Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

Abstract

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid‐enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA‐approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline‐treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Elucidating Residue-Level Determinants Affecting Dimerization of Ebola Virus Matrix Protein Using High-Throughput Site Saturation Mutagenesis and Biophysical Approaches

“…Along with WT VP40, these two mutants were expressed and purified from E. coli as previously described , and assessed for the VP40 dimer and octamer formation using an established size exclusion chromatography assay. ,, As shown in Figure , WT VP40 displayed the typical dimer and octamer peaks as shown for E. coli produced WT VP40. , In agreement with the computational predictions, the R52A and H61A mutants exhibited little detectable dimer formation and increased the relative levels of the VP40 octamer, a stable VP40 oligomer that forms when the N-terminal domain α-helical interface is disturbed, validating the calculations.…”

“…Mutations of VP40 were prepared by Gene Universal (Newark, DE) and verified by DNA sequencing. VP40 was expressed and purified as previously described in Escherichia coli in order to assess the VP40 dimer and octamer formation in vitro for WT VP40, Arg52Ala, and His61Ala. In brief, WT or eVP40 mutations were expressed and purified from RosettaTM 2 BL21 DE3 competent cells (Merck Millipore, Billerica, MA) using Ni-NTA affinity chromatography.…”

“…To assess the computational predictions on the VP40 dimer formation, we prepared two representative mutations of VP40 (R52A and H61A). Along with WT VP40, these two mutants were expressed and purified from E. coli as previously described 72,73 and assessed for the VP40 dimer and octamer formation using an established size exclusion chromatography assay. 9,25,73 As shown in Figure 7, WT VP40 displayed the typical dimer and octamer peaks as shown for E. coli produced WT VP40.…”

Minor changes in electrostatics robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles

“…To test this hypothesis, we prepared several mutations in this loop region: G198R, G198A, G198D, and G201R. Localization of WT VP40 and mutations in cells was assessed using a well-established EGFP-VP40 PM localization assay using confocal microscopy (26, 29). WT VP40 localized predominantly to the PM 24 hours post-transfection and formed extensions indicative of pre-assembled VLPs (10).…”

“…The VP40 dimer traffics to the inner leaflet of the PM (8) where it interacts with the PM through key lysine residues (especially Lys 221 , Lys 224 , Lys 225 , Lys 274 , and Lys 275 ) in its C-terminal domain (CTD) (8, 26). Once there, electrostatic interactions trigger the rearrangement of VP40 dimers into hexamers and larger oligomers (27–29), which construct a filamentous matrix structure that is critical for budding (11). The binding of VP40 to the PM is mediated by the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) in the membrane.…”