Studies of interactions between coxsackieviruses and their cellular receptors

The coxsackie B viruses are significant pathogens, causing myocarditis, especially in infants, aseptic meningitis, and various other illnesses of less proven etiology. They are classified in the family Picomaviridae , many of whose other members are equally dangerous, causing hepatitis, polio and foot and mouth disease. Understanding in detail how these pathogens infect cells and replicate could lead to treatments for these illnesses. The first step in infection is attachment of the virus to a cell surface receptor. Coxsackievirus B3 (CVB3) was understood (when these studies began) to have a minor receptor, DAF (decay accelerating factor) and an unidentified major receptor. We proposed to (1) identify the major receptor via a panning assay. (2) clone, produce and purify both receptors, (3) do cryo electron microscopy and image reconstruction on virus-receptor complexes to identify amino acid residues involved in the interactions, (4) verify conclusions from cryo EM by site-directed mutagenesis of the virus, and (5) reconcile the results with current understanding of picornavirus attachment and entry. Panning assays were highly variable and while some results were encouraging, other labs found the major receptor, CAR (coxsackievirus-Adenovirus receptor) first. DAF was produced in the baculovirus system and purified using anion exchange and hydrophobic interaction chromatography. Many tests were done to convince ourselves that authentic DAF was being expressed and that it was not aggregated. BDAF (baculovirus-produced DAF) was used in cryo EM with CVB3 and with coxsackievirus A21, which also binds DAR Both reconstructions resulted in images of bare virus particles. Several types of tests were done to try to detect interactions between BDAF and CVB3 to try to find conditions under which a stable complex could be formed for more cryo-EM, but no such conditions were found. The interaction is likely very weak. A cryo EM structure of CVB3 + CAR was done by He et al, which identified some of the important residues [Nature Struct. Biol., 8 874–878 (2001)]. Mutagenesis was started, and a preliminary result indicated that D1089 is important in interactions with the receptor, as predicted by the structure