Increasing the affinity of recombinant receptor fragments for common cold viruses: towards potential attachment inhibitory antivirals?

Research project P 14503 Common cold virus attachement inhibitors Dieter BLAAS 09.10.2000 Human rhinoviruses (HRVs), the main cause of common cold infections, circulate in the human population in fonn of more than one hundred different serotypes. Despite this diversity, only three receptors are being used for host cell entry. Ninety one serotypes bind to intercellular adhesion molecule I (the major group) and ten serotypes bind to members of the low-density lipoprotein receptor (LDLR) family (the minor group), the only exception (HRV87) binds to an uncharacterized glycoprotein. Although the viral serotypes are very similar to each other with respect to capsid structure, genome organization and symptoms of the disease caused, the receptors are structurally and functionally unrelated. In previous work we have defined the minimal structure requirements of LDLR for viral recognition and protection of cells against viral infection using recombinant soluble receptor fragments. Means have been devised to express minireceptors derived from various members of the LDLR family with high yield in Sf9 insect cells and in bacteria followed by in vitro folding to the native conformation. Within the frame of the proposed project small receptor fragments (encompassing one, two or three of the ligand binding complement type A repeats) derived from various members of the LDLR family will be expressed as fusion with the attachment protein P3 of M13 to allow for phage display. Suitable sites within these proteins will then be subjected to random mutagenesis and recombinant phage will be selected for highest affinity towards minor group viruses. By the same methodology, individual ligand binding repeats will be shuffled. and recombined randomly and binding parameters of these novel receptor molecules with respect to virus and other ligands will be investigated. Such receptor derivatives with artificially improved affinity might be taken as a starting point for the development of antivirally active compounds and will allow for the identification of contact sites between viral surface and receptor molecule. Determination of the parameters of the interaction with other ligands will help to define the basis of the specific recognition of various ligands.

More than 50% of all mild respiratory diseases summarized as common colds, are caused by human rhinoviruses (HRVs). Infection is initiated upon specific attachment to cellular receptors; virions are then transported into the cell, the RNA genome is released, and replication takes place in the cytosol. Upon destruction of the cell, progeny virus is set free and starts a new replication cycle in neighbouring cells. Therefore, inhibiting the very first encounter between virus and host cell is an efficient way of preventing infection. We have thus set out to investigate the mode of interaction between rhinoviruses and their respective receptors. Most of the work was carried out with HRV2 that belongs to the minor receptor group. This group of HRVs uses members of the low- density lipoprotein receptor (LDLR) family for cell entry. In contrast to other virus receptors, the ligand binding domain of the LDLR family is composed of various numbers of short modules with similar but not identical sequence. Using a recombinant receptor fragment including module 2 and 3 only we obtained the very first ever solved atomic structure of a virus-receptor complex at atomic resolution. The structure revealed an intricate network of hydrophobic and ionic interactions. In all minor group HRVs the key residue is a lysine within a surface loop of the capsid protein VP1 interacting with a tryptophan residue and an acidic cluster chelating a Ca-ion that are conserved in most of the ligand binding repeats of the LDLR-family. The arrangement of the modules on the viral surface strongly suggested that several modules within a single receptor molecule could attach simultaneously. This was confirmed using artificial recombinant concatemers of VLDLR repeat 3 (V33333) forming a ring-like structure around each of the 12 vertices of the icosahedral virus particle. This unique mode of interaction explains the extremely strong binding of V33333 resulting in very efficient blockage of cell attachment and viral infection. Recent data indicate the possibility that the binding strength can be further increased manifold by site-directed mutagenesis of the receptor and by modification of the length of the linkers between the modules. Thus, an extremely strong virus inhibitor will be created.