Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1––13.2 of human chromosome 19.

Poliovirus-sensitive transgenic mice were produced by introducing the human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic micejust as in tissues of humans. The transgenic mice are susceptible to all three poliovirus serotypes, and the mice inoculated with poliovirus show clinical symptoms similar to those observed in humans and monkeys. Rabbit antipoliovirus serum detects the antigens mainly in motor neurons in the anterior horn of the spinal cord and in nerve cells in the medulla oblongata and pons of the paralyzed transgenic mice. Therefore, cell types sensitive to poliovirus in the central nervous system of the transgenic mice appear to be identical to those of humans and monkeys. Furthermore, many more doses of oral poliovirus vaccine strains than ofthe virulent strains are required to cause paralysis in the transgenic mice. This may reflect the observation that the virulent strain multiplies more efficiently in the central nervous system than the attenuated strain. Thus, the transgenic mice may become an excellent new animal model to study molecular mechanisms of pathogenesis of poliovirus and to assess oral poliovirus vaccines.(5). Of these, the membrane-bound PVRa and PVR8, which correspond to H20A and H20B (4), are functional receptor molecules. Although it has been proved that PVRs are members of the immunoglobulin superfamily (4), physiological functions of these molecules are totally unknown at present.

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A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.Virus receptors are considered to have important roles in the early steps of viral infection such as binding to the cell surface, penetration, and uncoating of the virus. Therefore, elucidation offunctional regions ofthe virus receptors in each infection step is essential to an understanding of the molecular mechanisms of the interaction between cells and viruses in early infection. Indeed DNAs encoding several virus receptors have been isolated (1) and extensive studies have been performed to identify and characterize regions of the receptors required for infection, particularly those of the human immunodeficiency virus (2-8) and the major group human rhinovirus (9, 10).Poliovirus, the causative agent of poliomyelitis, infects primates and invades the target tissues including the central nervous system (11). The characteristic species specificity and tissue tropism of poliovirus are considered to be primarily determined by a unique cell surface receptor (12, 13). Although crystallographic studies on poliovirus revealed the precise three-dimensional structure of the virion and proposed a putative attachment site for the poliovirus receptor (PVR) that is a depression (called a "canyon") near the fivefold axis of the virion particle (14, 15), little is known about structure of the counterpart, the PVRs. To understand the specific interaction between poliovirus and PVRs that leads to the establishment of the infection, several monoclonal antibodies (mAbs) that block poliovirus infection have been isolated (16)(17)(18)(19). Furthermore, the genomic and complementary DNAs for human PVRs were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells (19,20). Mouse L cell transformants carrying these DNAs acquired susceptibility to poliovirus and were recognized by mAbs against PVRs. Transgenic mice carrying the human PVR gene are permissive for all three poliovirus serotypes (21,22). Thus, it is clear that the human PVR gene confers permissiveness for poliovirus to mice in vivo as well as mouse ce...

Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTgl, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dosedependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence.