Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

Abstract

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

Keywords: Marburg virus; VP24; antioxidative stress; hydrogen deuterium exchange mass spectrometry; viral subversion.

Figure 1. Dimerization of mVP24 is not required for Keap1 interactions

(A) Elution profiles of MBP-mVP24 on a Superdex 200 column at 3 (black), 6 (navy), 11 (royal blue), 22 (green), 38 (orange), 59 (magenta), 112 (yellow) µM concentrations. (B) SEC-MALS elution profiles of 60 µM MBP-mVP24 WT (purple) and MBP-mVP24_ΔNterm (black) from a Superdex 200 column. The theoretical monomeric molecular masses for MBP-mVP24 WT and MBP-mVP24_ΔNterm are 72.6 and 70.1 kDa, respectively. Molecular weights determined by SEC-MALS of the major peaks of MBP-mVP24 WT and MBP-mVP24_ΔNterm are 146 ± 1 and 78 ± 6 kDa, respectively. The minor peaks of MBP-mVP24 WT and MBP-mVP24_ΔNterm correspond to MBP contaminant and dimeric MBP-mVP24_ΔNterm, respectively. (C) ITC raw data and binding isotherm for MBP-mVP24_ΔNterm bound to Keap1 Kelch. Measured values are K_D = 77 ± 4 nM, ΔH = −2.0 ± 0.06 × 10⁻⁴ cal/mol, ΔS= −37 cal/mol/deg, and n (no. of sites) = 0.90 ± 0.02. (D) HEK293T cells were transfected with ARE luciferase reporter plasmid, a constitutively expressed Renilla luciferase plasmid, and pCAGGS (empty vector) or decreasing concentrations of mVP24 WT or mVP24_ΔNterm. At 18 h posttransfection, luciferase activity was assayed. Western blots of HA and β-tubulin are indicated.

Figure 2. mVP24 binds Keap1 Kelch domain with 1:1 stoichiometry

(A) Domain organization of Nrf2 and Keap1. Numbers and residues corresponding to Nrf2 Neh2 domain from the human species are shown. Regions containing the DLG and ETGE motifs are underlined. Keap1 is composed of N-Terminal Region (NTR), Bric-a-brac Tramtrack Broad-complex (BTB), intervening region (IVR), and Kelch/C-Terminal Region (CTR) domains. (B) Elution profiles of MBP-mVP24 (purple), Keap1 Kelch domain (blue), and MBP-mVP24/Keap1 Kelch domain complex (red) on a Superdex 200 column couple to MALS. Molecular weights determined by SEC-MALS of MBP-mVP24, Keap1 Kelch domain, and MBP-mVP24 WT/Kelch complex are 146 ± 1, 33 ± 3 and 106 ± 2 kDa, respectively. The theoretic monomeric molecular masses for Keap1 Kelch domain and MBP-mVP24 are 33.3 and 72.6 kDa, respectively. (C) Native mass spectra. The colored dots denote peaks for MBP-mVP24 monomer (green, +17 to 14), Keap1 Kelch domain (blue, +11 to 9), and MBP-mVP24/Keap1 Kelch domain complex (red, +21 to 16) corresponding to calculated MWs of 73, 34, and 106 kDa, respectively. Peaks surrounded by a blue box denotes peaks arising from the mVP24 dimer.

Figure 3. HDX-MS identifies solvent accessible regions within mVP24 and Keap1 Kelch domain

Cartoon representations of (A) Phyre2 model of mVP24 threaded with PDB 4OR8 and (B) Keap1 Kelch domain structure (PDB 1U6D). The percentage of deuterium incorporation for selected peptides of (C) mVP24 and (D) Kelch are mapped onto the structures for five incubation times (10 s, 1 min, 6 min, 15 min, 4 h); the color code legend is shown to the right of panels (C)-(D). Sequences that could not be detected by HDX-MS are colored in gray.

Figure 4. The molecular interface between mVP24 and Keap1 Kelch domain is defined by HDX-MS

(A) Differences in deuterium uptake induced by Kelch binding are displayed as a color gradient for each peptide at the indicated time points. The color code (see legend) represents the differential HDX; regions colored in white are not detected by HDX. (B) Comparison of deuterium uptake curves of mVP24 (black) and mVP24-Kelch complex (red). (C) Important binding regions are highlighted in the cartoon representation of mVP24. The color gradient represents the average amount of deuterium uptake of mVP24-Kelch complex subtracted from that of free mVP24 and is the same as in (A). (D) Differences in deuterium uptake induced by mVP24 (left column) and ETGE peptide (right column) binding are displayed as a color gradient for each peptide at the indicated time points. The color code (see legend) represents the differential HDX; regions colored in white are not detected by HDX. (E) Comparison of deuterium uptake curves of Kelch upon binding with mVP24 (top row, black and red curve) and ETGE peptide (bottom row, blue and purple curve), respectively. The average amount of deuterium uptake of (F) mVP24-Kelch and (G) Kelch-Nrf2 ETGE peptide complexes were subtracted from that of free Kelch and mapped onto the crystal structure of Kelch. Coloring follows the legend in (D). mVP24 and Keap1 Kelch structures have the same orientation as Fig. 3A and Fig. 3B, respectively.

Figure 5. Conserved cysteine residues within the K-Loop are critical for Kelch binding

(A) Representative ITC data for Keap1 Kelch binding to mVP24 K-Loop (K_D = not determined). Representative ITC data for Keap1 kelch binding to (B) MBP-mVP24_ΔNterm C209M/C210K (K_D = not determined) (C) MBP-mVP24_ΔNterm C209A/C210A (K_D = 550 ± 26 nM, ΔH = −1.45 ± 0.07E4 cal/mol, ΔS = −20.1 cal/mol/deg, and n = 0.850 ± 0.03) and (D) MBP-mVP24_ΔNterm G211A/E212A (K_D = not determined). Representative raw heats of reaction versus time (top panel) and the integrated heats of reaction versus molar ratio of ligand to receptor (bottom panels) are shown. (E) HEK293T cells were transfected with ARE luciferase reporter plasmid, a constitutively expressed Renilla luciferase plasmid, and pCAGGS (empty vector) or increasing concentrations of mVP24 WT, mVP24 C209A/C210A, or mVP24 C209M/C210K. At 18 h posttransfection, luciferase activity was assayed. Western blots of HA and β-tubulin are indicated.

Figure 6. NEM labeling reveals differential labeling of the mVP24 K-Loop

(A) mVP24 and (B) mVP24/Kelch complex at different labeling times. The relative fraction of unmodified mVP24 (black dots), + 1 NEM adduct (blue dots) and +2 NEM adduct (red dots) were calculated as the ratio of the intensity of each species to the sum of intensities of all labeling products. The sums of intensities of different NEM adducts of mVP24 were normalized to 1.