Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.
References
[1]
Cantin R, Methot S, Tremblay MJ (2005) Plunder and stowaways: incorporation of cellular proteins by enveloped viruses. J Virol 79: 6577–6587.
Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, et al. (2004) Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 78: 10960–10966.
[4]
Bechtel JT, Winant RC, Ganem D (2005) Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus. J Virol 79: 4952–4964.
[5]
Bortz E, Whitelegge JP, Jia Q, Zhou ZH, Stewart JP, et al. (2003) Identification of proteins associated with murine gammaherpesvirus 68 virions. J Virol 77: 13425–13432.
[6]
Johannsen E, Luftig M, Chase MR, Weicksel S, Cahir-McFarland E, et al. (2004) Proteins of purified Epstein-Barr virus. Proc Natl Acad Sci U S A 101: 16286–16291.
[7]
Kattenhorn LM, Mills R, Wagner M, Lomsadze A, Makeev V, et al. (2004) Identification of proteins associated with murine cytomegalovirus virions. J Virol 78: 11187–11197.
[8]
Zhu FX, Chong JM, Wu L, Yuan Y (2005) Virion proteins of Kaposi's sarcoma-associated herpesvirus. J Virol 79: 800–811.
[9]
O'Connor CM, Kedes DH (2006) Mass spectrometric analyses of purified rhesus monkey rhadinovirus reveal 33 virion-associated proteins. J Virol 80: 1574–1583.
[10]
Chung CS, Chen CH, Ho MY, Huang CY, Liao CL, et al. (2006) Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles. J Virol 80: 2127–2140.
[11]
Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, et al. (2006) Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 80: 9039–9052.
[12]
Saphire AC, Gallay PA, Bark SJ (2006) Proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins. J Proteome Res 5: 530–538.
[13]
Segura MM, Garnier A, Di Falco MR, Whissell G, Meneses-Acosta A, et al. (2008) Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations. J Virol 82: 1107–1117.
[14]
Demirov DG, Ono A, Orenstein JM, Freed EO (2002) Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc Natl Acad Sci U S A 99: 955–960.
[15]
Franke EK, Yuan HE, Luban J (1994) Specific incorporation of cyclophilin A into HIV-1 virions. Nature 372: 359–362.
[16]
Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, et al. (1994) Functional association of cyclophilin A with HIV-1 virions. Nature 372: 363–365.
[17]
Mariani R, Chen D, Schrofelbauer B, Navarro F, Konig R, et al. (2003) Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114: 21–31.
[18]
Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, et al. (2001) Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107: 55–65.
[19]
Braaten D, Luban J (2001) Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells. Embo J 20: 1300–1309.
[20]
Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, et al. (2003) Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99–103.
[21]
Mayer D, Molawi K, Martinez-Sobrido L, Ghanem A, Thomas S, et al. (2007) Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J Proteome Res 6: 672–682.
[22]
Palese P, Shaw M (2007) Orthomyxoviridae: The Viruses and Their Replication. In: Knipe D, Howley P, editors. Fields Virology, 5th Edition. Philadelphia: Lippincott, Williams and Wilkins. pp. 1647–1689.
[23]
Compans RW, Klenk HD, Caliguiri LA, Choppin PW (1970) Influenza virus proteins. I. Analysis of polypeptides of the virion and identification of spike glycoproteins. Virology 42: 880–889.
[24]
Schulze IT (1970) The structure of influenza virus. I. The polypeptides of the virion. Virology 42: 890–904.
[25]
Skehel JJ, Schild GC (1971) The polypeptide composition of influenza A viruses. Virology 44: 396–408.
[26]
Zebedee SL, Lamb RA (1988) Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol 62: 2762–2772.
[27]
Inglis SC, Carroll AR, Lamb RA, Mahy BW (1976) Polypeptides specified by the influenza virus genome I. Evidence for eight distinct gene products specified by fowl plague virus. Virology 74: 489–503.
[28]
Richardson JC, Akkina RK (1991) NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Arch Virol 116: 69–80.
[29]
Palese P, Ritchey MB, Schulman JL (1977) Mapping of the influenza virus genome. II. Identification of the P1, P2, and P3 genes. Virology 76: 114–121.
[30]
Ott DE, Coren LV, Johnson DG, Sowder RC 2nd, Arthur LO, et al. (1995) Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. AIDS Res Hum Retroviruses 11: 1003–1006.
[31]
Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, et al. (1996) Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 70: 7734–7743.
[32]
Wubbolts R, Leckie RS, Veenhuizen PT, Schwarzmann G, Mobius W, et al. (2003) Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J Biol Chem 278: 10963–10972.
[33]
Hegmans JP, Bard MP, Hemmes A, Luider TM, Kleijmeer MJ, et al. (2004) Proteomic analysis of exosomes secreted by human mesothelioma cells. Am J Pathol 164: 1807–1815.
