Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus of the Flaviviridae family. DENV is the causative agent of dengue fever disease, currently the world's fastest-spreading tropical disease. Dengue fever is a painful, debilitating mosquito-borne disease caused by any one of four closely related dengue viruses. Symptoms include sudden, high fever and severe headaches, and complications may progress to massive bleeding, shock, and death. At present, no specific treatments for Dengue infections are known. One crucial step in the viral life cycle is the amplification of the viral RNA genome in specialized replication complexes (RCs) that are localized inside invaginated vesicles at the endoplasmatic reticulum (ER). However, up to now the details of this RC assembly are only incompletely understood.
DENV non-structural protein 4A (NS4A) is an ER-localized membrane protein and essential component of the viral replication complex. NS4A may be involved in host membrane alterations resembling the virus-induced membrane structures. We confirmed the existence of a predicted amphipathic helix (AH) in the N-terminus of NS4A and demonstrated its ability to bind membranes. Our joint publication with the Sklan lab emphasizes the significance of NS4A's AH in the DENV life cycle and demarcate it as a target for novel anti-viral therapies. Our studies have been funded by a grant of MOI I.
Structure-function analysis of Dengue virus non-structural protein NS4A
Since antiviral drugs based on AH interfering small compounds have been successfully introduced for the treatment of the closely related hepatitis C virus, a similar approach might hold promise also for treating Dengue fever. Structural studies might represent a starting point for the development of such compounds. DENV NS4A is a protein with a conserved amphipathic helix within its N-terminal region. We will perform a detailed structural analysis of the full length NS4A protein using cell-free protein expression and co-translational insertion into model membranes such as nanodiscs or bicelles. Direct insertion into lipid membranes avoids potentially harmful non-native purification steps and detergent exposure. Subsequently, NMR spectra will be recorded followed by structure determination. Our goal is the identification/verification of structural elements within full-length NS4A that contribute to the two known activities of NS4A: membrane modification and oligomerization. Similar studies are planned for NS5 as it also is suggested to have conserved AH regions.
This project is carried out in tight collaboration with Bernd König. Cell functional studies will be done by the Sklan lab at Tel Aviv University.
Initial results were recently presented at the 59. Ann. Meeting of the Biophys. Soc., February 7 - 11, Baltimore, MD, USA:
Y.-F. Hung, M. Schwarten, S. Hoffmann, D. Willbold, E. Sklan and B. W. Koenig. 2015.
Cytoplasmic domain of Dengue virus protein NS4A preferentially binds highly curved