality with the virus expressing PB1-F2 compared with the PB1-F2 knock-out virus (13Conenello G.M. Palese P. Cell Host Microbe. 2007; 2: 207-209Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14McAuley J.L. Hornung F. Boyd K.L. Smith A.M. McKeon R. Bennink J. Yewdell J.W. McCullers J.A. Cell Host Microbe. 2007; 2: 240-249Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In addition, contemporary H1N1 strains that no longer express PB1-F2 seem to be less virulent. PB1-F2 is considered as one of the factors that contribute to IAV virulence (15Basler C.F. Aguilar P.V. Antiviral Res. 2008; 79: 166-178Crossref PubMed Scopus (91) Google Scholar).Compared with the other IAV proteins, PB1-F2 shows several distinct features. PB1-F2 is expressed differently among infected cells and independently of the expression level of other viral proteins. PB1-F2 has a short half-life and is rapidly degraded (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar). PB1-F2 is generally described as a proapoptotic factor that could facilitate evasion of host defenses (with NS1), allowing the virus to escape from the immune system by inducing apoptosis in macrophages and monocytes (1Lamb R.A. Takeda M. Nat. Med. 2001; 7: 1286-1288Crossref PubMed Scopus (48) Google Scholar, 7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar, 16Lowy R.J. Int. Rev. Immunol. 2003; 22: 425-449Crossref PubMed Scopus (79) Google Scholar, 17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar). Thus, PB1-F2 is thought to compromise the ability of the host to mobilize adaptive immune responses (18Coleman J.R. Virol. J. 2007; 4: 9Crossref PubMed Scopus (67) Google Scholar). Another specific feature is the mitochondrial tropism of PB1-F2, which is only partial because PB1-F2 can also be present in the nucleus and cytoplasm of infected cells, depending on the cell type (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar, 19Mazur I. Anhlan D. Mitzner D. Wixler L. Schubert U. Ludwig S. Cell Microbiol. 2008; 10: 1140-1152Crossref PubMed Scopus (125) Google Scholar). The carboxyl-terminal domain of the protein, containing the mitochondrial targeting signal, is capable by itself to interfere with mitochondrial function and cellular viability (20Gibbs J.S. Malide D. Hornung F. Bennink J.R. Yewdell J.W. J. Virol. 2003; 77: 7214-7224Crossref PubMed Scopus (182) Google Scholar, 21Yamada H. Chounan R. Higashi Y. Kurihara N. Kido H. FEBS Lett. 2004; 578: 331-336Crossref PubMed Scopus (113) Google Scholar). Studies of the interaction of a synthetic form of PB1-F2 with membrane showed that PB1-F2 was able to create pores of variable size in planar lipid membranes, suggesting that PB1-F2 could oligomerize to permeabilize membranes (22Chanturiya A.N. Basañez G. Schubert U. Henklein P. Yewdell J.W. Zimmerberg J. J. Virol. 2004; 78: 6304-6312Crossref PubMed Scopus (122) Google Scholar). PB1-F2 was also shown to interact with two proteins implicated in the formation of the mitochondrial permeability transition pore complex, ANT-3 (adenine nucleotide translocator 3), and VDAC-1 (voltage-dependent ion channel 1) (17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar).Although new insights have been gained in understanding the function of PB1-F2, very little structural information is available. Bruns and co-workers (23Henklein P. Bruns K. Nimtz M. Wray V. Tessmer U. Schubert U. J. Pept. Sci. 2005; 11: 481-490Crossref PubMed Scopus (24) Google Scholar, 24Röder R. Bruns K. Sharma A. Eissmann A. Hahn F. Studtrucker N. Fossen T. Wray V. Henklein P. Schubert U. J. Pept. Sci. 2008; 14: 954-962Crossref PubMed Scopus (9) Google Scholar) produced a full-length synthetic PB1-F2 and performed a study by CD and 1H NMR (25Bruns K. Studtrucker N. Sharma A. Fossen T. Mitzner