Structure and membrane interaction of the N-terminal region of Dengue virus NS4A protein

Abstract

Dengue virus (DENV) infection presents a serious public health threat with more than one third of the world population at risk. 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. However, the details of the assembly of this replication complex are incompletely understood. We focused on the DENV non-structural protein 4A (NS4A) which has been implicated in the formation of the viral RNA replication complex. Sequence analysis identified conserved regions in the N-terminal 48 amino acids of NS4A that might form amphipathic helices (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization [1]. However, little is known about the three dimensional structure of NS4A(1-48). We used solution state nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to study the structure of wild type NS4A(1-48) and of the double mutant NS4A(1-48, L6E;M10E) in the presence and absence of model membranes. The hydrodynamic radius of liposomes and detergent micelles was determined by dynamic light scattering (DLS). Both peptides were recombinantly produced in E.coli and are basically unstructured in aqueous buffer [2]. Addition of liposomes made of POPC or POPC/DOPS mixtures induced formation of α-helical secondary structure in case of the wild type NS4A(1-48) but not for the mutant peptide. The degree of helicity of wt NS4A(1-48) is sensitive to the lipid composition and to the size of the liposomes. Formation of α-helical secondary structure was observed for both wt and mutant NS4A(1-48) upon addition of various membrane mimicking detergent micelles (SDS, DPC, DHPC, DM). The degree of helix formation depends on the type and concentration of the detergent and reaches a maximum at about 100 mM SDS or DPC. Solution state NMR spectroscopy provided a detailed picture of the structure and micelle interaction for both peptides in presence of 100 mM SDS. Backbone resonance assignment followed by analysis of secondary chemical shifts allowed us to identify two α-helical segments in each peptide which cover amino acid residues 5-10 and 15-29 in NS4A(1-48) and residues 4-9 and 15-29 in NS4A(1-48, L6E;M10E). Analysis of paramagnetic relaxation enhancement after addition of paramagnetic Mn2+ to the SDS micelle-containing buffer allowed us to distinguish buffer exposed from buried amino acid residues.[1] O. Stern et al. (2013) J. Virol. 87:4080-85[2] Y.F. Hung et al. (2014) PLoS One. 9: e8648