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N-terminal region of DENV NS4A binds highly curved membranes

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.

 

 

This project is an ongoing cooperation with the lab of undefinedElla H. Sklan from Tel Aviv University and the teams undefinedHoffmann and undefinedSchwarten.

 

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