Identification and characterization of a 43Â kDa actin protein involved in the DENV-2 binding and infection of ECV304 cells

Characterization of the primary host factors associated with host-virus interaction is critical for understanding how a virus infects its host cell. In this study, a modified virus overlay protein binding assay was developed. Host factors with 34, 43, and 55 kDa proteins, which could interact with EDIII, a cell receptor-binding domain of Dengue virus (DENV)-enveloped E protein, were isolated from ECV304 cells. Mass spectrometry identified peptide masses of 43 kDa protein matched to actin, a cytoskeleton protein in eukaryotic cells. The interaction between 43 kDa actin and DENV-2 EDIII was further confirmed by competitive blocking and co-immunoprecipitation assays. Actin cytoskeleton rearrangement was observed within 1 h p.i. of DENV-2-infected ECV304 cells in the confocal immunofluorescent assay. The co-localization of DENV-2 E protein with the actin filaments occurred in the late stage of the DENV replication cycle. Finally, a docking complex was constructed, and the functional residues involved in the interaction of actin and DENV-2 EDIII protein were predicted. Our findings suggest that the direct contact of DENV E protein with 43 kDa actin protein may have a crucial function in DENV infection of ECV304 cells.