Background: Rhinoviruses belong to the picornavirus family and are small, icosahedral, non-enveloped viruses containing one positive RNA strand. Human rhinovirus 16 (HRV16) belongs to the major receptor group of rhinoviruses, for which the cellular receptor is intercellular adhesion molecule-1 (ICAM-1). In many rhinoviruses, one of the viral coat proteins (VP1) contains a hydrophobic pocket which is occupied by a fatty acid-like molecule, or so-called 'pocket factor'. Antiviral agents have been shown to bind to the hydrophobic pocket in VPI, replacing the pocket factor. The presence of the antiviral compound blocks uncoating of the virus and in some cases inhibits receptor attachment, A refined, high-resolution structure would be expected to provide further information on the nature of the pocket factor and other features previously not clearly identified.Results: The structure of native HRV16 has been refined to a resolution of 2.15 Angstrom. The hydrophobic pocket in VP1 is observed in two alternative conformations. In one of these, the pocket is filled by a pocket factor and the protein structure is similar to virus-antiviral compound complexes. In the other conformation, the hydrophobic pocket is collapsed and empty, RNA bases slack against both a tryptophan and a phenylalanine residue on the internal surface of the viral capsid. Site-directed mutagenesis of the tryptophan, which is conserved across the picornaviruses, to nonconservative residues results in non-viable virus. Five symmetry-related N termini of coat protein VP4 form a ten-stranded, antiparallel beta barrel around the base of the icosahedral fivefold axis. The N termini of VPI are amphipathic alpha helices, which stack on the outside of this beta barrel. The N termini of VP1 and VP4 have not been observed previously in rhinovirus structures.Conclusions: The observation of a partially occupied hydrophobic pocket in HRV16 forms a missing link between HRV14, which is always observed with no pocket factor in the native form, and rhinovirus 1A and other picornaviruses (e.g. poliovirus, coxsackievirus) which contain pocket factors. The pocket factor molecules probably regulate viral entry, uncoating and assembly. Picornavirus assembly is known to proceed via pentamers, therefore, the interaction of RNA with the conserved tryptophan residues across twofold axes between pentamers may play a role in picornavirus assembly. The positioning of a cation on the icosahedral fivefold axes and the structure of the N termini of VP4 and VP1 around these axes suggest a mechanism for the uncoating of rhinoviruses.

The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5 A resolution. The icosahedral CVB3 particles crystallize in the monoclinic space group, P2(1), (a = 574.6, b = 302.1, c = 521.6 A, beta = 107.7 degrees ) with two virions in the asymmetric unit giving 120-fold non-crystallographic redundancy. The crystals diffracted to 2.7 A resolution and the X-ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self-rotation function established pseudo R32 symmetry with each particle sitting on a 32 special position. This constrained the orientation and position of each particle in the monoclinic cell to near face-centered positions and allowed for a total of six possible monoclinic space-group settings. Correct interpretation of the high-resolution (3.0-3.2 A) self-rotation function was instrumental in determining the deviations from R32 orientations of the virus particles in the unit cell. Accurate particle orientations permitted the correct assignment of the crystal space-group setting amongst the six ambiguous possibilities and for the correct determination of particle positions. Real-space electron-density averaging and phase refinement, using human rhinovius 14 (HRV14) as an initial phasing model, have been carried out to 3.5 A resolution. The initial structural model has been built and refined to 3.5 A resolution using X-PLOR.

Structures have been determined of three human rhinovirus 14 (HRV14) compensation mutants that have resistance to the antiviral capsid binding compounds WIN 52035 and WIN 52084. In addition, the structure of HRV14 is reported, with a site-directed mutation at residue 1219 in VP1. A spontaneous mutation occurs at the same site in one of the compensation mutants. Some of the mutations are on the viral surface in the canyon and some lie within the hydrophobic binding pocket in VP1 below the ICAM footprint. Those mutant virus strains with mutations on the surface bind better to cells than does wild-type virus. The antiviral compounds bind to the mutant viruses in a manner similar to their binding to wild-type virus. The receptor and WIN compound binding sites overlap, causing competition between receptor attachment and antiviral compound binding. The compensation mutants probably function by shifting the equilibrium in favor of receptor binding. The mutations in the canyon increase the affinity of the virus for the receptor, while the mutations in the pocket probably decrease the affinity of the WIN compounds for the virus by reducing favorable hydrophobic contacts and constricting the pore through which the antiviral compounds are thought to enter the pocket. This is in contrast to the resistant exclusion mutants that block compounds from binding by increasing the bulk of residues within the hydrophobic pocket in VP1.

The three-dimensional structures of two human rhinovirus serotypes (HRV14 and HRV1A) are compared when complexed with various antiviral agents. Although these agents all bind into the same hydrophobic pocket, the exact viral-drug interactions differ. In the absence of drugs, the pocket is occupied by a fatty acid in HRV1A, but is empty in HRV14 except for two water molecules. The conformation of each drug is dependent upon the shape of the hydrophobic pocket. In HRV14 the major residues determining the shape of the binding site are Y1128, P1174 and M1224, corresponding to I1125, M1169 and I1220 in HRV1A. When there is no cofactor or a drug in the pocket, the entrance to the pocket is open. However, the entrance is closed when the pocket is occupied by a cofactor or a drug. There are relatively small conformational changes when the agents displace the natural cofactor in HRV1A. In contrast, there are much larger conformational changes on binding a drug in HRV14. These differences cause an inhibition of viral attachment in HRV14 but not in HRV1A. Binding of the drugs results in three additional interprotomer hydrogen bonds in HRV14 and one in HRV1A. These hydrogen bonds and a potential loss of flexibility upon efficient packing of the pocket may contribute to the inhibition of uncoating in both serotypes.

