Objectives: Characterization of the primary host factors associated with host-virus interaction is critical in understanding how a virus infects its host cell.Methods and Results: 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 the Dengue virus (DENV)-enveloped E protein, were isolated from ECV304 cells.Using mass spectrometry, peptide masses of the 43 kDa protein matched to actin, a cytoskeleton protein in eukaryotic cells, were identified.The interaction between the 43 kDa actin and Dengue virus serotype 2 (DENV-2) EDⅢ was further confirmed by competitive blocking assay and co-immunoprecipitation experiment using both self-made anti-43 kDa murine polyclonal sera and commercial mouse anti-actin polyclonal antibodies.Actin cytoskeleton rearrangement was observed remarkably 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, the docking models and functional residues of actin and DENV-2 EDⅢ protein were predicted by bioinformatics techniques.Conclusions: The present findings suggest that the direct contact of DENV E protein and the 43 kDa actin protein may play a crucial role in the DENV infection of ECV304 cells.Thus, the predicted models and functional residues were proposed.