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

Abstract

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer−dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV assembly.

Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation

“…The effect of mutations in the alpha-helical region were assessed for dimer and octamer abundance. As expected from the computational predictions and previously published work (Narkhede et al, 2023), T112A greatly reduced dimer formation and increased octamer abundance while L117A lead to near exclusive octamer formation with a small potential monomeric peak at 78-80 mL (Figure 5). In agreement with the computational predictions, F108A, D109A, and M116A extensively reduced dimer stability increasing octamer formation (Figure 5).…”

“…Most mutations tested prevented or reduced PM localization including R52A, I54A, A55L, D56K, I59A, D60A, and H61A (Figure 6). R52A and H61A were previously shown to inhibit VP40 dimer formation in vitro (Narkhede et al, 2023) and predicted to be destabilizing by computational studies presented above. The results for I54A, D56K, and I59A were consistent with computational predictions and size exclusion chromatography.…”

“…In contrast, interactions with PI(4,5)P 2 lead to dramatic reductions in deuterium incorporation in several regions of VP40 as shown in Figure 7b. Reductions in deuterium incorporation are typically due to protein-protein interactions (Parson et al, 2022), lipid-protein interactions (Vadas & Burke, 2015), conformational changes that alter the accessibility of regions/residues, or changes in secondary structure (Narang et al, 2020). Notably, cationic residues in two loop regions that were previously shown to interact with the anionic lipid membranes (del Vecchio et al, 2018;Johnson et al, 2024) had a reduction in deuterium incorporation (Figure 7b).…”

“…I G U R E 2 Hydrogen-bonding patterns for residues Arg52 (a), His61 (b), Asp109 (c), Ser110 (d), and Thr112 (e) from chain A (green) or chain D (blue). Adapted fromNarkhede et al, (2023).the contacts between Asp109 and Ser110. Residues involved in hydrogen bonding at the dimer interface are highlighted in the right panel of Figure3c.…”

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

“…VP40 harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) ( 19 , 20 ). NTD interactions (predominantly through an alpha-helical interface) are responsible for the formation and stability of the VP40 dimer ( 8 , 21 ) in the cytoplasm, and CTD interactions are important for trafficking of the dimer ( 22 , 23 ) and binding anionic PM lipids ( 19 , 20 , 24 ). VP40 binds to anionic lipids in the PM inner leaflet via electrostatic interactions to hijack the host membrane and form the viral lipid envelope ( 10 , 12 , 14 , 20 , 24 , 25 ).…”