The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.
References
[1]
Geisbert TW, Jahrling PB (2004) Exotic emerging viral diseases: progress and challenges. Nat Med 10: S110–121.
Richmond JK, Baglole DJ (2003) Lassa fever: epidemiology, clinical features, and social consequences. BMJ 327: 1271–1275.
[4]
McCormick JB, King IJ, Webb PA, Johnson KM, O'Sullivan R, et al. (1987) A case-control study of the clinical diagnosis and course of Lassa fever. J Infect Dis 155: 445–455.
[5]
Fisher-Hoch SP, Tomori O, Nasidi A, Perez-Oronoz GI, Fakile Y, et al. (1995) Review of cases of nosocomial Lassa fever in Nigeria: the high price of poor medical practice. BMJ 311: 857–859.
Jahrling PB, Peters CJ (1992) Lymphocytic choriomeningitis virus. A neglected pathogen of man. Arch Pathol Lab Med 116: 486–488.
[8]
Mets MB, Barton LL, Khan AS, Ksiazek TG (2000) Lymphocytic choriomeningitis virus: an underdiagnosed cause of congenital chorioretinitis. Am J Ophthalmol 130: 209–215.
[9]
Fischer SA, Graham MB, Kuehnert MJ, Kotton CN, Srinivasan A, et al. (2006) Transmission of lymphocytic choriomeningitis virus by organ transplantation. N Engl J Med 354: 2235–2249.
[10]
Peters CJ (2006) Lymphocytic choriomeningitis virus–an old enemy up to new tricks. N Engl J Med 354: 2208–2211.
[11]
Buchmeier MJ, de la Torre JC, Peters CJ (2007) Arenaviridae: the viruses and their replication. In: Knipe DL, Howley PM, editors. Fields Virology. 4th ed. Philadelphia: Lippincott-Raven. pp. 1791–1828.
[12]
Marsh M, Helenius A (2006) Virus entry: open sesame. Cell 124: 729–740.
[13]
Smith AE, Helenius A (2004) How viruses enter animal cells. Science 304: 237–242.
[14]
Cao W, Henry MD, Borrow P, Yamada H, Elder JH, et al. (1998) Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus [see comments]. Science 282: 2079–2081.
[15]
Barresi R, Campbell KP (2006) Dystroglycan: from biosynthesis to pathogenesis of human disease. J Cell Sci 119: 199–207.
[16]
Spiropoulou CF, Kunz S, Rollin PE, Campbell KP, Oldstone MB (2002) New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor. J Virol 76: 5140–5146.
[17]
Borrow P, Oldstone MB (1994) Mechanism of lymphocytic choriomeningitis virus entry into cells. Virology 198: 1–9.
[18]
Klewitz C, Klenk HD, ter Meulen J (2007) Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity. J Gen Virol 88: 2320–2328.
[19]
Quirin K, Eschli B, Scheu I, Poort L, Kartenbeck J, et al. (2008) Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes. Virology 378: 21–33.
[20]
Rojek JM, Perez M, Kunz S (2008) Cellular entry of lymphocytic choriomeningitis virus. J Virol 82: 1505–1517.
[21]
Rojek JM, Sanchez AB, Nguyen NT, de la Torre JC, Kunz S (2008) Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses. J Virol 82: 7677–7687.
[22]
Vonderheit A, Helenius A (2005) Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol 3: e233.
[23]
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.
[24]
Takada A, Robison C, Goto H, Sanchez A, Murti KG, et al. (1997) A system for functional analysis of Ebola virus glycoprotein. Proc Natl Acad Sci U S A 94: 14764–14769.
[25]
Kunz S, Rojek J, Perez M, Spiropoulou C, MB O (2005) Characterization of the interaction of Lassa fever virus with its cellular receptor α-dystroglycan. J Virol 79: 5979–5987.
[26]
Lyles DS, Rupprecht CE (2007) Rhabdoviridae. In: Knipe DM, Howley PM, editors. Fields Virology. 5th ed. Philadelphia: Lippincott. 1363 p.
[27]
Pinschewer DD, Perez M, Jeetendra E, Bachi T, Horvath E, et al. (2004) Kinetics of protective antibodies are determined by the viral surface antigen. J Clin Invest 114: 988–993.
[28]
Clemente R, de la Torre JC (2009) Cell entry of Borna disease virus follows a clathrin-mediated endocytosis pathway that requires Rab5 and microtubules. J Virol 83: 10406–10416.
[29]
Johannsdottir HK, Mancini R, Kartenbeck J, Amato L, Helenius A (2009) Host cell factors and functions involved in vesicular stomatitis virus entry. J Virol 83: 440–453.