D. Eissmann A. Tessmer U. Röder R. Henklein P. Wray V. Schubert U. J. Biol. Chem. 2007; 282: 353-363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The stability of the structured regions of PB1-F2 was shown to be dependent on the hydrophobicity of the solvent: random coil in aqueous solution and α-helical structure upon the addition of trifluoroethanol (TFE). This α-helical structure present in the positively charged C-terminal domain of PB1-F2 was described as not a true amphipathic helix and is more compact than previously predicted. In an effort to obtain deeper insight into the structure-function of PB1-F2 and to better understand its role in the virus cycle, we produced and compared PB1-F2 originating from seven IAV isolates, including the highly pathogenic strain H5N1, the 1918 Brevig Mission strain, and two apathogenic avian strains, in biochemical and biophysical assays. We showed that PB1-F2 can be classified in the newly described group of intrinsically disordered proteins, capable of conformation changes switching from a random to an α-helical or β-sheet secondary structure. PB1-F2 is able to oligomerize to form amyloid fibers upon different environmental conditions. The presence of amyloid PB1-F2 structures was also evidenced in infected cells. Moreover, functional tests showed that the seven variants of PB1-F2 efficiently permeabilized membranes.DISCUSSIONIn the present work, we have investigated both secondary and oligomeric structures of seven PB1-F2 variants to gain insight into their structural and functional relationships. We show that PB1-F2 has distinct folding pathways leading to the formation of either disordered conformations or α-helices or oligomeric β-enriched structures, depending on physico-chemical conditions (Fig. 7). As previously observed, our results confirm the disordered structure of PB1-F2 in aqueous solutions (acidic) as well as the formation of an α-helical secondary structure in TFE (25Bruns K. Studtrucker N. Sharma A. Fossen T. Mitzner D. Eissmann A. Tessmer U. Röder R. Henklein P. Wray V. Schubert U. J. Biol. Chem. 2007; 282: 353-363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). TFE is well known to induce α-helix folding by stabilizing hydrogen bonds in peptides (36Buck M. Q. Rev. Biophys. 1998; 31: 297-355Crossref PubMed Scopus (709) Google Scholar, 37Montserret R. Aubert-Foucher E. McLeish M.J. Hill J.M. Ficheux D. Jaquinod M. van der Rest M. Deléage G. Penin F. Biochemistry. 1999; 38: 6479-6488Crossref PubMed Scopus (29) Google Scholar) even if they do not have an intrinsic propensity to adopt α-helical structure. In contrast, all PB1-F2 variants exhibit β-sheet secondary structures in the presence of acetonitrile. Compared with TFE, acetonitrile is less polar, and thus it is a weaker hydrogen bond donor (38Kamlet M.J. Abboud J.L. Taft R.W. J. Am. Chem. Soc. 1977; 99: 6027-6037Crossref Scopus (1502) Google Scholar). Moreover, the β-sheet structural conformational switch of PB1-F2 was also observed with the addition of SDS. This mild detergent is commonly used for probing conformation of small proteins due to its ability to induce and stabilize ordered conformations in peptides having structure-forming potential (39Chang D.K. Cheng S.F. Chien W.J. J. Virol. 1997; 71: 6593-6602Crossref PubMed Google Scholar, 40Najbar L.V. Craik D.J. Wade J.D. Salvatore D. McLeish M.J. Biochemistry. 1997; 36: 11525-11533Crossref PubMed Scopus (46) Google Scholar, 41Zhong L. Johnson Jr., W.C. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 4462-4465Crossref PubMed Scopus (249) Google Scholar). Furthermore, SDS micelles have been used extensively for the structural investigations of membrane peptides because they provide an anionic, membrane-mimicking environment with a hydrophobic core and a polar head (42Montserret R. McLeish M.J. Böckmann A. Geourjon C. Penin F. Biochemistry. 