Molecular dynamics of human rhinoviruses in the context of receptor recognition and antiviral drug action

Research project P 14549 Virus Dynamics and Recognition Peter HINTERDORFER 09.10.2000 Development of effective drugs against human rhinoviruses (HRVs), which are a major cause of common cold infections, still remains a challenge because of the large numbers of different serotypes. This process could be significantly aided by an improved understanding of the binding mechanism of both (i) viruses to cells or cell- receptors and (ii) of drugs to viruses. A dynamic capsid is essential for the spatially and temporarily concerted sequence of events that determine the viral life cycle. Attachment of viruses to cells, cell entry, and nucleic acid release are necessarily accompanied by conformational changes of capsid proteins and/or by dynamic changes of the capsid structure. We will investigate molecular interactions mediating viral-cell recognition and drug-virus binding simultaneously with studies of the viral dynamics. This is expected to yield valuable insights into the processes of viral infection and drug action. Thereby, we will employ dynamic force microscopy (DFM), recently developed from atomic force microscopy (AFM), which is known as a powerful experimental method to study the molecular dynamics of biological processes on single bio-molecules in their native environment with high lateral resolution. The potential of high force resolution (pN) and fast response (ms) allows for the detection of molecule fluctuations and dynamic nanomechanical changes on soft biological samples. In addition, coupling of ligands and drugs to AFM tips opens the possibility of studying on the single molecule level receptor-ligand recognition and drug-receptor binding, respectively. This will lead to new results about the molecular dynamics of the recognition- and binding process.

In this project we have applied scanning probe microscopy (SPM) methods, i.e. topography imaging and recognition force microscopy spectroscopy/microscopy, to a cold virus, the human rhino virus 2 (HRV2). Hereby the development of surface chemistry protocols was the first step. We coupled antibodies covalently to SPM tips using the spacer technology and detected molecular recognition with the virus adsorbed to surfaces at the single molecule level. Antibody/virus interaction forces and the dynamics of binding were addressed in force spectroscopy experiments. For force spectroscopy experiments with cell receptors, different receptor constructs were coupled to AFM tips. Single molecular binding strengths were detected using recombinant receptor fragments with variable binding domains, i.e. V1-8 (with all 8 modules), V1-3 (with the first 3 modules), V33, V333 , and V333 (concatamers of module 3). V1-8 showed a stronger interaction than V333 and V33333 , the other constructs displayed very weak forces. From the dependence of the forces on the loading rate we were able to derive the thermal dissociation constant, koff , and xß, the length of the activation barrier. Binding of the receptor fragments to the virus was also measured in SPM topography images. This technique also allowed to follow RNA release from the virus. We obtained a lateral resolution of two nanometers. Thereby, we resolved substructers on the viral protein shell. Protein loops were arranged in a hexagonal pattern. At the same time we build up a statistical model for receptor/virus binding in order to compare the theoretical findings with the experimental data.