Identification of Critical Molecular Determinants of West Nile Virus PrM Protein: A Potential Site for Antiviral Targeting

get. This involves both oligomerization of the capsid (C) protein and packaging of the viral RNA. The RNA binding properties of the C protein is poorly understood. This study aims to characterize the C and RNA interaction. Method: Co-localization study was performed by transfecting in vitro transcribed WN virus RNA and C protein clones into BHK cells and visualized under fluorescence microscopy. RNA binding properties of C protein were further investigated with a Northwestern Blot assay and RNA pull-down assay. Synthesized C protein peptides were used to map out the RNA binding regions on C protein. In addition, C protein immunopurified from BHK cells were used to investigate the effect of phosphorylation of C protein on its RNA binding properties. Results: RNA and C protein have failed to show colocalization in BHK cells by immuno-fluorescence but interactions were observed at the molecular level. It showed that the first 465 and last 693 nucleotides of the WN virus RNA had specific affinity for the full length C protein. In addition, the aminoand carboxy-terminal of the C protein were shown to bind to the virus RNA. It was also found that the C protein had affinity for viral anti-sense RNA. Phosphorylated peptides of C protein and C protein expressed in BHK cells show attenuated binding to viral RNA. Conclusion: C protein interaction with anti-sense indicates that its interaction with viral sense RNA may not be specific. Phosphorylation of C protein could play a role in regulating C and RNA interaction and allow time for viral assembly. Understanding the interaction of C protein with viral RNA and role of phosphorylation in nucleocapsid assembly could help develop anti-viral strategies aimed at disrupting viral assembly.