Viruses with genomes composed of positive-sense, single-stranded RNA [(+) ssRNA] are believed to replicate within cells in remodeled host membranes called replication organelles (ROs). Picornaviridae, a family of (+) ssRNA viruses, contains several members (e.g. poliovirus, rhinovirus) in which the formation of replication organelles has been observed. The viral genome encodes a large polyprotein, consisting of structural and nonstructural proteins. Within the “P3” domain of the polyprotein is the 3CD protein, which is posited to be a master regulator of the formation of ROs and a key player in the reorganizing of lipids at these replication sites. Phosphoinositides (PIs) are often referred to as the zip codes of the cell as they facilitate sorting of proteins to specific subcellular locations. They only comprise a small percentage of cellular lipids, less than 1% the total lipidome, but become concentrated on ROs. Through computational binding studies of the poliovirus 3C protein, multiple residues were identified as being potentially involved with PI binding. To further explore viral protein-PI interactions, a microfluidics-based assay was developed to determine the dissociation constants for the binding of viral proteins to PI. The microfluidics-based assay mimics the lipid bilayer of the ROs, but can consist of any desired lipid composition. For example, to mimic the Golgi membrane, the vesicles are composed of 7.5 mol% phosphatidylinositol 4-phosphate. Poliovirus is known to reconstruct this subcellular membrane. The assay utilizes a pH-dependent fluorophore, ortho-sulforhodamine B conjugated to phosphoethanolamine. Ortho-sulforhodamine B is highly fluorescent at a low pH and quenched at a high pH. When a protein interacts with the bilayer, it brings with it counterions. Those counterions cause a local change in pH, which is detected as a change in fluorescence. Using this technique, the viral protein concentration can be varied and the fluorescence change can be plotted against concentration to yield a binding curve, from which a value for the apparent dissociation constant, Kd,app of the protein-membrane interaction can be determined. This technique can be applied to other picornaviral proteins to determine if PI4P binding is a concerted theme among Picornaviruses. If so, the PI-binding site may represent a target for the development of pan-picornaviral therapeutics.

PIP-on-a-chip assay: A microfluidics based approach to study viral protein – membrane interactions

255th National Meeting (2018)

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