Secondary structure and membrane topology of dengue virus NS4B N-terminal 125 amino acids

The transmembrane NS4B protein of dengue virus (DENV) is a validated antiviral target that plays important roles in viral replication and invasion of innate immune response. The first 125 amino acids of DENV NS4B are sufficient for inhibition of alpha/beta interferon signaling. Resistance mutations to NS4B inhibitors are all mapped to the first 125 amino acids. In this study, we expressed and purified a protein representing the first 125 amino acids of NS4B (NS4B(1-125)). This recombinant NS4B(1-125) protein was reconstituted into detergent micelles. Solution NMR spectroscopy demonstrated that there are five helices (alpha 1 to alpha 5) present in NS4B(1-125). Dynamic studies, together with a paramagnetic relaxation enhancement experiment demonstrated that four helices, alpha 2, alpha 3 alpha 4, and alpha 5 are embedded in the detergent micelles. Comparison of wild type and V63I mutant (a mutation that confers resistance to NS4B inhibitor) NS4B(1-125) proteins demonstrated that V63I mutation did not cause significant conformational changes, however, V63 may have a molecular interaction with residues in the alpha 5 transmembrane domain under certain conditions. The structural and dynamic information obtained in study is helpful to understand the structure and function of NS4B. (C) 2015 Elsevier B.V. All rights reserved.