2000; 39: 8362-8373Crossref PubMed Scopus (116) Google Scholar). Therefore, it clearly appears that the microenvironment surrounding PB1-F2 strongly affects its folding pathway. Finally, in the presence of small unilamellar asolectin liposomes of net negative charge, all PB1-F2 variants adopt a β-sheet conformation similar to those observed with acetonitrile and SDS.PB1-F2 oligomerizes in all of the conditions that favor β-sheet-enriched structure formation. In most cases, protein fibers emerge from an equilibrium of folded protein states with local unfolded or marginally stable structures that can be self-assembled into β-sheet oligomers, leading finally to the irreversible formation of insoluble fiber (43Dobson C.M. Semin. Cell Dev. Biol. 2004; 15: 3-16Crossref PubMed Scopus (713) Google Scholar). In contrast, oligomeric structures were not observed in TFE. The α-helical conformation evidenced with TFE may be considered as an end conversion pathway because only the PB1-F2 monomeric structured form was observed in this environmental condition. Fluorinated alcohols, such as TFE, have been shown to favor monomeric protein conformation by disrupting hydrophobic interaction in aggregated amyloid preparations (44Barrow C.J. Yasuda A. Kenny P.T. Zagorski M.G. J. Mol. Biol. 1992; 225: 1075-1093Crossref PubMed Scopus (605) Google Scholar). Only amorphous aggregates were observed upon the addition of acetonitrile. Amyloid fibers were generated in 0.01% SDS solution. Fibers were also observed in the presence of asolectin liposomes, as visualized by electronic microscopy. PB1-F2 variants form fibers of different lengths, from 100 nm to 1 μm. Because PB1-F2 variants exhibit differential abilities to form β-sheets and amyloid fibers, this process is likely to be sequence-specific. The structural switch leading to an amyloid pathway was observed for other viral proteins, such as the DNA binding domain of papillomavirus E2 proteins and EBNA-1 Epstein-Barr virus protein in vitro (45Wetzler D.E. Castaño E.M. de Prat-Gay G. Protein Sci. 2007; 16: 744-754Crossref PubMed Scopus (14) Google Scholar, 46Freire E. Oddo C. Frappier L. de Prat-Gay G. Proteins. 2008; 70: 450-461Crossref PubMed Scopus (11) Google Scholar).We also checked whether the structural switch between the unfolded state and oligomeric β-sheet-enriched structures may constitute a critical element for the regulation of PB1-F2 activities. PB1-F2 was previously reported to form pores of variable sizes in planar membranes (22Chanturiya A.N. Basañez G. Schubert U. Henklein P. Yewdell J.W. Zimmerberg J. J. Virol. 2004; 78: 6304-6312Crossref PubMed Scopus (122) Google Scholar). Otherwise, PB1-F2 was shown to be cytotoxic when added to cell medium at a low concentration (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar) and to promote loss of mitochondrial membrane potential by inducing morphological alterations (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar, 17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar). We show here that all of the variants used in this study permeabilize asolectin liposomes very efficiently, even at 5 nm nanomolar range concentrations. The fact that PB1-F2 adopts a β-sheet structure in the presence of synthetic membranes suggests a possible pore formation activity, as observed for many amyloid-forming proteins (33Lashuel H.A. Sci. Aging Knowledge Environ. 