Conformational changes in the capsid of a calicivirus upon interaction with its functional receptor

The interactions between viruses and receptors on the surface of host cells strongly influence viral pathogenesis and regulate morbidity and mortality in the host. Virus-receptor interactions determine the types of cells that can be infected, the pathway of entry into the cell, and the efficiency of productive infection. Interactions between nonenveloped virus capsids and their receptor(s) trigger one or more steps required for infectious entry. These steps can include interaction with other receptors, exposure to low pH or endosomal proteases, or other factors. Ultimately, one or more of these interactions induce changes in capsid conformation that result in the exposure of hydrophobic regions or release of a lipid-seeking factor that can interact with and disrupt the limiting cellular membrane to allow the capsid and/or the genome to be delivered to the interior of the cell (reviewed in reference 60). The Caliciviridae are small nonenveloped viruses containing a positive-sense RNA genome (∼7 to 8 kb). Several important disease-causing members of the Caliciviridae, including human noroviruses and rabbit hemorrhagic disease virus, cannot be propagated in tissue culture systems (19, 56). This has slowed progress on studies of the mechanisms of cellular entry of these viruses. In contrast, feline caliciviruses (FCVs) propagate readily in tissue culture, and two cell surface receptor molecules, feline junctional adhesion molecule A (fJAM-A) and α2,6 sialic acid, are known (29, 55). The FCV receptor, fJAM-A, is a type I transmembrane glycoprotein (molecular size of 36 to 41 kDa) member of the immunoglobulin superfamily (IgSF); it consists of an amino-terminal signal peptide, an extracellular domain (composed of two Ig-like domains—a membrane-distal D1 and a membrane-proximal D2), a transmembrane domain, and a short cytoplasmic domain that contains a type II PDZ domain-binding motif (11, 30). We have previously shown that the D1 domain of the fJAM-A ectodomain is necessary and sufficient for FCV binding and that preincubation of FCV with soluble fJAM-A (sfJAM-A) results in virus neutralization (35). Additional roles that fJAM-A might play in FCV entry, however, have not been investigated. Caliciviruses are composed of 180 copies of a single capsid protein. Atomic resolution structures of recombinant virus-like particles of Norwalk virus (genus Norovirus) and native San Miguel sea lion virus (SMSV) virions (genus Vesivirus) indicate that the virion consists of 90 dimers of the capsid protein arranged in T=3 icosahedral symmetry (5, 41). Each capsid monomer contains three structural domains—an N-terminal arm (NTA), the shell (S), and a protruding domain (P) that is further subdivided into P1 and P2 subdomains. The distal subdomain, P2, is structurally conserved between Norwalk virus and SMSV, but there is little sequence conservation. In the primary sequence of the FCV capsid, there are two hypervariable regions that contain neutralizing epitopes (18, 34, 58). The corresponding hypervariable regions (HVRs) of the SMSV capsid structure map to surface-exposed loops. Surface residues at the dimeric interface between two capsid monomers are conserved within individual calicivirus genera, and it has been suggested that this interface is involved in receptor binding (5). A cryo-electron microscopy (cryo-EM) reconstruction of the FCV vaccine strain F9 complexed with the ectodomain of fJAM-A (modeled on the crystal structures of SMSV and human JAM-A, respectively) shows that fJAM-A engages the top of the P2 domain and that binding causes a rotation in the P dimer (1). However, the relatively low resolution and the lack of atomic resolution structures of FCV and fJAM-A prevented precise identification of residues on the viral capsid that contact fJAM-A. A classical approach for identifying virus residues that directly bind or modulate the binding of a receptor is to select for mutant viruses resistant to neutralization with soluble receptors (6, 23, 46). Soluble receptor-resistant (srr) mutants of poliovirus revealed that both surface-exposed and internal residues regulate receptor attachment and conformational changes in the capsid (6, 42). Here, we report 24 srr mutants and the location of their capsid mutations on a 3.6-A structure of FCV. In addition, we describe the growth kinetics and receptor-binding properties of a subpanel of eight srr mutants and examine changes in capsid hydrophobicity concurrent with the interaction of FCV capsids with sfJAM-A.