Evolution of Rhinoviruses

Human rhinoviruses (HRVs) are the main causative agent of the common cold. Although identical in genome organization and very similar in 3D-structure twelve serotypes (the minor group) use members of the low-density lipoprotein receptor (LDLR) family and 87 serotypes (the major group) use intercellular adhesion molecule 1 (ICAM-1) for cell entry. It is unknown how these viruses evolved to discriminate between two structurally and functionally different receptors that also attach in dissimilar ways; sixty ICAM-1 molecules bind to 60 symmetry- related sites on the virion. The tips of their N-terminal domains reach into the canyon, a cleft encircling the vertex of the icosahedral particle. Conversely, LDL-receptors bind to the star-like mesa around the icosahedral five-fold axes in a unique way; more than one copy of their small ligand binding repeats attach to the symmetry related sites. In principle, a virion might accommodate five modules at each vertex. This results in binding of 12 copies of the receptor per virion. Not only receptor attachment is different. ICAM-1 destabilizes the viral capsid and facilitates release of the RNA genome whereas artificial multimeric LDL-receptors stabilize the virions. Since HRVs are internalized into endosomes the viral genome has to be transferred into the cytoplasm. This occurs via a pore in minor group HRVs and via disruption of the endosomal membrane in major group HRVs. The underlying mechanism is not understood. We thus want to elucidate the role of the amino acid residues at the virus - receptor interface in recognition and discrimination of the viral serotypes. We also want to determine which of the natural receptor modules are able to bind the virus and which ones are involved in the interaction. Finally, we want to find out how the viruses dissociate from their receptors at the low pH environment in the endosome and how they attach to the lipid membrane to either open a pore or to disrupt the membrane. Comparison between minor and major receptor group HRVs with respect to the principles of receptor discrimination is of imminent importance for the understanding of viral evolution. Information obtained in these studies might be valuable in the structure-based design of viral inhibitors.

About 115 human rhinovirus serotypes, species HRV A, -B, and C, are the main cause of the common cold. Despite this large diversity, roughly 85% (the major HRV receptor group), bind intercellular adhesion molecule 1 (ICAM 1) and about 10% (the minor HRV receptor group), bind members of the low-density lipoprotein receptor (LDLR) family for cell entry. The structural basis of receptor discrimination and differences between HRVs with respect to cellular uptake were largely unknown. Knowledge of common motives recognized by the receptors is essential for the design of binding inhibitors. We have calculated 3-D models of all HRVs with known genomic sequences by using the available X-ray coordinates from five HRV types as templates and a fragment of very-LDLR was docked by employing the X-ray structure of its complex with HRV2. The affinity of the receptor for each of the models was then predicted with various software tools. We thereby classified correctly all but one HRV-type (from sequence of the major capsid protein alone) as belonging to the minor or the major receptor group. Common structural features most important for receptor recognition and discrimination were extracted in this analysis. HRVs of the two receptor groups behave also differently with respect to entry. Therefore, we dissected the uptake pathways of HRV8 and HRV14 (both major group viruses) and identified a macropinocytosis-like mechanism. HRV8 uses heparan sulphate (HS) as an alternative receptor. Despite uptake via ICAM-1 and via HS exhibit very similar drug-inhibition profiles, the morphology of the invaginations of the plasma membrane accumulating the viruses was different suggesting the existence of various forms of macropinocytosis. In previous work we showed that uptake of the minor group virus HRV2 was clathrin dependent. We also carried out collaborative studies on: 1) the unbinding force of HRV2 when attached to LDLR on the cell surface by using atomic force microscopy, 2) on the use of photolithography for the production of switchable active surfaces, and 3) on antibodies against the `covalent linkage unit`, a specific structure at the 5`-end of viral RNA, for the immunolocalization of replication complexes. Finally, we published a review article on entry of human rhinoviruses including results of these studies.