The tissue tropism of a virus is a key determinant of viral pathogenicity which is often modulated by the presence or absence of appropriate molecules on the surface of a host cell that can be used by the virus to gain entry into that cell. Annexin II was seen to interact with dengue virus (DENV) and enhanced infection. Herein, we aimed to explore this interaction as a potential target for the design of anti-DENV therapeutics. We demonstrated annexin II extracellular translocation in Vero cells upon exposure to DENV, extracellular and intracellular colocalization assays as well as co-immunoprecipitation assay were performed to further confirmed protein interaction. Molecular docking and molecular dynamic (MD) simulation were employed to identify the interaction sites. The result showed extracellular translocation of annexin II upon DENV exposure to the cell, the result further showed annexin II colocalizing with DENV E-glycoprotein extracellularly and intracellularly. Furthermore, the result of co-immunoprecipitation assay shows DENV E glycoprotein pulling down annexin II, and the result of molecular docking showed strong interaction between the two proteins. MD simulations has proposed the binding of two regions of annexin II (i) Y274-K280 and (ii) K369-Q327 with BR3 E glycoprotein of DENV2 (residue 380-389), with potential of infections abrogation upon inhibition.

Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production relies on the polymerization of Gag protein at the inner leaflet of the plasma membrane. We previously generated an ankyrin repeat protein (Ank1D4) that specifically interacts with the CAp24 protein. This study aimed to improve the binding activity of Ank1D4 by generating two platforms for the Ank1D4 dimer. The design of these constructs featured a distinct orientation of monomeric Ank1D4 connected by a linker peptide (G 4S) 4. The binding surfaces in either dimer generated from the C-terminus of the Ank1D4 monomer linked with the N-terminus of another monomer (Ank1D4 NC-NC) or its inverted form (Ank1D4 NC-CN), similar to monomeric Ank1D4. The interaction of Ank1D4 NC-CN with CAp24 from capture ELISA was significantly greater than that of Ank1D4 NC-NC and the parental Ank1D4. The bifunctional characteristic of Ank1D4 NC-CN was further demonstrated using sandwich ELISA. The binding kinetics of these ankyrins were evaluated using bio-layer interferometry analysis. The K D of Ank1D4 NC-CN, Ank1D4 NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively. The dynamics of the interdomain linker and the behavior of ankyrin dimers were investigated in silico. Upon the binding distance calculation from the candidate structures, the achievement in obtaining double active sites is more possible in Ank1D4 NC-CN. The CD spectroscopic data indicated that secondary structure of dimer forms resemble Ank1D4 monomer α-helical content. This finding confers the strategy to generate dimer from rigid scaffold for acquiring the binding avidity.