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Published in: Virology Journal 1/2010

Open Access 01-12-2010 | Short report

HIV-1 is budded from CD4+ T lymphocytes independently of exosomes

Published in: Virology Journal | Issue 1/2010

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Abstract

The convergence of HIV-1 budding and exosome biogenesis at late endosomal compartments called multivesicular bodies has fueled the debate on whether HIV-1 is budded from its target cells and transmitted in the form of exosomes. The point of contention appears to primarily derive from the types of target cells in question and lack of a well-defined protocol to separate exosomes from HIV-1. In this study, we adapted and established a simplified protocol to define the relationship between HIV-1 production and exosome biogenesis. Importantly, we took advantage of the newly established protocol to unequivocally show that HIV-1 was produced from CD4+ T lymphocytes Jurkat cells independently of exosomes. Thus, this study not only presents a simplified way to obtain highly purified HIV-1 virions for identification of host proteins packaged into virions, but also provides a technical platform that can be employed to define the relationship between exosome biogenesis and budding of HIV-1 or other viruses and its contributions to viral pathogenesis.
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Literature
1.
go back to reference Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C: Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 1987, 262: 9412-9420.PubMed Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C: Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 1987, 262: 9412-9420.PubMed
2.
go back to reference Simpson RJ, Jensen SS, Lim JW: Proteomic profiling of exosomes: current perspectives. Proteomics 2008, 8: 4083-4099. 10.1002/pmic.200800109PubMedCrossRef Simpson RJ, Jensen SS, Lim JW: Proteomic profiling of exosomes: current perspectives. Proteomics 2008, 8: 4083-4099. 10.1002/pmic.200800109PubMedCrossRef
3.
go back to reference Pan BT, Johnstone RM: Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983, 33: 967-978. 10.1016/0092-8674(83)90040-5PubMedCrossRef Pan BT, Johnstone RM: Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983, 33: 967-978. 10.1016/0092-8674(83)90040-5PubMedCrossRef
4.
go back to reference Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G: The biogenesis and functions of exosomes. Traffic 2002, 3: 321-330. 10.1034/j.1600-0854.2002.30502.xPubMedCrossRef Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G: The biogenesis and functions of exosomes. Traffic 2002, 3: 321-330. 10.1034/j.1600-0854.2002.30502.xPubMedCrossRef
5.
go back to reference Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ: B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996, 183: 1161-1172. 10.1084/jem.183.3.1161PubMedCrossRef Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ: B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996, 183: 1161-1172. 10.1084/jem.183.3.1161PubMedCrossRef
6.
go back to reference Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998, 4: 594-600. 10.1038/nm0598-594PubMedCrossRef Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998, 4: 594-600. 10.1038/nm0598-594PubMedCrossRef
7.
go back to reference Johnstone RM, Adam M, Pan BT: The fate of the transferrin receptor during maturation of sheep reticulocytes in vitro. Can J Biochem Cell Biol 1984, 62: 1246-1254. 10.1139/o84-159PubMedCrossRef Johnstone RM, Adam M, Pan BT: The fate of the transferrin receptor during maturation of sheep reticulocytes in vitro. Can J Biochem Cell Biol 1984, 62: 1246-1254. 10.1139/o84-159PubMedCrossRef
8.
go back to reference Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ: Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol 2000, 165: 1259-1265.PubMedCrossRef Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ: Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol 2000, 165: 1259-1265.PubMedCrossRef
9.
go back to reference Hwang I, Shen X, Sprent J: Direct stimulation of naive T cells by membrane vesicles from antigen-presenting cells: distinct roles for CD54 and B7 molecules. Proc Natl Acad Sci USA 2003, 100: 6670-6675. 10.1073/pnas.1131852100PubMedPubMedCentralCrossRef Hwang I, Shen X, Sprent J: Direct stimulation of naive T cells by membrane vesicles from antigen-presenting cells: distinct roles for CD54 and B7 molecules. Proc Natl Acad Sci USA 2003, 100: 6670-6675. 10.1073/pnas.1131852100PubMedPubMedCentralCrossRef
10.
