VIROLOGY 189, 350-353 (1992) A 44,000 Glycoprotein Is Involved in the Attachment of Echovirus-ll onto Susceptible Cells ANDRE D. MBIDA, BRUNO POZZETTO, ODETTE G. GAUDIN,’ FLORENCE GRATTARD, JEAN-CLAUDE LE BIHAN, YVES AKONO, AND ALAIN Ros Department of Virology, Faculty of Medicine J. Lisfranc, 15, rue A. Park, 42023 Saint-Etienne Cedex 2, France Received January 2, 1992; accepted March 23, 1992 Cellular receptors play an important role in viral pathogenesis. Until now little was known on echovirus (EV) receptor. Using detergent-treated KB cell extracts as immunogen, a mouse monoclonal antibody (Mab 143) was produced that selectively blocks the attachment of EV-11 to KB and other susceptible cells. By immunoblotting, Mab 143 detected a 44,000 protein on susceptible cell lines but not on cell lines from nonprimate origin. The receptor protein complex, purified from KB cell membranes by immunoaffinity using Mab 143 as ligand, was shown to contain a single glycopro- tein with apparent molecular weight of 44,000 (gp44). The role of gp44 in the attachment of EV-11 onto KB cells was demonstrated by the ability (i) of affinity-purified gp44 to reduce the infectivity of EV-11 and (ii) of rabbit polyclonal antisera raised against gp44 to protect cells from the replication of various EV, as did Mab 143. o 1992 Academic PESS, IX. Cellular receptors for viruses are involved in the first steps of viral infection of susceptible cells. An ap- proach of the structure of these receptors may help for the comprehension of viral diseases’ pathogenesis. Different strategies have been proposed for studying picornavirus cell receptors. The first one requires the virus itself as ligand to the solubilized cell material and was applied to the purification of coxsackievirus and encephalomyocarditis virus receptors (1-3). However, this approach is not easy because the virus affinity for receptor monomers is decreased after solubilization. Alternatively, immunoaffinity with monoclonal antibod- ies (Mab) as ligand has been successfully used for the isolation and characterization of poliovirus and rhino- virus receptor proteins (4-8). Until now, little was known about the cellular recep- tor of echoviruses (EVs), a subgenus of the picorna- virus family responsible for a great variety of clinical symptoms in humans (reviewed in 9, 10). We previ- ously reported (11) the production of a Mab-called Mab 143-that specifically inhibits the attachment of Echovirus type 11 (EV-1 1) onto susceptible cells as demonstrated by the following data: (i) Mab 143 pro- tected different cell lines of primate origin against the cytopathic effect of EV-1 1 and of other EV types; (ii) by cytometry analysis, Mab 143 was shown to bind to susceptible cells but not to cells of nonprimate origin. On the basis of these results, the present report de- ’ To whom reprint requests should be addressed. scribes the isolation and the preliminary characteriza- tion of a putative receptor of EVs on human KB cells using the Mab technology. The technique used for cell solubilization was de- signed from previous studies (2, 12, 13). KB cells were harvested with a mixture of trypsine 0.25% in sodium versenate (EDTA), pooled, and washed three times at 4’ with cold phosphate buffer saline (PBS) pH 7.2 by low-speed centrifugation. The cells were then sus- pended in PBS containing 1% Triton Xl00 (TX), 0.5% sodium desoxycholate (DOC), and protease inhibitors (2 mM phenylmethylsulfonyl-fluoride, 2 mM ethyl-ma- leimide, and 5 mM EDTA) for 1 hr in an ice bath with gentle agitation. The preparation was centrifuged at 100,000 g at 4” for 2 hr. After removal of detergent by dialysis against PBS for 48 hr, the pellet and the super- natant of solubilized KB cell extracts were assayed for receptor activity: 450 ~1 of each sample containing 6.4 and 3.6 mg of protein respectively were mixed with 50 ~1 of Minimum Essential Medium (MEM) containing about 100 TCID,, of EV-1 1 (Gregory strain) and incu- bated for 30 min at 4”. The residual virus activity was titrated in 96-well microplates seeded with KB cells. As a mean of six experiments, the virus titers were 108.64 in the pellet and 107.75in the supernatant (not significant and P < 0.01, respectively, as compared to the titer of untreated EV-1 1 (1 O*.7’) by Mann-Whitney test), dem- onstrating that the main receptor activity was con- tained in the supernatant fraction. This supernatant extract was analyzed by high pres- sure liquid chromatography (HPLC) and six fractions were collected (Fl to F6). As shown in Fig. 1, the 0042.6822/92 $5.00 350 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.