Cytoplasmic Domain of Dengue Virus Protein NS4A Preferentially Binds Highly Curved Membranes

Abstract

Dengue virus (DENV) is a mosquito-transmitted virus that causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. There is no vaccine available against DENV and no specific treatment for dengue fever. DENV is believed to replicate its RNA genome in association with modified intracellular membranes. DENV non-structural protein 4A (NS4A) has been implicated in the formation of the viral RNA replication complex (RC). However, the details of RC assembly are incompletely understood. We have previously identified a conserved region in the N-terminal 48 amino acids of NS4A containing putative amphipathic helices (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization [1]. Solution state NMR spectroscopy was used to study the structure of recombinant wild type NS4A a.a. 1-48 peptide and a double mutant NS4A(1-48, L6E;M10E) peptide in the presence of membrane mimicking SDS micelles. The peptides are basically unstructured in aqueous buffer. However, two α-helical segments separated by a non-helical linker are observed for both peptides in presence of SDS micelles. Addition of liposomes induced formation of α-helical secondary structure in the wild type NS4A(1-48) but not in the mutant peptide. We used surface plasmon resonance, floatation assays, and circular dichroism spectroscopy to analyze the binding of recombinant NS4A(1-48) peptides to liposomes. We found that NS4A(1-48) binds to liposomes in a membrane curvature-dependent manner. The AH mutations reduced the affinity of NS4A(1-48) for lipid membranes. These results suggest that the two AHs in the N-terminus of NS4A may be crucial for membrane binding, curvature sensing and stabilization. Better understanding of the molecular details of the DENV RC formation might lead to novel anti-DENV strategies.[1] O. Stern et al. (2013) J. Virol. 87:4080-85