2005; 2005: pe28Crossref PubMed Scopus (38) Google Scholar). Furthermore, the net positive charge of PB1-F2 in acidic conditions may promote its incorporation into negatively charged asolectin liposomes by electrostatic interaction. Lysis assays showed that there is no direct correlation between the length of fibers and PB1-F2 activity. Interestingly, the addition of preformed fibers to liposomes did not lead to liposome permeabilization. This suggests that a prefibrillar protein but not amyloid may be the active lytic form. The ordered prefibrillar aggregates may form nonspecific pores perturbing cell ionic homeostasis. Frequently, prefibrillar aggregates display much higher toxicity than mature fibrils (47Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Crossref PubMed Scopus (1321) Google Scholar, 48Kayed R. Sokolov Y. Edmonds B. McIntire T.M. Milton S.C. Hall J.E. Glabe C.G. J. Biol. Chem. 2004; 279: 46363-46366Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar, 49Simoneau S. Rezaei H. Salès N. Kaiser-Schulz G. Lefebvre-Roque M. Vidal C. Fournier J.G. Comte J. Wopfner F. Grosclaude J. Schätzl H. Lasmézas C.I. PLoS Pathog. 2007; 3: e125Crossref PubMed Scopus (179) Google Scholar). The fast leakage kinetics observed here (within a few seconds) suggests the formation of β-amyloid pore structures rather than the generation of long fiber aggregates. The high activity of PB1-F2 on membranes (with pores formed at nanomolar concentrations) suggests that PB1-F2 within an infected cell may contribute to perturbation of the intracellular ionic homeostasis, leading ultimately to cell death.Our results do not rule out the possibility that free or membrane-bound PB1-F2 interacts with cellular and/or viral proteins. Actually, PB1-F2 was shown to interact with the mitochondrial proteins VDAC-1 and ANT-3 and to enhance the opening of the permeability transition pore complex (17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar). It has also been recently proposed that PB1-F2 could be involved in interactions with members of the Bcl-2 family known to regulate mitochondrially mediated apoptosis (50McLean J.E. Datan E. Matassov D. Zakeri Z.F. J. Virol. 2009; 83: 8233-8246Crossref PubMed Scopus (71) Google Scholar). On the other hand, PB1-F2 was shown to regulate viral polymerase activity by interaction with the PB1 protein in the nucleus (19Mazur I. Anhlan D. Mitzner D. Wixler L. Schubert U. Ludwig S. Cell Microbiol. 2008; 10: 1140-1152Crossref PubMed Scopus (125) Google Scholar). The natively disordered state of PB1-F2 in aqueous solutions could allow interactions with all of these proteins and lipids. Proteins other than PB1-F2 have also been shown to require a high degree of structural disorder to fulfill their functions and need to undergo disorder-order transition during or prior to their biological function (51Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1492) Google Scholar). Such proteins are frequently involved in some of the most important regulatory functions in the cell, and the lack of intrinsic structure in many cases is relieved when the protein binds to its target molecule (52Wright P.E. Dyson H.J. J. Mol. Biol. 1999; 293: 321-331Crossref PubMed Scopus (2303) Google Scholar).A critical finding of our work is that PB1-F2 forms amyloid-like fibers in infected cells. Although amyloidosis is mainly implicated in neurodegenerative diseases, fibrillization of some viral or cellular proteins has been evidenced in viral infections. Recently, semen-derived amyloid fibrils were shown to drastically enhance human immunodeficiency virus infection (53Münch J. Rücker E. Ständker L. Adermann K. Goffinet C. Schindler M. Wildum S. Chinnadurai R. Rajan D. Specht A. Giménez-Gallego G. Sánchez P.C. Fowler D.M. Koulov A. Kelly J.W. Mothes W. Grivel J.C. Margolis L. Keppler O.T. Forssmann W.G. Kirchhoff F. Cell. 2007; 131: 1059-1071Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). This capability is not restricted to retroviruses and suggests a possible role of amyloids in the transmission and pathogenesis of other enveloped viruses (54Wojtowicz W.M. Farzan M. Joyal J.L. Carter K. Babcock G.J. Israel D.I. Sodroski J. Mirzabekov T. J. Biol. Chem. 2002; 277: 35019-35024Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Viral proteins by themselves can also initiate protein aggregation and regulate amyloid formation (55Pulliam L. J. Neuroimmune Pharmacol. 