[34]
Mears R, Craven RA, Hanrahan S, Totty N, Upton C, et al. (2004) Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 4: 4019–4031.
[35]
Chazal N, Gerlier D (2003) Virus entry, assembly, budding, and membrane rafts. Microbiol Mol Biol Rev 67: 226–237, table of contents.
[36]
Nguyen DH, Hildreth JE (2000) Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol 74: 3264–3272.
[37]
Scheiffele P, Rietveld A, Wilk T, Simons K (1999) Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 274: 2038–2044.
[38]
Nayak DP, Hui EK, Barman S (2004) Assembly and budding of influenza virus. Virus Res 106: 147–165.
[39]
Ono A, Freed EO (2001) Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci U S A 98: 13925–13930.
[40]
Brugger B, Glass B, Haberkant P, Leibrecht I, Wieland FT, et al. (2006) The HIV lipidome: a raft with an unusual composition. Proc Natl Acad Sci U S A 103: 2641–2646.
[41]
Foster LJ, De Hoog CL, Mann M (2003) Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A 100: 5813–5818.
[42]
Li N, Shaw AR, Zhang N, Mak A, Li L (2004) Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry. Proteomics 4: 3156–3166.
[43]
Li N, Mak A, Richards DP, Naber C, Keller BO, et al. (2003) Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation. Proteomics 3: 536–548.
[44]
Blonder J, Hale ML, Lucas DA, Schaefer CF, Yu LR, et al. (2004) Proteomic analysis of detergent-resistant membrane rafts. Electrophoresis 25: 1307–1318.
[45]
von Haller PD, Donohoe S, Goodlett DR, Aebersold R, Watts JD (2001) Mass spectrometric characterization of proteins extracted from Jurkat T cell detergent-resistant membrane domains. Proteomics 1: 1010–1021.
[46]
Radtke K, Dohner K, Sodeik B (2006) Viral interactions with the cytoskeleton: a hitchhiker's guide to the cell. Cell Microbiol 8: 387–400.
[47]
Smith GA, Enquist LW (2002) Break ins and break outs: viral interactions with the cytoskeleton of Mammalian cells. Annu Rev Cell Dev Biol 18: 135–161.
[48]
Moyer SA, Baker SC, Horikami SM (1990) Host cell proteins required for measles virus reproduction. J Gen Virol 71 (Pt 4): 775–783.
[49]
Bukrinskaya A, Brichacek B, Mann A, Stevenson M (1998) Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton. J Exp Med 188: 2113–2125.
[50]
Gupta S, De BP, Drazba JA, Banerjee AK (1998) Involvement of actin microfilaments in the replication of human parainfluenza virus type 3. J Virol 72: 2655–2662.
[51]
Burke E, Dupuy L, Wall C, Barik S (1998) Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus. Virology 252: 137–148.
[52]
Sun X, Whittaker GR (2007) Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells. Cell Microbiol 9: 1672–1682.
[53]
Simpson-Holley M, Ellis D, Fisher D, Elton D, McCauley J, et al. (2002) A functional link between the actin cytoskeleton and lipid rafts during budding of filamentous influenza virions. Virology 301: 212–225.
[54]
Avalos RT, Yu Z, Nayak DP (1997) Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells. J Virol 71: 2947–2958.
[55]
Digard P, Elton D, Bishop K, Medcalf E, Weeds A, et al. (1999) Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments. J Virol 73: 2222–2231.
[56]
Hayes MJ, Rescher U, Gerke V, Moss SE (2004) Annexin-actin interactions. Traffic 5: 571–576.
[57]
Jacob R, Heine M, Eikemeyer J, Frerker N, Zimmer KP, et al. (2004) Annexin II is required for apical transport in polarized epithelial cells. J Biol Chem 279: 3680–3684.
[58]
Ryzhova EV, Vos RM, Albright AV, Harrist AV, Harvey T, et al. (2006) Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages. J Virol 80: 2694–2704.
[59]
Raynor CM, Wright JF, Waisman DM, Pryzdial EL (1999) Annexin II enhances cytomegalovirus binding and fusion to phospholipid membranes. Biochemistry 38: 5089–5095.
[60]
Derry MC, Sutherland MR, Restall CM, Waisman DM, Pryzdial EL (2007) Annexin 2-mediated enhancement of cytomegalovirus infection opposes inhibition by annexin 1 or annexin 5. J Gen Virol 88: 19–27.
[61]
Mailliard WS, Haigler HT, Schlaepfer DD (1996) Calcium-dependent binding of S100C to the N-terminal domain of annexin I. J Biol Chem 271: 719–725.