[30]
Ohkuma S, Poole B (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A 75: 3327–3331.
[31]
Ohkuma S, Poole B (1981) Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances. J Cell Biol 90: 656–664.
[32]
Shah WA, Peng H, Carbonetto S (2006) Role of non-raft cholesterol in lymphocytic choriomeningitis virus infection via alpha-dystroglycan. J Gen Virol 87: 673–678.
Benmerah A, Lamaze C, Begue B, Schmid SL, Dautry-Varsat A, et al. (1998) AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J Cell Biol 140: 1055–1062.
[35]
Nichols BJ (2002) A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex. Nat Cell Biol 4: 374–378.
[36]
Damke H, Baba T, Warnock DE, Schmid SL (1994) Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127: 915–934.
[37]
Oh P, McIntosh DP, Schnitzer JE (1998) Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. J Cell Biol 141: 101–114.
[38]
van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ, Meyerowitz EM, et al. (1993) Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol 122: 553–563.
[39]
Gruenberg J (2001) The endocytic pathway: a mosaic of domains. Nat Rev Mol Cell Biol 2: 721–730.
[40]
Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2: 107–117.
[41]
Raiborg C, Stenmark H (2009) The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458: 445–452.
[42]
Matsuo H, Chevallier J, Mayran N, Le Blanc I, Ferguson C, et al. (2004) Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 303: 531–534.
[43]
Henry MD, Campbell KP (1998) A role for dystroglycan in basement membrane assembly. Cell 95: 859–870.
[44]
Hall K, Blair Zajdel ME, Blair GE (2010) Unity and diversity in the human adenoviruses: exploiting alternative entry pathways for gene therapy. Biochem J 431: 321–336.
[45]
Meier O, Greber UF (2004) Adenovirus endocytosis. J Gene Med 6: Suppl 1S152–163.
[46]
Bache KG, Brech A, Mehlum A, Stenmark H (2003) Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. J Cell Biol 162: 435–442.
[47]
Babst M, Odorizzi G, Estepa EJ, Emr SD (2000) Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 1: 248–258.
[48]
Razi M, Futter CE (2006) Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation. Mol Biol Cell 17: 3469–3483.
[49]
Bache KG, Stuffers S, Malerod L, Slagsvold T, Raiborg C, et al. (2006) The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor. Mol Biol Cell 17: 2513–2523.
[50]
Malerod L, Stuffers S, Brech A, Stenmark H (2007) Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation. Traffic 8: 1617–1629.
[51]
Perez M, Craven RC, de la Torre JC (2003) The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci U S A 100: 12978–12983.
[52]
Kanagawa M, Saito F, Kunz S, Yoshida-Moriguchi T, Barresi R, et al. (2004) Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 117: 953–964.
[53]
Scheuring S, Rohricht RA, Schoning-Burkhardt B, Beyer A, Muller S, et al. (2001) Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking. J Mol Biol 312: 469–480.
[54]
Bishop N, Woodman P (2000) ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. Mol Biol Cell 11: 227–239.
[55]
Fujita H, Yamanaka M, Imamura K, Tanaka Y, Nara A, et al. (2003) A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells. J Cell Sci 116: 401–414.
Kunz S, Sevilla N, Rojek JM, Oldstone MB (2004) Use of alternative receptors different than alpha-dystroglycan by selected isolates of lymphocytic choriomeningitis virus. Virology 325: 432–445.
[58]
Pires R, Hartlieb B, Signor L, Schoehn G, Lata S, et al. (2009) A crescent-shaped ALIX dimer targets ESCRT-III CHMP4 filaments. Structure 17: 843–856.
[59]
McCullough J, Fisher RD, Whitby FG, Sundquist WI, Hill CP (2008) ALIX-CHMP4 interactions in the human ESCRT pathway. Proc Natl Acad Sci U S A 105: 7687–7691.
[60]
Abrami L, Lindsay M, Parton RG, Leppla SH, van der Goot FG (2004) Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway. J Cell Biol 166: 645–651.
[61]
Schmidt MH, Hoeller D, Yu J, Furnari FB, Cavenee WK, et al. (2004) Alix/AIP1 antagonizes epidermal growth factor receptor downregulation by the Cbl-SETA/CIN85 complex. Mol Cell Biol 24: 8981–8993.
[62]
Chatellard-Causse C, Blot B, Cristina N, Torch S, Missotten M, et al. (2002) Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J Biol Chem 277: 29108–29115.
[63]
Strack B, Calistri A, Craig S, Popova E, Gottlinger HG (2003) AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114: 689–699.
[64]
Chen C, Vincent O, Jin J, Weisz OA, Montelaro RC (2005) Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding. J Biol Chem 280: 40474–40480.
