| To identify the cellular receptor for Enterovirus 70 (EV70), mice were immunized with HeLa cells or HeLa cell membranes. Hybridomas were generated and screened for the production of antibody that protected HeLa cells against infection by EV70, and one monoclonal antibody (mAb), EVR1, was isolated. EVR1 recognized a trypsin-resistant HeLa cell surface epitope, and interfered with EV70 binding to HeLa cell monolayers. The specificity of EVR1 for EV70 was demonstrated by the inability of this mAb to protect HeLa cells against infection by poliovirus (PV) or coxsackievirus B3 (CVB3). Furthermore, EVR1 did not bind to monkey kidney (LLC-MK;In Western blots and immunoprecipitations, EVR1 recognized a HeLa cell glycoprotein of approximately 75 kDa. The properties of this protein suggested that it may be decay-accelerating factor (DAF/CD55). DAF is a 70-75 kDa glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that is a regulator of complement activation, and that has also been shown to act as a receptor for a number of human enteroviruses. Several experiments were performed to demonstrate that EVR1 recognized DAF. HeLa cells treated with phosphatidylinositol-specific phospholipase C (PI-PLC) had a greatly reduced ability to bind EVR1, indicating that the ligand of EVR1 is attached to cells via a GPI anchor. Furthermore, EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF, and DAF-specific mAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Using a vaccinia virus based system, human DAF was transiently expressed in murine NIH/3T3 cells. Transient expression of human DAF conferred virus binding activity to these cells, and a stably transformed NIH/3T3 cell line expressing human DAF supported low levels of EV70 replication.;To map the EV70 binding site of DAF, chimeric DAF/membrane cofactor protein (MCP) molecules were transiently expressed in NIH/3T3 cells and tested for their ability to bind EV70. Expression of chimeric receptors, in which individual extracellular short consensus repeat (SCR) domains of DAF were exchanged with those of MCP indicated that DAF SCR1 contained sequences essential for EV70 binding. Sequences within DAF SCR2 may also play a minor role in EV70 binding, but SCRs 3 and 4 were not specifically required to retain binding activity of the molecule. Similarly, the juxtamembrane domain and the GPI anchor of DAF were not specifically required for EV70 binding. Chimeric receptor expression was also used to identify DAF SCR1 as the binding site for EVR1.;These results indicate that DAF acts as a HeLa cell receptor for EV70. However, whether or not DAF is necessary or sufficient to mediate EV70 infection of cells is not yet clear. The widespread in vivo distribution of DAF and the restricted in vivo tropism of EV70, as well as the discovery that other human enteroviruses also use DAF as a receptor indicate that DAF expression is not the sole determinant of EV70 tropism. In addition, it remains to be determined if EV70 uses DAF homologs or entirely different surface molecules as receptors on cells of non-human origin. The identification of co-receptors or accessory factors that govern the specificity of EV70 infection awaits further investigation. |
