Atomic force microscopy-derived nanoscale chip for the detection of human pathogenic viruses.

Native-protein nanolithography (NPNL) was used to fabricate stable bioactive arrays of viral receptor spots. The arrays were specific for the cognate virus and devoid of nonspecific protein and virus adsorption under physiologic conditions. The spot size ranged from 200 nm x 200 nm to 2 microm x 2 microm and up to 3 x 3 spots were arranged per array. With proper force adjustment in the patterning experiments, His(6)-tagged bovine serum albumin (BSA) molecules were selectively removed from the underlying self-assembled monolayer (SAM) while leaving the latter intact. Injection of His(6)-tagged very low density lipoprotein receptor (VLDLR-His(6)) constructs resulted in specific, oriented binding to the Ni(2+)-loaded bis-(nitrolotriacetic acid) (bis-NTA) groups to the re-exposed SAM areas. The arrays of viral receptors were used for the detection of human rhinovirus particles (serotype 2; HRV2) under native conditions by topographical imaging at high signal-to-noise ratio. The kinetic on-rate of the HRV2-VLDLR interaction was derived from the time-dependent binding of the virions to the VLDL receptor spots. No significant binding was observed for the major group virus HRV14 that uses the unrelated receptor ICAM-1.

Gas-phase electrophoretic molecular mobility analysis of size and stoichiometry of complexes of a common cold virus with antibody and soluble receptor molecules.

Attachment of a nonaggregating monoclonal antibody and of a soluble recombinant receptor molecule to the icosahedral nonenveloped human rhinovirus serotype 2 was studied with a nanoelectrospray ionization gas-phase electrophoretic molecular mobility analyzer (nESI-GEMMA). The virus mass, as determined via nESI-GEMMA, was within instrument accuracy (+/-6%) close to the theoretical value (8 x 10(6) Da) calculated from the sum of all constituents of one virus particle (60 copies of each of the four viral capsid proteins, the RNA genome, and one copy of the RNA-linked protein VpG). The formation of virus-antibody complexes of different stoichiometries (up to a mass 12.5 x 10(6) Da corresponding to 30 attached antibodies) and virus-receptor complexes (up to a mass 8.8 x 10(6) Da corresponding to 12 attached receptor molecules) was monitored. Via the volume derived from the electrophoretic mobility diameter (EMD), the stoichiometry of the HRV complexes was calculated. The accuracy of the EMD was within +/-0.5 nm, which corresponds to an accuracy of +/-4 antibodies and +/-5 receptor molecules in the respective complexes. For the first time, we here demonstrate the use of nESI-GEMMA for the analysis of the size and stoichiometry of biomolecules in high-order complexes in real time under normal pressure conditions.

Attachment of VLDL receptors to an icosahedral virus along the 5-fold symmetry axis: multiple binding modes evidenced by fluorescence correlation spectroscopy.

Human rhinoviruses (HRVs) are composed of 60 identical subunits, each comprising one copy of the viral capsid proteins VP1, 2, 3, and 4. Consequently, 60 symmetry-related epitopes are available for binding of antibodies or receptors. The minor receptor group of HRVs uses members of the low-density lipoprotein receptor family for cell entry. The ligand binding domains of these receptors are composed of various numbers of ligand binding repeats, and several of these modules within a single molecule are believed to attach simultaneously to the star-shaped dome at the 5-fold symmetry axis of the virus. Using fluorescence correlation spectroscopy (FCS), we have now determined the equilibrium binding constants and the mode of attachment of recombinant concatemers of ligand binding module 3 of the human very-low-density lipoprotein receptor to HRV2. We demonstrate that the avidity of the interaction drastically increases with the number of concatenated modules. For the trimer, the binding isotherm was biphasic, indicating that attachment of two and of three modules within the same molecule was resolved. The receptor consisting of seven repeats was found to bind most strongly, but a complete binding isotherm could not be established due to cross-linking of virions. The values of the dissociation constants were about 1 order of magnitude higher than those previously determined by using surface plasmon resonance techniques reflecting the different presentation of the binding partners. As compared to the concatemers, the natural receptors are composed of similar but not identical repeats; thus, cooperativity and different specificity of the ligand-binding modules allow for recognition of many ligands and viral serotypes. Due to the low concentrations and amounts of sample required, FCS is ideally suited for the determination of receptor binding parameters of viruses difficult to produce in high quantities and/or concentrations.

A mutation in the first ligand-binding repeat of the human very-low-density lipoprotein receptor results in high-affinity binding of the single V1 module to human rhinovirus 2.

Minor group human rhinoviruses (HRVs) bind members of the low-density lipoprotein receptor family for cell entry. The ligand-binding domains of these membrane proteins are composed of various numbers of direct repeats of about 40 amino acids in length. Residues involved in binding of module 3 (V3) of the very-low-density lipoprotein receptor (VLDLR) to HRV2 have been identified by X-ray crystallography (N. Verdaguer, I. Fita, M. Reithmayer, R. Moser, and D. Blaas, Nat. Struct. Mol. Biol. 11:429-434, 2004). Sequence comparisons of the eight repeats of VLDLR with respect to the residues implicated in the interaction between V3 and HRV2 suggested that (in addition to V3) V1, V2, V5, and V6 also fulfill the requirements for interacting with the virus. Using a highly sensitive binding assay employing phage display, we demonstrate that single modules V2, V3, and V5 indeed bind HRV2. However, V1 does not. A single mutation from threonine 17 to proline converted the nonbinding wild-type form of V1 into a very strong binder. We interpret the dramatic increase in affinity by the generation of a hydrophobic patch between virus and receptor; in the presence of threonine, the contact area might be disturbed. This demonstrates that the interaction between virus and its natural receptors can be strongly enhanced by mutation.

Neutralization of a common cold virus by concatemers of the third ligand binding module of the VLDL-receptor strongly depends on the number of modules.

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.