2009; 4: 213-217Crossref PubMed Scopus (51) Google Scholar). Recent work suggests that highly pathogenic H5N1 influenza virus or other neurotropic influenza viruses could initiate central nervous system disorders, including Parkinson and Alzheimer diseases (56Jang H. Boltz D. Sturm-Ramirez K. Shepherd K.R. Jiao Y. Webster R. Smeyne R.J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14063-14068Crossref PubMed Scopus (304) Google Scholar). Our observation that PB1-F2 is able to form amyloidal fibers in virus-infected cells may be related to the nervous system disorders observed with influenza neurotropic viruses and opens the way for further studies of the role of PB1-F2 in the pathogenesis of the influenza virus.Overall, our results provide new insights into structural behavior of PB1-F2 able to switch from a disordered state to α-helix and to β-sheet structures in membranes. We demonstrate that PB1-F2 forms amyloid fibers in infected cells, an observation that explains the pathogenicity associated with this protein. These observations open the way for further studies to elucidate the role of PB1-F2 in the influenza virus cycle. IntroductionInfluenza A virus (IAV) 3The abbreviations used are: IAVinfluenza A virusTFEtrifluoroethanolDLSdynamic light scatteringThTthioflavin TThSthioflavin SSPRsurface plasmon resonance. is a major pathogen of humans and animals responsible for three main pandemics of the last century: 1957, 1968, and the most severe in 1918 with 20–40 million deaths reported (1Lamb R.A. Takeda M. Nat. Med. 2001; 7: 1286-1288Crossref PubMed Scopus (48) Google Scholar). Every year, influenza epidemics cause considerable illness and death. Since 2003, one of the greatest worldwide pandemic threats remains the highly pathogenic H5N1 avian viruses that display a high lethality rate (60% of the 438 cases resulted in death) (2Lipatov A.S. Govorkova E.A. Webby R.J. Ozaki H. Peiris M. Guan Y. Poon L. Webster R.G. J. Virol. 2004; 78: 8951-8959Crossref PubMed Scopus (203) Google Scholar, 3Gambotto A. Barratt-Boyes S.M. de Jong M.D. Neumann G. Kawaoka Y. Lancet. 2008; 371: 1464-1475Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Since its identification in April 2009, a novel swine origin influenza A virus H1N1 is currently causing a pandemic as declared by the WHO in June 2009 (around 1% of the 300,000 cases resulted in death) (4Garten R.J. Davis C.T. Russell C.A. Shu B. Lindstrom S. Balish A. Sessions W.M. Xu X. Skepner E. Deyde V. Okomo-Adhiambo M. Gubareva L. Barnes J. Smith C.B. Emery S.L. Hillman M.J. Rivailler P. Smagala J. de Graaf M. Burke D.F. Fouchier R.A. Pappas C. Alpuche-Aranda C.M. López-Gatell H. Olivera H. López I. Myers C.A. Faix D. Blair P.J. Yu C. Keene K.M. Dotson Jr., P.D. Boxrud D. Sambol A.R. Abid S.H. St George K. Bannerman T. Moore A.L. Stringer D.J. Blevins P. Demmler-Harrison G.J. Ginsberg M. Kriner P. Waterman S. Smole S. Guevara H.F. Belongia E.A. Clark P.A. Beatrice S.T. Donis R. Katz J. Finelli L. Bridges C.B. Shaw M. Jernigan D.B. Uyeki T.M. Smith D.J. Klimov A.I. Cox N.J. Science. 2009; 325: 197-201Crossref PubMed Scopus (1986) Google Scholar). IAV belongs to the Orthomyxoviridae viral family, and its genome is composed of eight negative-strand RNA gene segments encoding 11 proteins (5Palese P. Cell. 1977; 10: 1-10Abstract Full Text PDF PubMed Scopus (173) Google Scholar). The virulence of IAV is complex and can be influenced by each of the eight viral segments and notably the HA, NA, and PB1 segments (6Pappas C. Aguilar P.V. Basler C.F. Solórzano A. Zeng H. Perrone L.A. Palese P. García-Sastre A. Katz J.M. Tumpey T.