Anchoring Energy of Dengue E Protein into Host Membranes and Quaternary Assembly Depend upon Lipid Composition

Dengue virus is a devastating human pathogen responsible for millions of infections each year. No antiviral therapies for Dengue currently exist, making effective treatment of the virus challenging. Dengue is taken into the cell through endocytosis. Low-pH mediated structural rearrangements of the envelope protein E leads to the formation of fusogenic E trimers that facilitate membrane fusion with late endosomes. The fusion mechanism is not fully understood, but poses as a key target for inhibiting the viral infection pathway. An important aspect of fusion is the requirement of anionic lipids in the endosomal membrane. This study aims to characterize the biophysical reasons for this dependence by examining the role of anionic lipids in anchoring E to the membrane. E anchoring was investigated by sucrose gradient coflotation. We discuss the results of coflotation studies that were used to probe the unbinding of E trimers from liposomes of various compositions. We showed that E protein became unbound from liposomes over time when the membrane lacked anionic lipids, but remained bound to liposomes containing anionic lipids. This demonstrates that anionic lipids facilitate greater anchoring of E to the membrane, which is essential for successful membrane fusion. Coflotation is commonly used to assess protein binding to membranes, but in this study coflotation is utilized as a novel methodology to probe weakly associated protein-membrane unbinding. To our knowledge, this is the first method of its kind. In addition, we also begin to investigate the role of anionic lipids in facilitating oligomerization of E trimers. We present preliminary results from sucrose gradient sedimentation and chemical crosslinking studies comparing trimer formation in the presence of liposomes with and without anionic lipids. Though further refinement of the trimer detection assays is necessary, sedimentation results suggest that trimerization occurred to a greater extent in the presence of anionic lipids. Taken together, our results reveal important functions of anionic lipids in facilitating membrane fusion useful for development of new therapeutic approaches.Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000