poliovirus type 1 (P1/Mahoney) is unable to grow at 25°C, but cold-adapted (ca) mutants are readily selected at this temperature. Ca mutants replicate efficiently at 25°C without forming 135S particles (they do form 135S particles at 37°C). The Ca phenotype maps to a central region of the viral RNA encoding nonstructural proteins, suggesting that the block to replication in wild-type P1/Mahoney is past the stage of cell entry— possibly RNA replication, proteolytic processing, or even assembly. In support of this hypothesis, when the entry steps are bypassed by transfection of viral RNA into cells, ca viral RNA replicates at 25°C, but wt RNA does not.
These results suggest that the formation of 135S particles is not required for poliovirus replication. The altered particle might be a stable end product that is readily detected; the true intermediate in RNA uncoating might be an earlier particle, perhaps less stable than 135S particles, that represents a less-drastic PVR-induced conformational change. The ability to study a productive poliovirus infection at 25°C, in the absence of 135S particle formation, should enable the identification of such structural changes. We are left with the question of why so many nonfunctional 135S particles are formed. The answer is not known, but 135S particle formation has been studied mainly in cultured cells; in vivo, where the accessibility and/or level of cell receptors might be limited, fewer A particles may be generated.
To fully understand how the poliovirus-PVR interaction initiates cell entry, a detailed picture of how the virus and receptor combine is required. Ultimately, resolution of a virus-receptor complex will be needed, but the results of genetic analyses have provided some insight into the interaction. The binding site for poliovirus appears to be contained within domain 1, which can bind poliovirus when expressed on the cell surface either alone or linked to other domains from CD4, the intracellular cell adhesion molecule ICAM-1, or MPH (for review, see ref. 16). Virus does not bind as well to domain 1 as it does to native PVR, suggesting that domains 2 and 3 contribute to the interaction, either directly or by influencing the structure of domain 1. Several laboratories have mutagenized PVR domain 1 to identify the putative contact point, and the results show that three main sites are important for poliovirus binding (Fig. 1): (i) the C-C′ loop through the C″ strand, (ii) the border of the D strand and the D-E loop, and (iii) the G strand. A mutation at the beginning of the F strand also reduces virus binding, probably by altering domain structure. Mutagenesis of other loops and strands has not revealed other regions that are important for binding.
These studies indicate that the C′-C″ ridge is likely to be the main part of PVR that contacts poliovirus. The homologous part of CD4 plays a major role in the interaction with human immunodeficiency virus type 1 (for review, see ref. 18). The D-E loop of domain 1 may also contact poliovirus, but the G strand is more distant and not likely to be directly involved with the binding site. Consistent with this hypothesis is the observation that substitution of PVR residues 70–100, which contains the C′-C″ ridge (Fig. 1) into the corresponding region of MPH produces a chimeric receptor that can be recognized by type 1 but not types 2 and 3 poliovirus (Y.Lin and V.R.R., unpublished data). This result suggests that the poliovirus binding site on PVR is contained with this 30-amino acid segment, although contribution of conserved MPH residues to poliovirus binding cannot be excluded. The three serotypes of poliovirus contact PVR slightly differently, a conclusion also drawn from studies of a G-strand mutation (Fig. 1) that abrogates binding of types 1 and 2 but not type 3 poliovirus (19).
When the three-dimensional structures of rhinovirus and poliovirus were solved, a 1.2-nm-deep 1.5-nm-wide channel was noted surrounding the prominent peak at the fivefold axis of symmetry of the particle (6, 20). This channel was called the canyon and was proposed to be the receptor binding site for rhinovirus 14 (20). A model of the interaction of HRV-16 with its soluble receptor, ICAM-1, indicates that ICAM-1 does bind in the canyon (21). Evidence that the canyon is the receptor binding site in poliovirus comes from the study of two types of viral mutants: soluble receptor resistant (srr) mutants and viruses adapted to utilize mutant PVRs. Detergent-solubilized PVR expressed in insect cells converts poliovirus to 135S particles, neutralizing its infectivity (13). Poliovirus mutants resistant to neutralization with soluble PVR have been selected that possess a range of binding defects to PVR (22, 23). Each srr mutant contains a single mutation, located on the surface or the interior of the capsid. The surface mutations (Fig. 2) are located in the canyon and may form part of the contact site for PVR. Mutation at any one of eight residues decreases the binding affinity of poliovirus for PVR, indicating that multiple points in the virus-receptor interface contribute to binding. Mutations at internal capsid residues also reduce binding affinity. These residues are not likely to contact the receptor directly but may affect the ability of the virus to bind to PVR with high affinity by altering the flexibility of the capsid. The proximity of several of the internal mutations near a hydrocarbon binding pocket that appears to contain sphingosine (24) is consistent with this hypothesis. This pocket is believed to regulate the ability of the capsid to undergo receptor-mediated structural transitions (24).