go back to reference Pelchen-Matthews A, Raposo G, Marsh M: Endosomes, exosomes and Trojan viruses. Trends Microbiol 2004, 12: 310-316. 10.1016/j.tim.2004.05.004PubMedCrossRef Pelchen-Matthews A, Raposo G, Marsh M: Endosomes, exosomes and Trojan viruses. Trends Microbiol 2004, 12: 310-316. 10.1016/j.tim.2004.05.004PubMedCrossRef
11.
go back to reference Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ: Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999, 94: 3791-3799.PubMed Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ: Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999, 94: 3791-3799.PubMed
12.
go back to reference Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007, 9: 654-659. 10.1038/ncb1596PubMedCrossRef Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007, 9: 654-659. 10.1038/ncb1596PubMedCrossRef
14.
go back to reference Thery C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002, 2: 569-579.PubMed Thery C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002, 2: 569-579.PubMed
15.
go back to reference Raposo G, Moore M, Innes D, Leijendekker R, Leigh-Brown A, Benaroch P, Geuze H: Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 2002, 3: 718-729. 10.1034/j.1600-0854.2002.31004.xPubMedCrossRef Raposo G, Moore M, Innes D, Leijendekker R, Leigh-Brown A, Benaroch P, Geuze H: Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 2002, 3: 718-729. 10.1034/j.1600-0854.2002.31004.xPubMedCrossRef
16.
go back to reference Leng Q, Bentwich Z, Magen E, Kalinkovich A, Borkow G: CTLA-4 upregulation during HIV infection: association with anergy and possible target for therapeutic intervention. Aids 2002, 16: 519-529. 10.1097/00002030-200203080-00002PubMedCrossRef Leng Q, Bentwich Z, Magen E, Kalinkovich A, Borkow G: CTLA-4 upregulation during HIV infection: association with anergy and possible target for therapeutic intervention. Aids 2002, 16: 519-529. 10.1097/00002030-200203080-00002PubMedCrossRef
17.
go back to reference Pelchen-Matthews A, Kramer B, Marsh M: Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 2003, 162: 443-455. 10.1083/jcb.200304008PubMedPubMedCentralCrossRef Pelchen-Matthews A, Kramer B, Marsh M: Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 2003, 162: 443-455. 10.1083/jcb.200304008PubMedPubMedCentralCrossRef
18.
go back to reference Nguyen DG, Booth A, Gould SJ, Hildreth JE: Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J Biol Chem 2003, 278: 52347-52354. 10.1074/jbc.M309009200PubMedCrossRef Nguyen DG, Booth A, Gould SJ, Hildreth JE: Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J Biol Chem 2003, 278: 52347-52354. 10.1074/jbc.M309009200PubMedCrossRef
19.
go back to reference Kramer B, Pelchen-Matthews A, Deneka M, Garcia E, Piguet V, Marsh M: HIV interaction with endosomes in macrophages and dendritic cells. Blood Cells Mol Dis 2005, 35: 136-142. 10.1016/j.bcmd.2005.06.006PubMedCrossRef Kramer B, Pelchen-Matthews A, Deneka M, Garcia E, Piguet V, Marsh M: HIV interaction with endosomes in macrophages and dendritic cells. Blood Cells Mol Dis 2005, 35: 136-142. 10.1016/j.bcmd.2005.06.006PubMedCrossRef
20.
go back to reference Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW Jr, Sowder RC, Barsov E, Hood BL, Fisher RJ, et al.: Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 2006, 80: 9039-9052. 10.1128/JVI.01013-06PubMedPubMedCentralCrossRef Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW Jr, Sowder RC, Barsov E, Hood BL, Fisher RJ, et al.: Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 2006, 80: 9039-9052. 10.1128/JVI.01013-06PubMedPubMedCentralCrossRef
21.
go back to reference Wiley RD, Gummuluru S: Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection. Proc Natl Acad Sci USA 2006, 103: 738-743. 10.1073/pnas.0507995103PubMedPubMedCentralCrossRef Wiley RD, Gummuluru S: Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection. Proc Natl Acad Sci USA 2006, 103: 738-743. 10.1073/pnas.0507995103PubMedPubMedCentralCrossRef
22.
