Targeting Lipid Metabolism to Inhibit Ebola VP40 Mediated Viral Budding

In 2014, an international public health emergency was declared when an Ebola virus outbreak claimed the lives of thousands, resulting in the most fatal, widespread and prolonged outbreak of Ebola virus recorded in history. Although 40 years have passed since the initial outbreak, numerous vaccines and post‐exposure therapeutics have entered phase I, II and III clinical trials, yet all have failed to gain FDA approval. Clinical case studies have highlighted that a measurable immune response is the key difference between fatal cases and survivors of Ebola infections. Therefore slowing down viral spread through inhibition of viral budding or intracellular viral replication could be the key to developing an efficacious treatment. The Ebola virus is a filamentous, lipid enveloped filovirus, which acquires its lipid coat when budding from the host cell plasma membrane (PM). The viral matrix protein VP40 is the driving force of viral budding. Strikingly, independent expression of VP40 in mammalian cells leads to the production of virus like particles (VLPs) which are nearly indistinguishable from authentic virions. This project investigates strategies to exploit host‐virus lipid‐protein interactions to hinder viral egress. Initial association between VP40 and the PM has been attributed to phosphatidyl serine (PS), therefore reducing cellular levels of PS could slow down viral egress. Moreover, once at the plasma membrane significant conformational changes within VP40 are hypothesized to aid in generating negative curvature of the PM, a fundamental prerequisite to viral budding. Phosphatidylinositol‐4,5‐bisphosphate (PIP 2 ) has been shown to cluster around filamentous VP40. Additionally, PIP 2 is a necessary cofactor for the phospholipase D (PLD) cleavage of phosphatidyl choline to produce phosphatidic acid (PA). Clustering of PIP 2 as well as the intrinsic properties of PA both favor negative curvature. Moreover, preliminary data suggests that activating PLD through treatment of phorbol 12‐myristate 13‐acetate (PMA) in cells expressing VP40 enhanced VLP formation. Therefore, our goal is to reduce PA levels through PLD inhibition, in order to reduce negative curvature at sites of viral egress and slow down VLP egress. Previous research and preliminary work in the Stahelin lab suggests that two FDA approved drugs, Fendiline and Amitriptyline reduce levels of PS within the plasma membrane. Additionally, preliminary data from the Stahelin lab has shown that small molecule inhibitors specific for PLD‐1 and PLD‐2, as well as a mixed inhibitor FIPI, reduces levels of PA at the PM in PMA treated cells. Each of the proposed compounds have displayed minimal toxicity in cells, at concentrations up to 50 uM. Therefore, live cell imaging to measure VP40 association with the PM and VLP budding efficiency assays will be performed to determine if treatment of cells expressing VP40 with either Fendiline, Amitriptyline, or PLD inhibitor have a significant effect on VLP formation. Support or Funding Information This work has been supported by NIH training grant T32GM075762