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3064-3069Crossref PubMed Scopus (123) Google Scholar).PB1-F2, the 11th discovered IAV protein, is translated from an alternative reading frame in the PB1 gene (+1 reading frame of PB1, PB1-Frame 2) (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar). PB1-F2 is expressed by many IAV isolates from a wide range of hosts and in nearly all avian and human strains (8Pancuchárová H. Russ G. Acta Virol. 2006; 50: 269-272PubMed Google Scholar, 9Zell R. Krumbholz A. Wutzler P. Emerg. Infect. Dis. 2006; 12 (author reply 1608–1609): 1607-1608Crossref PubMed Scopus (19) Google Scholar, 10Zell R. Krumbholz A. Eitner A. Krieg R. Halbhuber K.J. Wutzler P. J. Gen Virol. 2007; 88: 536-546Crossref PubMed Scopus (124) Google Scholar). Recent studies have described the influence of PB1-F2 on viral pathogenesis in mouse models (11Zamarin D. Ortigoza M.B. Palese P. J. Virol. 2006; 80: 7976-7983Crossref PubMed Scopus (255) Google Scholar, 12Conenello G.M. Zamarin D. Perrone L.A. Tumpey T. Palese P. PLoS Pathog. 2007; 3: 1414-1421Crossref PubMed Scopus (389) Google Scholar, 13Conenello G.M. Palese P. Cell Host Microbe. 2007; 2: 207-209Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14McAuley J.L. Hornung F. Boyd K.L. Smith A.M. McKeon R. Bennink J. Yewdell J.W. McCullers J.A. Cell Host Microbe. 2007; 2: 240-249Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Loss of PB1-F2 expression results in attenuation of the virus and in a more rapid clearance of the virus from the lungs of infected mice. Insertion of a single N66S mutation present in the PB1-F2 of the 1918 pandemic strain is sufficient to increase virulence of a low pathogenic IAV in mice (12Conenello G.M. Zamarin D. Perrone L.A. Tumpey T. Palese P. PLoS Pathog. 2007; 3: 1414-1421Crossref PubMed Scopus (389) Google Scholar). Moreover, a mouse model of secondary Streptococcus pneumoniae pneumonia following A/PR/834 virus infection shows greater weight loss of infected mice, more severe pneumonia, and a higher lethality with the virus expressing PB1-F2 compared with the PB1-F2 knock-out virus (13Conenello G.M. Palese P. Cell Host Microbe. 2007; 2: 207-209Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14McAuley J.L. Hornung F. Boyd K.L. Smith A.M. McKeon R. Bennink J. Yewdell J.W. McCullers J.A. Cell Host Microbe. 2007; 2: 240-249Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In addition, contemporary H1N1 strains that no longer express PB1-F2 seem to be less virulent. PB1-F2 is considered as one of the factors that contribute to IAV virulence (15Basler C.F. Aguilar P.V. Antiviral Res. 2008; 79: 166-178Crossref PubMed Scopus (91) Google Scholar).Compared with the other IAV proteins, PB1-F2 shows several distinct features. PB1-F2 is expressed differently among infected cells and independently of the expression level of other viral proteins. PB1-F2 has a short half-life and is rapidly degraded (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar). PB1-F2 is generally described as a proapoptotic factor that could facilitate evasion of host defenses (with NS1), allowing the virus to escape from the immune system by inducing apoptosis in macrophages and monocytes (1Lamb R.A. Takeda M. Nat. Med. 2001; 7: 1286-1288Crossref PubMed Scopus (48) Google Scholar, 7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar, 16Lowy R.J. Int. Rev. Immunol. 