Pocket Factor Research Articles

Picornaviruses: Epitopes, Canyons, and Pockets

This chapter focuses on focuses on studies of human rhinovirus 14 and some recent results that have begun to clarify interrelationships among the modes of action of antibodies, receptors, and antiviral drugs. The structures of several picornaviruses are now known in atomic detail. Together with virological and biochemical examination, these structures have revealed antigenic sites and possible receptor contact regions. For rhino-, polio-, and coxsackie viruses, studies using antiviral drugs have illuminated the process of viral uncoating and have shown how these nonenveloped viruses exist in a precarious state, poised between premature uncoating and inability to release their genomic payload. Finally, structural and immunological techniques have been used to study a number of antibody-virion complexes to ascertain how antibodies, the major protective agent in the immunological arsenal against picornaviruses, can neutralize viral infectivity. Discriminating among the various models may prove to be difficult because any of a large number of small hydrophobic compounds in the cell could act as pocket factors.

The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses.

Coxsackievirus A9 (CAV9), a human pathogen causing symptoms ranging from common colds to fatal infections of the central nervous system, is an icosahedral single-stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. One of the four capsid proteins, VP1, includes the arginine-glycine-aspartate (RGD) motif within its C-terminal extension. This region binds to integrin alpha v beta 3, the only receptor for CAV9 to be conclusively identified to date. The crystal structure of CAV9 in complex with the antiviral compound WIN 51711 has been solved to 2.9 A resolution. The structures of the four capsid proteins, VP1 to VP4, resemble those of other picornaviruses. The antiviral compound is bound in the VP1 hydrophobic pocket, and it is possible that the pocket entrance contains a second WIN 51711 molecule. Continuous electron density for the VP1 N terminus provides a complete picture of the structure close to the fivefold axis. The VP1 C-terminal portion is on the outer surface of the virus and becomes disordered five-residues N-terminal to the RGD motif. The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.

Human rhinovirus 3 at 3.0 å resolution

Background The over 100 serotypes of human rhinoviruses (HRV) are major causative agents of the common cold in humans. These HRVs can be roughly divided into a major and minor group according to their cellular receptors. They can also be divided into two antiviral groups, A and B, based on their sensitivity to different capsid-binding antiviral compounds. The crystal structures of HRV14 and HRV16, major-receptor group rhinoviruses, as well as HRV1A, a minor-receptor group rhinovirus, were determined previously. Sequence comparisons had shown that HRV14 seemed to be an outlier among rhinoviruses. Furthermore, HRV14 was the only virus with no cellular ‘pocket factor’ in a hydrophobic pocket which is targeted by many capsid-binding antiviral compounds and is thought to regulate viral stability. HRV3, another major-receptor group virus, was chosen for study because it is one of a subset of serotypes that best represents the drug sensitivity of most rhinovirus serotypes. Both HRV3 and HRV14 belong to antiviral group A, while HRV16 and HRV1A belong to antiviral group B. Results HRV3 was found to be very similar to HRV14 in sequence and structure. Like HRV14, crystallized HRV3 also has no bound pocket factor. The structure of HRV3 complexed with an antiviral compound, WIN56291, was also determined and found to be similar to the same antiviral compound complexed with HRV14. Conclusions The amino-acid sequence and structural similarity between HRV3 and HRV14 suggests that rhinoviruses in the same antiviral group have similar amino-acid sequences and structures. The similar amino-acid composition in the pocket region and the viral protein VP1 N termini in all known group B HRV sequences suggests that these viruses may all contain pocket factors and ordered N-terminal amphipathic helices in VP1. Both of these factors contribute to viral stability, which is consistent with the observations that group B rhinoviruses have a higher chance of successful transmission from one host to another and is a possible explanation for the observed higher pathogenicity of these rhinoviruses.

Implications for viral uncoating from the structure of bovine enterovirus

We have determined the crystal structure of a bovine enterovirus, revealing that the topologies of the major capsid proteins and the overall architecture of the virion are similar to those of related picornaviruses. The external loops joining beta-strands are truncated and the canyon region is partially filled by an extension of the VP3 G-H loop giving the viral capsid a relatively smooth appearance. These changes may have implications for cell attachment. In spite of these differences the virus maintains a hydrophobic pocket within VP1, occupied by a specific 'pocket factor' which appears to be myristic acid. These observations support the proposal that a kinetic equilibrium exists between occupied and unoccupied pocket states, with occupation inhibiting uncoating.

Viral cell recognition and entry.

Rhinovirus infection is initiated by the recognition of a specific cell-surface receptor. The major group of rhinovirus serotypes attach to intercellular adhesion molecule-1 (ICAM-1). The attachment process initiates a series of conformational changes resulting in the loss of genomic RNA from the virion. X-ray crystallography and sequence comparisons suggested that a deep crevice or canyon is the site on the virus recognized by the cellular receptor molecule. This has now been verified by electron microscopy of human rhinovirus 14 (HRV14) and HRV16 complexed with a soluble component of ICAM-1. A hydrophobic pocket underneath the canyon is the site of binding of various hydrophobic drug compounds that can inhibit attachment and uncoating. This pocket is also associated with an unidentified, possibly cellular in origin, "pocket factor." The pocket factor binding site overlaps the binding site of the receptor. It is suggested that competition between the pocket factor and receptor regulates the conformational changes required for the initiation of the entry of the genomic RNA into the cell.

WIN 51711-dependent mutants of poliovirus type 3: evidence that virions decay after release from cells unless drug is present.

Twenty-two spontaneous mutants of the Sabin strain of poliovirus type 3 were selected for drug resistance by plating on HeLa cell monolayers in the presence of WIN 51711, an uncoating inhibitor. When replated in the presence and absence of drug, two classes of mutants were observed; mutants displayed either a drug-dependent or a non-drug-dependent phenotype, in the proportion 14:8. Non-drug-dependent mutants plaqued with equal efficiency in the presence or absence of drug. By contrast, drug-dependent mutants made no plaques in the absence of drug, except for revertants. In single-step growth curve experiments, however, drug-dependent mutants grew as well in the absence of drug as in its presence. This paradoxical behavior of dependent mutants was traced to extreme thermolability at 37 degrees C (12- to 30-s half-life) in the absence of drug. Thermolability was exhibited only after the virus was released from the cell, implying the presence of a cell-associated protective factor, possibly pocket factor. Thus, in the absence of a thermostabilizing drug, drug-dependent mutants decayed too rapidly after release to permit spread in the plaque assay. The thermodecay product was shown to consist of 135S particles lacking VP4.

Novel generation of heteroarylthio, sulfoxy, or sulfonyl alkyl azoles as antipicornavirus agents : M. Daneshtalab, R.G. Micetich, D.Q. Nguyen, C.M. Ha, H.T. Luu, M.L. Tempest and C. Koski. SynPhar Laboratories, Inc., #24 Taiho Alberta Center, 4290-91A Street, Edmonton, Alberta, Canada T6E 5V2

Background: Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs.Results The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 å. The β-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 β-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes.Conclusion The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.