No abstract

We demonstrate the detection and characterization of ligand binding to viruses via NMR. To illustrate the methodology, the interaction of an antiviral compound with human rhinovirus serotype 2 (HRV2) was investigated. Specific interaction of a capsid-binding inhibitor and native HRV2 was monitored utilizing saturation transfer difference (STD) NMR. STD NMR experiments at atomic resolution allowed those regions of the ligand that are involved in the interaction with the virus to be determined. The approach allows for (i) the fast and robust assessment of binding, (ii) the determination of the ligand binding epitope at atomic resolution without the necessity to crystallize virus-ligand complexes, and (iii) the reuse of the virus in subsequent assays. This methodology enables one to easily identify binding of drugs, peptides, and receptor or antibody fragments to the viral capsid.

In-gel protein kinase assays using myelin basic protein (MBP) as substrate have been used to demonstrate that abscisic acid (ABA) activates an MBP kinase (AMBP kinase) in epidermal peels prepared from leaves of the Argenteum mutant of pea, Pisum sativum L. AMBP kinase has the characteristics of a mitogen-activated protein kinase (MAPK): it utilizes MBP preferentially as an artificial substrate, it is rapidly and transiently activated, it is of the appropriate size (molecular weight c. 45 kDa), requires tyrosine phosphorylation for activity and is tyrosine phosphorylated upon activation. Reverse transcription-PCR was used to generate a previously-cloned MAPK from guard cells, epidermis and mesophyll and immunoblotting using an antibody raised against a mammalian MAPK detected MAPK-related proteins, including one of 45 kDa, in epidermal peels, mesophyll and guard cells. Inhibition of AMBP kinase activation by PD98059, a specific inhibitor of MAPK kinase, and thus MAPK activation, correlated with PD98059-inhibition of ABA-induced stomatal closure and dehydrin gene expression, suggesting that ABA effects in pea epidermal peels require MAPK activation. AMBP kinase was not activated by ABA in guard cells isolated by enzyme treatment. However, a protein kinase of c. 43 kDa was activated by ABA in isolated guard cells, but not in mesophyll or epidermal tissue.

Minor group human rhinoviruses (HRVs) use members of the low-density lipoprotein receptor family for cell entry. To investigate the utility of receptor fragments as viral inhibitors, various polypeptide segments derived from the ligand binding domain of human very-low-density lipoprotein receptor (VLDLR) were expressed in a soluble form in bacteria. Whereas none of the fragments was active in virus binding immediately after recovery from the cell lysates, constructs encompassing complement type repeats 1-3, 1-6, and 1-8 spontaneously acquired virus binding activity by incubation at 4 degrees C in buffer containing Ca(2+) ions and lacking any redox system. When immobilized receptor-associated protein (RAP), a specific chaperone for VLDLR, was present during the incubation, the yield of protein active in ligand binding was substantially increased. A VLDLR fragment with repeats 4-6 failed to bind virus; however, it bound RAP. Bacterial expression of truncated VLDLR 1-3 at high yield, easy purification, and folding together with high inhibitory activity toward HRV2 makes this protein a promising starting point for the development of an oligopeptide-based antiviral agent. Using sucrose density gradient centrifugation, we demonstrate the formation of virus-receptor complexes. The recombinant receptors can thus be used for structure determination by electron cryo-microscopy.

Complex formation between monoclonal antibodies or soluble receptor fragments and a human rhinovirus is quantified by relating the concentration of the antibody or receptor under equilibrium conditions to the initial concentration of the virus. Within a given concentration range of the reactants, the shape of the resulting curve depends only on the value of the dissociation constant of the particular system studied. Using antibodies and receptor fragments, cases for high, low, and intermediate affinity were investigated. For high-affinity systems, the curve approximates a decaying straight line and the binding stoichiometry can be accurately determined from the intercept with the x-axis. For the case of intermediate affinity, the curve can be linearized at low virus concentrations with the receptors present in large excess. Extrapolation of this line allows derivation of the binding stoichiometry from the intercept with the x-axis, although with less accuracy. For intermediate affinities, an estimate of the dissociation constant can be obtained from fitting the curve to the data points measured. Finally, in the case of low affinity none of the binding parameters can be quantified, although a rough estimate of the lower limit of the dissociation constant is possible. The method was applied for two different monoclonal antibodies, a Fab fragment and a receptor fragment, binding to human rhinovirus serotype 2. Thirty copies of the monoclonal antibody 8F5 were found to bind to the virion, which is in agreement with data from electron cryomicroscopy. The complex between monovalent human very-low-density lipoprotein receptor encompassing repeats 2 and 3 and human rhinovirus serotype 2 showed 60 receptor molecules bound per virion.

