Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.
References
[1]
La Scola B, Audic S, Robert C, Jungang L, de Lamballerie X, et al. (2003) A giant virus in amoebae. Science 299: 2033.
[2]
Raoult D, La Scola B, Birtles R (2007) The discovery and characterization of Mimivirus, the largest known virus and putative pneumonia agent. Clin Infect Dis 45: 95–102.
[3]
Khan M, La Scola B, Lepidi H, Raoult D (2007) Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus. Microb Pathog 42: 56–61.
[4]
Xiao C, Chipman PR, Battisti AJ, Bowman VD, Renesto P, et al. (2005) Cryo-electron microscopy of the giant Mimivirus. J Mol Biol 353: 493–496.
[5]
Suzan-Monti M, Scola BL, Barrassi L, Espinosa L, Raoult D (2007) Ultrastructural Characterization of the Giant Volcano-like Virus Factory of Acanthamoeba polyphaga Mimivirus. PLoS ONE 2: e328. doi:10.1371/journal.pone.0000328.
[6]
Conner SD, Schmid SL (2003) Regulated portals of entry into the cell. Nature 422: 37–44.
[7]
Pelkmans L, Helenius A (2003) Insider information: what viruses tell us about endocytosis. Curr Opin Cell Biol 15: 414–422.
[8]
Pelkmans L (2005) Viruses as probes for systems analysis of cellular signalling, cytoskeleton reorganization and endocytosis. Curr Opin Microbiol 8: 331–337.
[9]
Le Blanc I, Luyet PP, Pons V, Ferguson C, Emans N, et al. (2005) Endosome-to-cytosol transport of viral nucleocapsids. Nat Cell Biol 7: 653–664.
[10]
Marsh M, Helenius A (2006) Virus entry: open sesame. Cell 124: 729–740.
[11]
Li J, Scherl A, Medina F, Frank PG, Kitsis RN, et al. (2005) Impaired phagocytosis in caveolin-1 deficient macrophages. Cell Cycle 4: 1599–1607.
[12]
Veiga E, Cossart P (2006) The role of clathrin-dependent endocytosis in bacterial internalization. Trends Cell Biol 16: 499–504.
Kee SH, Cho EJ, Song JW, Park KS, Baek LJ, et al. (2004) Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells. Microbiol Immunol 48: 823–829.
[15]
West MA, Bretscher MS, Watts C (1989) Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol 109: 2731–2739.
[16]
Schnatwinkel C, Christoforidis S, Lindsay MR, Uttenweiler-Joseph S, Wilm M, et al. (2004) The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol 2: e261. doi:10.1371/journal.pbio.0020261.
[17]
Locker JK, Kuehn A, Schleich S, Rutter G, Hohenberg H, et al. (2000) Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV. Mol Biol Cell 11: 2497–2511.
[18]
Marechal V, Prevost MC, Petit C, Perret E, Heard JM, et al. (2001) Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis. J Virol 75: 11166–11177.
[19]
Aderem A, Underhill DM (1999) Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17: 593–623.
[20]
Gold ES, Underhill DM, Morrissette NS, Guo J, McNiven MA, et al. (1999) Dynamin 2 is required for phagocytosis in macrophages. J Exp Med 190: 1849–1856.
[21]
Clement C, Tiwari V, Scanlan PM, Valyi-Nagy T, Yue BY, et al. (2006) A novel role for phagocytosis-like uptake in herpes simplex virus entry. J Cell Biol 174: 1009–1021.
[22]
Sun X, Yau VK, Briggs BJ, Whittaker GR (2005) Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells. Virology 338: 53–60.
[23]
Benmerah A, Bayrou M, Cerf-Bensussan N, Dautry-Varsat A (1999) Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J Cell Sci 112 (Pt 9): 1303–1311.
[24]
Boleti H, Benmerah A, Ojcius DM, Cerf-Bensussan N, Dautry-Varsat A (1999) Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth. J Cell Sci 112 (Pt 10): 1487–1496.
Cao H, Chen J, Awoniyi M, Henley JR, McNiven MA (2007) Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells. J Cell Sci 120: 4167–4177.
[27]
Velasco-Velazquez MA, Barrera D, Gonzalez-Arenas A, Rosales C, Agramonte-Hevia J (2003) Macrophage-Mycobacterium tuberculosis interactions: role of complement receptor 3. Microb Pathog 35: 125–131.
[28]
Smith AE, Helenius A (2004) How viruses enter animal cells. Science 304: 237–242.
[29]
Greub G, Raoult D (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17: 413–433.
[30]
Kojic LD, Joshi B, Lajoie P, Le PU, Cox ME, et al. (2007) Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells. J Biol Chem 282: 29305–29313.
[31]
Steenbergen JN, Shuman HA, Casadevall A (2001) Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc Natl Acad Sci USA 98: 15245–15250.
[32]
La Scola B, Mezi L, Weiller PJ, Raoult D (2001) Isolation of Legionella anisa using an amoebic coculture procedure. J Clin Microbiol 39: 365–366.
[33]
Ghigo E, Honstettre A, Capo C, Gorvel JP, Raoult D, et al. (2004) Link between impaired maturation of phagosomes and defective Coxiella burnetii killing in patients with chronic Q fever. J Infect Dis 190: 1767–1772.
[34]
Cook P, Totemeyer S, Stevenson C, Fitzgerald KA, Yamamoto M, et al. (2007) Salmonella-induced SipB-independent cell death requires Toll-like receptor-4 signalling via the adapter proteins Tram and Trif. Immunology 122: 222–229.
[35]
Pelkmans L, Kartenbeck J, Helenius A (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3: 473–483.
[36]
Chu JJ, Ng ML (2004) Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J Virol 78: 10543–10555.
[37]
Kartenbeck J, Stukenbrok H, Helenius A (1989) Endocytosis of simian virus 40 into the endoplasmic reticulum. J Cell Biol 109: 2721–2729.
[38]
Bantel-Schaal U, Hub B, Kartenbeck J (2002) Endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the Golgi compartment. J Virol 76: 2340–2349.
[39]
Wong WR, Chen YY, Yang SM, Chen YL, Horng JT (2005) Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71. Life Sci 78: 82–90.
[40]
Barker LP, George KM, Falkow S, Small PL (1997) Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect Immun 65: 1497–1504.
[41]
Ullrich HJ, Beatty WL, Russell DG (2000) Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway. J Immunol 165: 6073–6080.