Background: Rhinoviruses and the homologous polioviruses have hydrophobic pockets below their receptor-binding sites, which often contain unidentified electron density ('pocket factors'). Certain antiviral com pounds also bind in the pocket, displacing the pocket factor and inhibiting uncoating. However, human rhinovirus (HRV)14, which belongs to the major group of rhinoviruses that use intercellular adhesion molecule-1 (ICAM-1) as a receptor, has an empty pocket. When antiviral compounds bind into the empty pocket of HRV14, the roof of the pocket, which is also the floor of the receptor binding site (the canyon), is deformed, preventing receptor attachment. The role of the pocket in aral infectivity is not known.Results: We have determined the structure of HRV16, another major receptor group rhinovirus serotype, to atomic resolution. Unlike HRV14, the pockets contain electron density resembling a fatty acid, eight or more carbon atoms long. Binding of the antiviral compound WIN 56231 does not cause deformation of the pocket, although it does prevent receptor attachment.Conclusions: We conjecture that the binding of the receptor to HRV16 can occur only when the pocket is temporarily empty, when it is possible for the canyon floor to be deformed downwards into the pocket. We further propose that the role of the pocket factor is to stabilize virus in transit from one host cell to the next, and that binding of ICAM-1 traps the pocket in the empty state, destabilizing the virus as required for uncoating.

A series of conformationally restricted analogs of disoxaril has been synthesized and evaluated against human rhinovirus types (HRV) 14 and 1A. The sensitivity of these serotypes to this series varied and was dependent upon the length of the molecule as well as upon the flexibility of the aliphatic chain. Minimum energy conformations of these compounds were overlaid with the X-ray structure of a closely related analog 9 bound to the capsid protein of both HRV-14 and -1A and then modeled in the compound-binding site of both serotypes. A comparative sweep volume of these compounds about the isoxazole ring revealed an inaccessible region of space for the cis-olefin 8b, which is not the case for either the trans-olefin 8a or the acetylene 5. This region may be important to the binding of the compounds to the HRV-14 site particularly during entry into the pocket.

X-ray diffraction data were collected from human rhinovirus 14 crystals a few minutes after exposure to acid vapor and prior to excessive crystalline disorder. Conformational changes occurred (i) in the GH loop of viral protein (VP) 1, (ii) at the ion binding site on the outer surface of the pentamer center, and (iii) in VP3 and VP4 on the virion's interior in the vicinity of the fivefold axis. Amino acid substitutions in mutants resistant to low pH, or to drugs that inhibit uncoating, were concentrated in the vicinity of the GH loop. It is proposed that the acid-induced changes reflect processes that trigger uncoating.

The binding of the antirhinoviral agent R 61837 to human rhinovirus 14 has been examined by X-ray crystallographic methods. The compound R 61837 binds in the same pocket (underneath the canyon floor) as the "WIN" antirhinoviral agents. It does not penetrate as far into the pocket but causes similar conformational changes in the virus capsid. The movement of residues 1217 to 1221 of viral protein 1 (in the "FMDV loop") is more pronounced for R 61837 than for WIN compounds. Although both R 61837 and WIN antiviral agents partially fill the same hydrophobic pocket, atomic binding interactions differ, showing that considerable diversity in the nature of antiviral agents is possible.

The structure of Mengo virus had been determined from crystals grown in the presence of 100 mM phosphate buffer at pH 7.4. It is shown that Mengo virus is poorly infectious at the phosphate concentration similar to that in which it was crystallized. Maximal infectivity is achieved at 10 mM phosphate or less in physiological saline. The phosphate effect is ameliorated when the pH is lowered to 4.6. Although it has not been possible to study the crystal structure of the virus at low phosphate concentrations, it is shown that increasing the Cl- concentration at pH 6.2 or decreasing the pH to 4.6 causes substantial conformational changes confined to the "pit," a deep surface depression. These structural changes involve a movement of the "FMDV loop" (GH loop) in VP1, an ordering of the "VP3 loop" (GH loop in VP3) between 3176 and 3182, the displacement of a bound phosphate near the "FMDV loop" (GH loop in VP1), and movement of the carboxy terminus of VP2. The changes in conformation are correlated with the dissociation of the virion into pentamers at pH 6.2 and 150 mM Cl-. The localization of the conformational changes and the correlated role of the phosphate in controlling infectivity support the hypothesis that the "pit" is the receptor attachment site.

X-Ray diffraction data have been obtained for nine related antiviral agents ("WIN compounds") while bound to human rhinovirus 14 (HRV14). These compounds can inhibit both viral attachment to host cells and uncoating. To calculate interpretable electron density maps it was necessary to account for (1) the low (approximately 60%) occupancies of these compounds in the crystal, (2) the large (up to 7.9 A) conformational changes induced at the attachment site, and (3) the incomplete diffraction data. Application of a density difference map technique, which exploits the 20-fold noncrystallographic redundancy in HRV14, resulted in clear images of the HRV14:WIN complexes. A real-space refinement procedure was used to fit atomic models to these maps. The binding site of WIN compounds in HRV14 is a hydrophobic pocket composed mainly from residues that form the beta-barrel of VP1. Among rhinoviruses, the residues associated with the binding pocket are far more conserved than external residues and are mostly contained within regular secondary structural elements. Molecular dynamics simulations of three HRV14:WIN complexes suggest that portions of the WIN compounds and viral protein near the entrance of the binding pocket are more flexible than portions deeper within the beta-barrel.