FRET and FCS Measurements of HRV / Receptor Interactions

The recently solved X-ray structure of a complex between the common cold virus HRV2 (human rhinovirus serotype 2) and a very-low density lipoprotein receptor (VLDLR) fragment including ligand binding repeats 2 and 3 shows attachment of five copies of repeat 3 (V3) around the five-fold axis of icosahedral symmetry. V2 is not visible and possibly does not contribute to binding. The N-termini and the C-termini of adjacent V3 modules in the complex lie within a distance of 10 Å suggesting that, upon concatemerization, five V3 modules within a single molecule might attach simultaneously. This view is supported by the finding that artificial concatemers show an increase of cell protective activity with the number of repeats contained in the molecule when added together with virus to HeLa cells. This might indicate that their antiviral effect stems from inhibition of viral uncoating by stabilizing the virion via crosslinking the capsid subunits. Our findings so far also raise the question of whether interaction of the virus with the native membrane proteins, such as the low-density lipoprotein receptor (LDLR) and LDLR-related protein (LRP), occurs via the multiple modules within the ligand binding domains of the receptor molecules. In order to assess the geometry of attachment of the receptors and their derivatives, fluorescence resonance energy transfer (FRET) methods will be used. N-termini and C-termini of artificial concatemers and of soluble natural receptor fragments will be labeled with suitable FRET couples. Occurrence of FRET in the presence of virus then demonstrates that C- and N-termini lie within the Förster distance when the molecule is bound to the viral surface. In order to complement the data on spatial relationships, the affinities of the interactions will be measured by fluorescence correlation spectrometry. The minimal structural unit recognizing virus and the influence of length and sequence of the linkers between the modules on virus binding will be determined. This might eventually lead to highly efficient inhibitors of viral infection. Results of this work will also reveal the basis of virus neutralization, lead to a better understanding of principles of protein-protein interaction, and point to novel strategies to inhibit viral infection.

The recently solved X-ray structure of a complex between the common cold virus HRV2 (human rhinovirus serotype 2) and a very-low density lipoprotein receptor (VLDLR) fragment including ligand binding repeats 2 and 3 shows attachment of five copies of repeat 3 (V3) around the five-fold axis of icosahedral symmetry. V2 is not visible and possibly does not contribute to binding. The N-termini and the C-termini of adjacent V3 modules in the complex lie within a distance of 10 Å suggesting that, upon concatemerization, five V3 modules within a single molecule might attach simultaneously. This view is supported by the finding that artificial concatemers show an increase of cell protective activity with the number of repeats contained in the molecule when added together with virus to HeLa cells. This might indicate that their antiviral effect stems from inhibition of viral uncoating by stabilizing the virion via crosslinking the capsid subunits. Our findings so far also raise the question of whether interaction of the virus with the native membrane proteins, such as the low-density lipoprotein receptor (LDLR) and LDLR-related protein (LRP), occurs via the multiple modules within the ligand binding domains of the receptor molecules. In order to assess the geometry of attachment of the receptors and their derivatives, fluorescence resonance energy transfer (FRET) methods will be used. N-termini and C-termini of artificial concatemers and of soluble natural receptor fragments will be labeled with suitable FRET couples. Occurrence of FRET in the presence of virus then demonstrates that C- and N-termini lie within the Förster distance when the molecule is bound to the viral surface. In order to complement the data on spatial relationships, the affinities of the interactions will be measured by fluorescence correlation spectrometry. The minimal structural unit recognizing virus and the influence of length and sequence of the linkers between the modules on virus binding will be determined. This might eventually lead to highly efficient inhibitors of viral infection. Results of this work will also reveal the basis of virus neutralization, lead to a better understanding of principles of protein-protein interaction, and point to novel strategies to inhibit viral infection.