Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6245-6248, June 1994 Medical Sciences Decay-accelerating factor (CD55), a glycosylphosphatidylinositol- anchored complement regulatory protein, is a receptor for several echoviruses (virus receptor/picornavirus/echovirus 7) JEFFREY M. BERGELSON*, MELVIN CHAN, KEITH R. SOLOMON, NICOLE F. ST. JOHN, HUAMAO LIN, AND ROBERT W. FINBERG Laboratory of Infectious Diseases, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 Communicated by Baruj Benacerraf, March 29, 1994 ABSTRACT Echoviruses are human pathogens belonging autologous complement (4). Deficient expression ofDAF and to the picornavirus family. Decay-accelerating factor (DAF) is other GPI-anchored complement regulatory proteins results a glycosylphosphatidylhinostol (GPI)-anchored surface protein in the hematologic disorder paroxysmal nocturnal hemoglo- that protects cells from lysis by autologous complement. Anti- binuria. In this report, we show that DAF also mediates cell DAF monoclonal antibodies prevented echovirus 7 attachment surface attachment and infection by several echovirus sero- to susceptible cells and protected cells from infection. HeLa types. cells specifically lost the capacity to bind echovirus 7 when treated with phosphatidylinositol-specific phospholipase C, an MATERIALS AND METHODS enzyme that releases GPI-anchored proteins from the cell surface, indicating that the virus receptor, like DAF, is a Viruses and Virus Assays. Echoviruses 1 (Farouk), 6 GPI-anchored protein. Although Chinese hamster ovary cells (D'Amori), 7 (Wallace), 8 (Bryson), 11 (Gregory), 12 (Travis do not bind echovirus 7, transfectants expressing human DAF 2-85), 20 (JV-1), and 21 (Farina) were obtained from the bound virus efficiently, and binding was prevented by pre- American Type Culture Collection and grown in HeLa cells. treatment with an anti-DAF monoclonal antibody. Anti-DAF Antibody inhibition ofviral cytopathic effect and inhibition of antibodies prevented infection by at least six echovirus sero- plaque formation were measured as described (2). ypes. These results indicate that DAF is the receptor mediating Monoclonal Antibodies (mAbs). To obtain mAb IF7, attachment and infection by several echoviruses. BALB/c mice were immunized with live HeLa cells, their splenocytes were fused to P3x63.Ag8U.1 myeloma cells, and Despite their importance in the pathogenesis of viral illness, hybridomas were selected in hypoxanthine/aminopterin/ specific receptors have been identified for only a few of the thymidine medium. Culture supernatants were assessed for viruses that cause human disease. Among the echoviruses, their ability to protect HeLa cell monolayers from infection nonenveloped RNA viruses responsible for febrile illnesses by echovirus 7 using a colorimetric assay as described for including aseptic meningitis, echoviruses 1 and 8 initiate echovirus 1 (1). Anti-DAF mAbs 1H4, 8D11, and 11D7 (5) infection by attaching to the integrin VLA-2 (1, 2). However, were obtained from Wendell Rosse (Duke University), and these two closely related serotypes account for only a small IA10 (6) was from Victor Nussenzweig (New York Univer- percentage of echovirus infections, and anti-VLA-2 antibod- sity). The anti-DAF antibody MEM 118 and the anti-CD-59 ies have no effect on infection by many of the 30 echovirus mAb MEM 125 were from the 5th International Conference serotypes. Similarly, although echoviruses 1 and 8 bind to on Human Leukocyte Differentiation Antigens (November and infect Chinese hamster ovary (CHO) cells transfected 3-7, 1993, Boston). The anti-5'-nucleotidase (CD73) mAb with human VLA-2, otherechoviruses do not, suggesting that 1E9 was provided by Linda Thompson (Oklahoma Medical other echoviruses may bind other receptors (1, 2). Foundation). mAb D2 recognizes CDw109 (7), and DE9 Most cell surface proteins are anchored in the cell mem- recognizes the integrin f31 subunit (CD29) (1). brane by a hydrophobic transmembrane peptide domain. Immunoprecipitation and Immunodepletion. lodination of Some, however, lack a classic transmembrane domain and HeLa cells by the lactoperoxidase/glucose oxidase method, are instead anchored to the membrane by a fatty acid tail, extraction in buffer containing 1% Nonidet P-40, and precip- attached to the protein C terminus by a glycosylphosphati- itation with mAbs and rabbit-anti-mouse immunoglobulin- dylinositol (GPI) linkage (reviewed in ref. 3). Studies ofthese coated protein A-Sepharose beads were performed essen- proteins have been facilitated by identification ofthe enzyme tially as described (1). In some experiments, cell extracts phosphatidylinositol-specific phospholipase C (PI-PLC), were immunodepleted three times with mAb IA10 or a which, by cleaving the anchor, releases GPI-linked proteins control mAb before final immunoprecipitation. Because we from the cell surface. Since the first report of the GPI-linked found that mAb IF7 failed to bind cells in the absence of trypanosomal variant surface glycoprotein, an increasing divalent cations, all procedures