This grant seeks to identify liver cell expressed dengue virus receptor protein(s) using affinity column chromatography. In earlier work we have used the virus overlay protein binding assay (VOPBA) technique to identify two dengue virus receptor proteins. VOPBA relies upon the electrophoretic separation of membrane proteins through an SDS-PAGE gel and transfer to a solid matrix support followed by hybridization (overlay) with the dengue virus. Virus protein bands can be visualized by subsequent analysis with a dengue specific antibody. This methodology works relatively well, probably based upon a degree of protein re-naturation during the hybridization process, but suffers from the primary disadvantage that the electrophoretic separation of the proteins would preclude any binding that results from a complex of several proteins. As such, the binding of the dengue virus to the membrane proteins is in a less physiologically relevant system.
To isolate dengue receptor proteins under physiologically relevant conditions, this grant will employ affinity column chromatography. Affinity chromatography is a method of selectively and reversibly binding proteins to a solid matrix based on the exploitation of known affinities between molecules. The interaction between the molecules (in this case the dengue virus and its cognate receptor protein or proteins) is not based upon general properties of the molecules in question, but rather relies upon individual structural properties of the molecules leading to a specific binding.
In this specific case, biologically active (infectious) dengue virus will be bound to a sepharose matrix using cyanogen bromide activated cross linking. This matrix will be used to specifically bind dengue virus binding proteins prepared from membrane fractions of liver (HepG2) cells under low salt conditions. The remaining proteins that are not able to bind dengue virus proteins will be eluted from the column under low salt conditions. Specific dengue virus binding proteins will be eluted from the column under increasingly high salt concentrations and analyzed by SDS-PAGE. Purified dengue virus binding proteins will be analyzed by peptide mass finger printing and their role as receptor proteins confirmed by a combination of natural ligand inhibition (where possible) or antibody mediated interference of infection as previously used in our earlier studies. This methodology should enable the identification of receptor molecules previously identified by VOPBA (and as such will act as a positive internal control) as well as novel proteins that either do not bind to the denatured proteins used in the VOPBA analysis, or proteins that act together as a receptor complex to