[62]
Hemler ME (2005) Tetraspanin functions and associated microdomains. Nat Rev Mol Cell Biol 6: 801–811.
[63]
Le Naour F, Andre M, Boucheix C, Rubinstein E (2006) Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts. Proteomics 6: 6447–6454.
[64]
Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, et al. (2004) CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol 78: 1448–1455.
[65]
Cormier EG, Tsamis F, Kajumo F, Durso RJ, Gardner JP, et al. (2004) CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 101: 7270–7274.
[66]
Ho SH, Martin F, Higginbottom A, Partridge LJ, Parthasarathy V, et al. (2006) Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1. J Virol 80: 6487–6496.
[67]
Gordon-Alonso M, Yanez-Mo M, Barreiro O, Alvarez S, Munoz-Fernandez MA, et al. (2006) Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion. J Immunol 177: 5129–5137.
[68]
de Parseval A, Lerner DL, Borrow P, Willett BJ, Elder JH (1997) Blocking of feline immunodeficiency virus infection by a monoclonal antibody to CD9 is via inhibition of virus release rather than interference with receptor binding. J Virol 71: 5742–5749.
[69]
Schmid E, Zurbriggen A, Gassen U, Rima B, ter Meulen V, et al. (2000) Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion. J Virol 74: 7554–7561.
[70]
Jolly C, Sattentau QJ (2007) Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains. J Virol 81: 7873–7884.
[71]
Khurana S, Krementsov DN, de Parseval A, Elder JH, Foti M, et al. (2007) HIV-1 and influenza virus exit via different membrane microdomains. J Virol 81: 12630–12640.
[72]
Sokolskaja E, Sayah DM, Luban J (2004) Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity. J Virol 78: 12800–12808.
[73]
Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD (2005) Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells. J Virol 79: 176–183.
[74]
Towers GJ (2007) The control of viral infection by tripartite motif proteins and cyclophilin A. Retrovirology 4: 40.
[75]
Luban J (2007) Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J Virol 81: 1054–1061.
[76]
Bose S, Mathur M, Bates P, Joshi N, Banerjee AK (2003) Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype. J Gen Virol 84: 1687–1699.
[77]
Luo C, Luo H, Zheng S, Gui C, Yue L, et al. (2004) Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A. Biochem Biophys Res Commun 321: 557–565.
[78]
Castro AP, Carvalho TM, Moussatche N, Damaso CR (2003) Redistribution of cyclophilin A to viral factories during vaccinia virus infection and its incorporation into mature particles. J Virol 77: 9052–9068.
[79]
Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, et al. (2005) Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 19: 111–122.
[80]
Saifuddin M, Parker CJ, Peeples ME, Gorny MK, Zolla-Pazner S, et al. (1995) Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1. J Exp Med 182: 501–509.
[81]
Spear GT, Lurain NS, Parker CJ, Ghassemi M, Payne GH, et al. (1995) Host cell-derived complement control proteins CD55 and CD59 are incorporated into the virions of two unrelated enveloped viruses. Human T cell leukemia/lymphoma virus type I (HTLV-I) and human cytomegalovirus (HCMV). J Immunol 155: 4376–4381.
[82]
Vanderplasschen A, Mathew E, Hollinshead M, Sim RB, Smith GL (1998) Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope. Proc Natl Acad Sci U S A 95: 7544–7549.
[83]
Ogino T, Yamadera T, Nonaka T, Imajoh-Ohmi S, Mizumoto K (2001) Enolase, a cellular glycolytic enzyme, is required for efficient transcription of Sendai virus genome. Biochem Biophys Res Commun 285: 447–455.
[84]
Kim JW, Dang CV (2005) Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 30: 142–150.
[85]
Choudhary S, De BP, Banerjee AK (2000) Specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro. J Virol 74: 3634–3641.
[86]
De BP, Gupta S, Zhao H, Drazba JA, Banerjee AK (1996) Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cis-acting RNAs of human parainfluenza virus type 3. J Biol Chem 271: 24728–24735.
[87]
Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, et al. (1998) Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine 16: 960–968.
[88]
Romanova J, Katinger D, Ferko B, Vcelar B, Sereinig S, et al. (2004) Live cold-adapted influenza A vaccine produced in Vero cell line. Virus Res 103: 187–193.
[89]
Shifman MA, Li Y, Colangelo CM, Stone KL, Wu TL, et al. (2007) YPED: a web-accessible database system for protein expression analysis. J Proteome Res 6: 4019–4024.
[90]
Gurer C, Cimarelli A, Luban J (2002) Specific incorporation of heat shock protein 70 family members into primate lentiviral virions. J Virol 76: 4666–4670.
[91]
Ott DE, Coren LV, Copeland TD, Kane BP, Johnson DG, et al. (1998) Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus. J Virol 72: 2962–2968.
