Dengue virus (DENV), the causative agent of dengue fever, is responsible for millions of disease cases and thousands of deaths annually. Non-structural protein 4A (NS4A) plays a major role in the formation of host cell membrane-derived structures that provide a crucial scaffold for virus replication. Two transmembrane helices anchor the mature NS4A to the membrane. The N-terminal 48 amino acids of NS4A reside in the cytoplasm, contain two amphipathic helices, and may serve as a second membrane anchor of NS4A. NS4A(1-48) selectively binds to highly curved, small liposomes. Two point mutations L6E;M10E in NS4A, which inhibit DENV replication, do strongly reduce binding of NS4A(1-48) to small liposomes.
We studied the structure of recombinant NS4A(1-48) and NS4A(1-48, L6E;M10E) in SDS micelles and the interaction of the two peptides with liposomes. Both peptides are unstructured in buffer and form two amphipathic helices in SDS micelles. However, only wild type NS4A(1-48) shows helix formation in presence of liposomes. In addition, binding of NS4A(1-48) to liposomes requires high membrane curvature (hydrodynamic radius of ca. 26 nm). No liposome binding was observed for the mutant. Solution NMR data suggest that the N-terminal amphipathic helix in NS4A(1-48) is the major lipid binding site.
Liposome floatation assay. Alexa Fluor 488-labeled wild type (A) or mutant NS4A(1-48) (B), or free Alexa 488 dye (C) was loaded together with small, sonicated liposomes in the 35% sucrose layer. Only wt NS4A(1-48) is found in the liposome containing low density layer on top of the gradient after 14 h of centrifugation.
- Hung et al. (2014) PLoS One 9:e86482
- Hung et al. (2015) Biochim Biophys Acta 1848:1119-26
- Hung et al. (2015) Viruses 7:4119-30