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Several mouse cell lines expressing hybrid human poliovirus receptors (hPVRs) bearing mutations in the first immunoglobulin-like domain were previously characterized for their defective binding and replication of poliovirus type 1 Mahoney (G. Bernhardt, J. Harber, A. Zibert, M. DeCrombrugghe, and E. Wimmer, Virology, 203, 344-356, 1994). Here we report that these mutant hPVRs were utilized to explore differences in the binding behavior of the three serotypes of poliovirus. Type 3 polioviruses (both Sabin and the neurovirulent Leon strain) clearly bound to the hPVR mutant Q130G/GD, but were incapable of initiating infection. Also, binding at 25 degrees of poliovirus types 2 and 3 to cell lines expressing the hPVR mutants P84SYS/HPGA, L99GAE/AAAA, and D117F was greater than type 1 poliovirus. Further study of the serotype-specific interaction with mutant hPVRs was accomplished with antigenic hybrid viruses. Improved binding by antigenic hybrid viruses demonstrated that serotype-specific binding to mutant hPVRs is, in part, determined by the amino acid sequence of neutralization antigenic sites (NAgs) and the probable conformational rearrangement of amino acids adjacent to the NAg sites. Finally, site-directed mutants of poliovirus were utilized to determine the relative contributions, to hPVR interactions, of individual amino acids with solvent accessible side chains in the viral canyon. Of the 18 viable virus mutants produced, 3 (D1226A, I1089A, and VPEK1166HPGA) expressed impaired replication phenotypes on the mutant hPVR cell lines P84SYS/HYSA and D117F. A location at the rim of the poliovirus canyon was implicated for the interaction of the amino terminal domain of the poliovirus receptor with conserved and serotype-specific viral surface amino acids. The possible involvement of elements of neutralization antigenic sites in receptor binding may explain, in part, why poliovirus exists in only three serotypes.

The N-terminal domain 1 of the human poliovirus receptor (hPVR), a three-domain, immunoglobulin-like molecule, was previously shown to be necessary and sufficient to confer poliovirus (PV) susceptibility to mouse cells. However, studies with truncated versions of hPVR suggested that the C-terminal hPVR domains may contribute to receptor function. We describe sets of hybrid receptors, constructed between hPVR and hICAM-1 (human intercellular adhesion molecule-1) that were tested in mouse cells for hPVR functionally. Whereas the context in which hPVR is expressed is of minor importance, all three domains of hPVR are required to reach wild-type function. Single and multiple amino acid exchanges were introduced into the first hPVR domain in order to localize regions that were involved in virus-receptor interactions. The mutations were analyzed for their ability to bind PV1 (Mahoney) or monoclonal antibodies as well as their ability to support viral replication in either the hPVR alpha or hybrid hPVR-hICAM-1 receptor context. When placed into a model of the V domain of hPVR, the effect of the mutations indicated that the C'C"D as well as the DE region harbored amino acids that contacted the PV1(M) surface in the process of receptor-virus complex formation. The binding of the virus to the receptor and subsequent uptake into the cells were linked; no hPVR mutants were observed that bound the virus but blocked infection. N-glycosylation of the four sites in domains 1 and 2 is not required for hPVR function, but glycosylation in domain 1 has a greater effect on receptor function than that of domain 2.

Poliovirus and foot-and-mouth disease virus (FMDV) initiate infection by binding to specific cell surface receptors, which is followed by a poorly understood disassembly process. To probe these early steps of infection, the ability of poliovirus and FMDV to infect cells following binding through an alternative receptor was examined. For these studies, a Chinese hamster ovary (CHO) cell line expressing the B2 isoform of the murine Fc receptor (FcR) was used. Both viruses were able to bind to this cell line in an antibody-dependent manner, but only FMDV was able to productively infect these cells following binding through the FcR. These results suggest that the natural poliovirus receptor has dual functions in binding and destabilizing the virus particle, whereas the putative FMDV receptor may only be necessary for virion binding. These findings are consistent with differences in virion architecture which predict a more intimate virion-receptor association for poliovirus than for FMDV.

In order to study the properties of foreign antigenic sites expressed on poliovirus a hybrid was constructed in which neutralization antigenic site IA of poliovirus type 1 strain Mahoney [PV1(M)] was replaced by neutralization immunogenic site IA (NImIA) of human rhinovirus 14 (HRV14). The resulting hybrid was viable, but growth was impaired in comparison to PV1(M). The hybrid expressed both PV1(M) and HRV14 antigenic determinants. When inoculated into rabbits it elicited neutralizing antibodies against both PV1(M) and HRV14. Furthermore, the hybrid was efficiently neutralized by polyclonal antisera specific for either PV1(M) or HRV14 and by three out of five monoclonal antibodies directed to NImIA. The monoclonal antibodies also blocked binding of the hybrid to the cellular receptor for poliovirus. One of them is thought to neutralize rhinovirus in this manner, and it appears that NImIA is expressed in a sufficiently favorable context on the hybrid for the same mechanism to be effective. This can be interpreted to mean that the interactions between the parental viruses and their respective cellular receptors are very similar.