[65]
Zhai Q, Fisher RD, Chung HY, Myszka DG, Sundquist WI, et al. (2008) Structural and functional studies of ALIX interactions with YPX(n)L late domains of HIV-1 and EIAV. Nat Struct Mol Biol 15: 43–49.
[66]
Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, et al. (2000) Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J Exp Med 192: 1249–1260.
[67]
Smelt SC, Borrow P, Kunz S, Cao W, Tishon A, et al. (2001) Differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics. J Virol 75: 448–457.
[68]
Rojek JM, Pasqual G, Sanchez AB, Nguyen NT, de la Torre JC, et al. (2010) Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses. J Virol 84: 573–584.
[69]
Cosset FL, Marianneau P, Verney G, Gallais F, Tordo N, et al. (2009) Characterization of Lassa virus cell entry and neutralization with Lassa virus pseudoparticles. J Virol 83: 3228–3237.
[70]
Puig-Kroger A, Serrano-Gomez D, Caparros E, Dominguez-Soto A, Relloso M, et al. (2004) Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages. J Biol Chem 279: 25680–25688.
[71]
Kunz S, Rojek JM, Perez M, Spiropoulou CF, Oldstone MB (2005) Characterization of the interaction of lassa fever virus with its cellular receptor alpha-dystroglycan. J Virol 79: 5979–5987.
[72]
Reignier T, Oldenburg J, Noble B, Lamb E, Romanowski V, et al. (2006) Receptor use by pathogenic arenaviruses. Virology 353: 111–120. Epub 2006 Jun 21.
[73]
Kunz S, Sevilla N, McGavern DB, Campbell KP, Oldstone MB (2001) Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan. J Cell Biol 155: 301–310.
[74]
Teng MN, Borrow P, Oldstone MB, de la Torre JC (1996) A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome. J Virol 70: 8438–8443.
[75]
Trombetta ES, Mellman I (2005) Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol 23: 975–1028.
[76]
Burgdorf S, Kurts C (2008) Endocytosis mechanisms and the cell biology of antigen presentation. Curr Opin Immunol 20: 89–95.
[77]
Abrami L, Reig N, van der Goot FG (2005) Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol 13: 72–78.
[78]
Khor R, McElroy LJ, Whittaker GR (2003) The ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells. Traffic 4: 857–868.
Mahanty S, Hutchinson K, Agarwal S, McRae M, Rollin PE, et al. (2003) Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses. J Immunol 170: 2797–2801.
[81]
Baize S, Kaplon J, Faure C, Pannetier D, Georges-Courbot MC, et al. (2004) Lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells. J Immunol 172: 2861–2869.
[82]
Beutler B, Jiang Z, Georgel P, Crozat K, Croker B, et al. (2006) Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 24: 353–389.
[83]
Buchmeier MJ, Lewicki HA, Tomori O, Oldstone MB (1981) Monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization, and cross-reactivity with other arenaviruses. Virology 113: 73–85.
[84]
Kobayashi T, Stang E, Fang KS, de Moerloose P, Parton RG, et al. (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392: 193–197.
[85]
Lefrancois L, Lyles DS (1982) The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. II. Monoclonal antibodies of nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes. Virology 121: 168–174.
[86]
Ahmed R, Salmi A, Butler LD, Chiller JM, Oldstone MB (1984) Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J Exp Med 160: 521–540.
[87]
Dutko FJ, Oldstone MB (1983) Genomic and biological variation among commonly used lymphocytic choriomeningitis virus strains. J Gen Virol 64(Pt 8): 1689–1698.
[88]
Cao W, Henry MD, Borrow P, Yamada H, Elder JH, et al. (1998) Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science 282: 2079–2081.
[89]
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.
[90]
Sieczkarski SB, Whittaker GR (2002) Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol 76: 10455–10464.
[91]
Sieczkarski SB, Whittaker GR (2003) Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic 4: 333–343.
[92]
Rojek JM, Lee AM, Nguyen N, Spiropoulou CF, Kunz S (2008) Site 1 protease is required for proteolytic processing of the glycoproteins of the South American hemorrhagic fever viruses Junin, Machupo, and Guanarito. J Virol 82: 6045–6051.
[93]
Bishop N, Horman A, Woodman P (2002) Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates. J Cell Biol 157: 91–101.
[94]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(？Delta Delta C(T)) Method. Methods 25: 402–408.
[95]
Kim S, Kim H, Chang B, Ahn N, Hwang S, et al. (2006) Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability. FASEB J 20: 2399–2401.
[96]
Li L, Liao J, Ruland J, Mak TW, Cohen SN (2001) A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control. Proc Natl Acad Sci U S A 98: 1619–1624.