2003; 22: 425-449Crossref PubMed Scopus (79) Google Scholar, 17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar). Thus, PB1-F2 is thought to compromise the ability of the host to mobilize adaptive immune responses (18Coleman J.R. Virol. J. 2007; 4: 9Crossref PubMed Scopus (67) Google Scholar). Another specific feature is the mitochondrial tropism of PB1-F2, which is only partial because PB1-F2 can also be present in the nucleus and cytoplasm of infected cells, depending on the cell type (7Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (803) Google Scholar, 19Mazur I. Anhlan D. Mitzner D. Wixler L. Schubert U. Ludwig S. Cell Microbiol. 2008; 10: 1140-1152Crossref PubMed Scopus (125) Google Scholar). The carboxyl-terminal domain of the protein, containing the mitochondrial targeting signal, is capable by itself to interfere with mitochondrial function and cellular viability (20Gibbs J.S. Malide D. Hornung F. Bennink J.R. Yewdell J.W. J. Virol. 2003; 77: 7214-7224Crossref PubMed Scopus (182) Google Scholar, 21Yamada H. Chounan R. Higashi Y. Kurihara N. Kido H. FEBS Lett. 2004; 578: 331-336Crossref PubMed Scopus (113) Google Scholar). Studies of the interaction of a synthetic form of PB1-F2 with membrane showed that PB1-F2 was able to create pores of variable size in planar lipid membranes, suggesting that PB1-F2 could oligomerize to permeabilize membranes (22Chanturiya A.N. Basañez G. Schubert U. Henklein P. Yewdell J.W. Zimmerberg J. J. Virol. 2004; 78: 6304-6312Crossref PubMed Scopus (122) Google Scholar). PB1-F2 was also shown to interact with two proteins implicated in the formation of the mitochondrial permeability transition pore complex, ANT-3 (adenine nucleotide translocator 3), and VDAC-1 (voltage-dependent ion channel 1) (17Zamarin D. García-Sastre A. Xiao X. Wang R. Palese P. PLoS Pathog. 2005; 1: e4Crossref PubMed Scopus (301) Google Scholar).Although new insights have been gained in understanding the function of PB1-F2, very little structural information is available. Bruns and co-workers (23Henklein P. Bruns K. Nimtz M. Wray V. Tessmer U. Schubert U. J. Pept. Sci. 2005; 11: 481-490Crossref PubMed Scopus (24) Google Scholar, 24Röder R. Bruns K. Sharma A. Eissmann A. Hahn F. Studtrucker N. Fossen T. Wray V. Henklein P. Schubert U. J. Pept. Sci. 2008; 14: 954-962Crossref PubMed Scopus (9) Google Scholar) produced a full-length synthetic PB1-F2 and performed a study by CD and 1H NMR (25Bruns K. Studtrucker N. Sharma A. Fossen T. Mitzner D. Eissmann A. Tessmer U. Röder R. Henklein P. Wray V. Schubert U. J. Biol. Chem. 2007; 282: 353-363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The stability of the structured regions of PB1-F2 was shown to be dependent on the hydrophobicity of the solvent: random coil in aqueous solution and α-helical structure upon the addition of trifluoroethanol (TFE). This α-helical structure present in the positively charged C-terminal domain of PB1-F2 was described as not a true amphipathic helix and is more compact than previously predicted. In an effort to obtain deeper insight into the structure-function of PB1-F2 and to better understand its role in the virus cycle, we produced and compared PB1-F2 originating from seven IAV isolates, including the highly pathogenic strain H5N1, the 1918 Brevig Mission strain, and two apathogenic avian strains, in biochemical and biophysical assays. We showed that PB1-F2 can be classified in the newly described group of intrinsically disordered proteins, capable of conformation changes switching from a random to an α-helical or β-sheet secondary structure. PB1-F2 is able to oligomerize to form amyloid fibers upon different environmental conditions. The presence of amyloid PB1-F2 structures was also evidenced in infected cells. Moreover, functional tests showed that the seven variants of PB1-F2 efficiently permeabilized membranes.
Referência(s)