go back to reference Izquierdo-Useros N, Naranjo-Gomez M, Archer J, Hatch SC, Erkizia I, Blanco J, Borras FE, Puertas MC, Connor JH, Fernandez-Figueras MT, et al.: Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway. Blood 2009, 113: 2732-2741. 10.1182/blood-2008-05-158642PubMedPubMedCentralCrossRef Izquierdo-Useros N, Naranjo-Gomez M, Archer J, Hatch SC, Erkizia I, Blanco J, Borras FE, Puertas MC, Connor JH, Fernandez-Figueras MT, et al.: Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway. Blood 2009, 113: 2732-2741. 10.1182/blood-2008-05-158642PubMedPubMedCentralCrossRef
23.
go back to reference Izquierdo-Useros N, Naranjo-Gomez M, Erkizia I, Puertas MC, Borras FE, Blanco J, Martinez-Picado J: HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse? PLoS Pathog 6: e1000740. 10.1371/journal.ppat.1000740 Izquierdo-Useros N, Naranjo-Gomez M, Erkizia I, Puertas MC, Borras FE, Blanco J, Martinez-Picado J: HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse? PLoS Pathog 6: e1000740. 10.1371/journal.ppat.1000740
24.
go back to reference Jouvenet N, Neil SJ, Bess C, Johnson MC, Virgen CA, Simon SM, Bieniasz PD: Plasma membrane is the site of productive HIV-1 particle assembly. PLoS Biol 2006, 4: e435. 10.1371/journal.pbio.0040435PubMedPubMedCentralCrossRef Jouvenet N, Neil SJ, Bess C, Johnson MC, Virgen CA, Simon SM, Bieniasz PD: Plasma membrane is the site of productive HIV-1 particle assembly. PLoS Biol 2006, 4: e435. 10.1371/journal.pbio.0040435PubMedPubMedCentralCrossRef
25.
go back to reference Gousset K, Ablan SD, Coren LV, Ono A, Soheilian F, Nagashima K, Ott DE, Freed EO: Real-time visualization of HIV-1 GAG trafficking in infected macrophages. PLoS Pathog 2008, 4: e1000015. 10.1371/journal.ppat.1000015PubMedPubMedCentralCrossRef Gousset K, Ablan SD, Coren LV, Ono A, Soheilian F, Nagashima K, Ott DE, Freed EO: Real-time visualization of HIV-1 GAG trafficking in infected macrophages. PLoS Pathog 2008, 4: e1000015. 10.1371/journal.ppat.1000015PubMedPubMedCentralCrossRef
26.
go back to reference Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, et al.: Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001, 107: 55-65. 10.1016/S0092-8674(01)00506-2PubMedCrossRef Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, et al.: Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001, 107: 55-65. 10.1016/S0092-8674(01)00506-2PubMedCrossRef
27.
go back to reference Morita E, Sundquist WI: Retrovirus budding. Annu Rev Cell Dev Biol 2004, 20: 395-425. 10.1146/annurev.cellbio.20.010403.102350PubMedCrossRef Morita E, Sundquist WI: Retrovirus budding. Annu Rev Cell Dev Biol 2004, 20: 395-425. 10.1146/annurev.cellbio.20.010403.102350PubMedCrossRef
28.
go back to reference Coren LV, Shatzer T, Ott DE: CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway. Retrovirology 2008, 5: 64. 10.1186/1742-4690-5-64PubMedPubMedCentralCrossRef Coren LV, Shatzer T, Ott DE: CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway. Retrovirology 2008, 5: 64. 10.1186/1742-4690-5-64PubMedPubMedCentralCrossRef
29.
go back to reference Ono A, Freed EO: Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. J Virol 2004, 78: 1552-1563. 10.1128/JVI.78.3.1552-1563.2004PubMedPubMedCentralCrossRef Ono A, Freed EO: Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. J Virol 2004, 78: 1552-1563. 10.1128/JVI.78.3.1552-1563.2004PubMedPubMedCentralCrossRef
30.
go back to reference Jolly C, Sattentau QJ: Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains. J Virol 2007, 81: 7873-7884. 10.1128/JVI.01845-06PubMedPubMedCentralCrossRef Jolly C, Sattentau QJ: Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains. J Virol 2007, 81: 7873-7884. 10.1128/JVI.01845-06PubMedPubMedCentralCrossRef
31.