were performed in buffers number of GPI-anchored mammalian proteins have been containing 2 mM CaCl2 and 2 mM MgCl2, and EDTA and shown to function in cell adhesion, enzymatic processes, EGTA were omitted. small molecule transport, and transmembrane signaling. No Radiolabeled Virus Binding Assays. Viruses were radiola- identified virus receptor has been shown to possess a GPI beled by growth in medium containing [35S]methionine and anchor. purified by velocity sedimentation in sucrose gradients as Decay-accelerating factor (DAF; CD55) is a GPI-anchored described (2). HeLa cell monolayers in 24-well plates or glycoprotein that functions in protecting cells from lysis by Abbreviations: DAF, decay-accelerating factor; GPI, glycosylphos- The publication costs of this article were defrayed in part by page charge phatidylinositol; PI-PLC, phosphatidylinositol-specific phospholi- payment. This article must therefore be hereby marked "advertisement" pase C; mAb, monoclonal antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6245 Downloaded by guest on September 26, 2021 6246 Medical Sciences: Bergelson et al. Proc. Natl. Acad. Sci. USA 91 (1994) monolayers of CHO cells transfected with human DAF (8) A B C (provided by Douglas M. Lublin, Washington University, St. Louis) or CHO cells transfected with the a2 subunit of VLA-2 (2) were preincubated with mAbs and then washed 100- 4W- and exposed to radiolabeled echoviruses (10,000-30,000 cpm) for 1 hr at room temperature. Monolayers were then 70- - washed and dissolved for determination of cell-bound radio- activity as described (9). Purified antibodies and hybridoma 49- - supernatants were used at 10-20 gg/ml and ascites fluids were diluted 1:100. 1 2 9 10 11 12 For measurements of virus binding after PI-PLC treat- 3 4 5 6 7 8 13 ment, HeLa cells were dispersed with EDTA, suspended in FIG. 1. Immunoprecipitation and immunodepletion with IF7 and PI-PLC buffer (RPMI 1640 medium/0.2% bovine serum anti-DAF mAb IA10. Surface-iodinated HeLa cells were extracted albumin/50 tkM 2-mercaptoethanol/l0 mM Hepes/0.1% with 1% Nonidet P-40 and precipitation was performed as described. azide), and divided in two portions, one of which received (A) Immunoprecipitation. Lanes: 1, control mAb MOPC 195; 2, IF7; PI-PLC (0.5 unit per 106 cells; Oxford Glycosystems, 3, IA10. (B) Immunoprecipitation after immunodepletion with mAb Rosedale, NY). Both treated and mock-treated cells were D2 (anti-CDwlO9), which recognizes 180- and 150-kDa GPI- anchored proteins on HeLa cells. Lanes: 4, MOPC 195; 5, IA10; 6, incubated for 1 hr at 370C and then washed and dispensed into IF7; 7, D2; 8, DE9, which recognizes the 130-kDa integrin f31 Eppendorf tubes before addition of radiolabeled virus. subunit. (C) Immunoprecipitation after immunodepletion with anti- DAF mAb IA10. Lanes: 9, MOPC 195; 10, IF7; 11, IA10; 12, D2; 13, RESULTS DE9. Numbers on left are kDa. A mAb That Prevents Cell Attachment by Echovirus 7. To HeLa cells (Fig. 1, lane 2). When HeLa cells were incubated identify potential receptors for echovirus 7, we took an with PI-PLC, IF7 expression detected by indirect immuno- approach that was successful for identification of VLA-2 as fluorescence decreased dramatically, indicating that IF7 rec- the receptor for echovirus 1 (1). Mice were immunized with ognized a GPI-anchored protein (data not shown). We there- HeLa cells and their splenocytes were fused to myeloma cells fore suspected that IF7 might recognize DAF, a 70-kDa to generate hybridomas. Hybridoma supernatants were then GPI-anchored protein highly expressed on HeLa cells. IF7 screened for the capacity to protect susceptible cells from and the well-characterized anti-DAF antibody IAl0 (6) rec- infection by echovirus 7. Three fusions were screened, and ognized proteins of identical mobility (Fig. lA). Immunode- one protective antibody, mAb IF7, was obtained. pletion with a control antibody recognizing CDw1O9, another mAb IF7 Recognizes DAF. IF7 immunoprecipitated a 70- GPI-anchored protein expressed on HeLa cells, did not affect kDa protein from detergent extracts of surface-iodinated immunoprecipitation of the 70-kDa proteins by IF7 or IAl0 A B Preincubated with: 8- * ControlAb -o E2 IF7 4000 c 2000- .8 - a 1000 - 2000 -o-i CZ z o _0 __Echo Eco- a: 0- ---I 0 - Echo 7 Echo 1 Echo 6 Echo 1 C anti- VLA-2 anti- 5'-NT anti- CD59 IAl 0 MEM 118 -T 8D11 11D7 anti-DAF MAbs 1 H4 IF7 No MAb 0 4000 8000 Radiolabeled virus bound (cprm) FIG. 2. Inhibition of radiolabeled virus binding by mAb IF7 and other anti-DAF antibodies. (A) IF7 inhibits attachment of echovirus 7. (B) IF7 inhibits attachment of echovirus 6. (C) Other anti-DAF antibodies inhibit attachment of echovirus 7. HeLa cell monolayers in 24-well tissue culture plates were incubated with mAbs for 1 hr at room temperature and then antibody was removed and radiolabeled viruses were added (10,000-20,000 cpm). After 1 hr, monolayers were washed and dissolved for determination of cell-bound virus by scintillation counting. Detailed conditions for assays have been described (9).