Concatemers of various numbers of the third ligand binding repeat of human very-low density lipoprotein receptor arranged in tandem were fused to maltose-binding protein and expressed as soluble polypeptides. These artificial receptors protected HeLa cells against infection with human rhinovirus serotype 2 (HRV2) to a degree that strongly increased with the number of repeats present; maximal protection was seen for the pentameric concatemer (MBP-V33333). This V3 pentamer neutralized HRV2 more efficiently than a recombinant protein with the entire ligand binding domain of the native receptor encompassing all 8 non-identical repeats. A concatemer of seven V3 modules (MBP-V3333333) was also less neutralizing. Neutralization was correlated with the degree of inhibition of virus binding to the cell surface. The results were in agreement with kinetic measurements using Biacore instrumentation demonstrating an increase in avidity with the number of modules present. At low concentrations of the receptor fragments, a 1:1 Langmuir kinetics was observed which became of complex type in the higher concentration range. This is most likely a consequence of receptor molecules simultaneously binding via several modules. Since there is no viral aggregation, neutralization of viral infectivity results from blockage of the receptor binding sites and possibly from inhibition of viral uncoating by crosslinking the viral capsid subunits via multi-module binding. Finally, the low affinity of the single V3 module allowed demonstrating the possibility of mapping the binding epitope of the V3 receptor fragment by saturation transfer difference nuclear magnetic resonance methodology.

The formation of complexes between the minor receptor group human rhinovirus HRV2 and two recombinant soluble receptor fragments derived from the human very low density lipoprotein receptor (VLDLR) and containing ligand-binding repeats 1-3 (MBP⅐VLDLR 1-3 ) or 1-8 (MBP⅐VLDLR 1-8 ) fused to the carboxyl terminus of the maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the determination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versus total virus concentration. Extrapolation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components present in the complex. Using this method, we determined that about 60 molecules of MBP⅐VLDLR 1-3 but only about 30 molecules of MBP⅐VLDLR 1-8 were bound per virion.Human rhinoviruses (HRVs), 1 members of the picornavirus family, are small (ϳ30 nm in diameter) icosahedral particles composed of 60 copies each of the viral capsid proteins VP1 through VP4 and a positive strand RNA genome of about 7200 nucleotides in length (1). The 102 serotypes recognized to date use three different classes of receptors for cell entry. 91 serotypes (the major group) bind to intercellular adhesion molecule 1 (ICAM-1, Refs. 2-4), 10 serotypes (the minor group) bind to several members of the low density lipoprotein receptor (LDLR) family (5-7), and 1 serotype (HRV87) binds to a glycoprotein of so far unknown function (8). ICAM-1 is a member of the immunoglobulin superfamily with five immunoglobulinlike domains making up its extracellular part. The LDLR family comprises a number of membrane proteins all having various numbers of highly conserved complement type A repeats of about 40 amino acids in length containing six cysteines each, which exhibit extensive disulfide bridging (for review, see Ref. 9). Whereas the structure of ICAM-1 is known at atomic resolution (10, 11), only the structures of single ligandbinding repeats have been determined by NMR (12) and x-ray crystallography (13).The binding site of ICAM-1 on HRV14 and on HRV16 has been characterized by electron cryo-microscopy followed by image reconstruction techniques (14,15). This confirmed the earlier prediction of receptor attachment occurring within the canyon, a cleft encircling the 5-fold axes of the viral icosahedral symmetry (16,17). In an attempt to determine the binding site of LDL receptors on minor group viruses, we had previously expressed soluble fragments of LDLR in Sf9 insect cells using the baculovirus system (18,19). Although these recombinant minireceptors are extremely potent in protecting HeLa cells against infection wit...

The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-Å-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor "staples" the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.