go back to reference Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C: Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods 2008, 338: 21-30. 10.1016/j.jim.2008.07.007PubMedCrossRef Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C: Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods 2008, 338: 21-30. 10.1016/j.jim.2008.07.007PubMedCrossRef
32.
go back to reference Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ: Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 2006, 172: 923-935. 10.1083/jcb.200508014PubMedPubMedCentralCrossRef Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ: Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 2006, 172: 923-935. 10.1083/jcb.200508014PubMedPubMedCentralCrossRef
33.
go back to reference Fang Y, Wu N, Gan X, Yan W, Morrell JC, Gould SJ: Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes. PLoS Biol 2007, 5: e158. 10.1371/journal.pbio.0050158PubMedPubMedCentralCrossRef Fang Y, Wu N, Gan X, Yan W, Morrell JC, Gould SJ: Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes. PLoS Biol 2007, 5: e158. 10.1371/journal.pbio.0050158PubMedPubMedCentralCrossRef
34.
go back to reference Grigorov B, Arcanger F, Roingeard P, Darlix JL, Muriaux D: Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines. J Mol Biol 2006, 359: 848-862. 10.1016/j.jmb.2006.04.017PubMedCrossRef Grigorov B, Arcanger F, Roingeard P, Darlix JL, Muriaux D: Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines. J Mol Biol 2006, 359: 848-862. 10.1016/j.jmb.2006.04.017PubMedCrossRef
35.
go back to reference Bess JW Jr, Gorelick RJ, Bosche WJ, Henderson LE, Arthur LO: Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 1997, 230: 134-144. 10.1006/viro.1997.8499PubMedCrossRef Bess JW Jr, Gorelick RJ, Bosche WJ, Henderson LE, Arthur LO: Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 1997, 230: 134-144. 10.1006/viro.1997.8499PubMedCrossRef
36.
go back to reference Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ: Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 1997, 230: 125-133. 10.1006/viro.1997.8453PubMedCrossRef Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ: Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 1997, 230: 125-133. 10.1006/viro.1997.8453PubMedCrossRef
37.
go back to reference Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, Sowder RC, Chertova EN, Arthur LO, Henderson LE: Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 1996, 70: 7734-7743.PubMedPubMedCentral Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, Sowder RC, Chertova EN, Arthur LO, Henderson LE: Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 1996, 70: 7734-7743.PubMedPubMedCentral
38.
go back to reference Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, Amigorena S: Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 2001, 166: 7309-7318.PubMedCrossRef Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, Amigorena S: Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 2001, 166: 7309-7318.PubMedCrossRef
39.
go back to reference Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 1999, 147: 599-610. 10.1083/jcb.147.3.599PubMedPubMedCentralCrossRef Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 1999, 147: 599-610. 10.1083/jcb.147.3.599PubMedPubMedCentralCrossRef
40.
go back to reference Lancaster GI, Febbraio MA: Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins. J Biol Chem 2005, 280: 23349-23355. 10.1074/jbc.M502017200PubMedCrossRef Lancaster GI, Febbraio MA: Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins. J Biol Chem 2005, 280: 23349-23355. 10.1074/jbc.M502017200PubMedCrossRef
41.
go back to reference Liu Y, Shah SV, Xiang X, Wang J, Deng ZB, Liu C, Zhang L, Wu J, Edmonds T, Jambor C, et al.: COP9-associated CSN5 regulates exosomal protein deubiquitination and sorting. Am J Pathol 2009, 174: 1415-1425. 10.2353/ajpath.2009.080861PubMedPubMedCentralCrossRef Liu Y, Shah SV, Xiang X, Wang J, Deng ZB, Liu C, Zhang L, Wu J, Edmonds T, Jambor C, et al.: COP9-associated CSN5 regulates exosomal protein deubiquitination and sorting. Am J Pathol 2009, 174: 1415-1425. 10.2353/ajpath.2009.080861PubMedPubMedCentralCrossRef
Metadata
Title
HIV-1 is budded from CD4+ T lymphocytes independently of exosomes
Publication date
01-12-2010
Published in
Virology Journal / Issue 1/2010
Electronic ISSN: 1743-422X
DOI
https://doi.org/10.1186/1743-422X-7-234

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