The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold

Abstract

Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

Figure 1. Purified truncated VP24s, SUDV_1–233 and EBOV_1–233, were determined to bind to purified STAT1_1–683 using an ELISA assay.

Either SUDV_1–233 or EBOV_1–233 was coated onto the ELISA plate at 0.01 mg/ml as described in the Materials and methods section. Upon subsequent incubation with STAT1_1–683, binding was detected with HRP conjugated secondary antibody and O.D. was read at 450 nm. BSA was used as a negative control.

Figure 2. Alternate views of VP24 secondary structure.

The overall shape of VP24 resembles a three-sided pyramid with Faces 1 (a), 2 (b), and 3 (c) as illustrated. Only SUDV_1–233 is shown for clarity. Arrows indicate conserved pockets on Faces 1 and 3.

Figure 3. Conservation map of VP24.

(a) Sequence conservation in VP24 among Ebola (Zaire), Sudan, Reston, Taï Forest, and Bundibugyo viruses mapped onto the structure of SUDV_1–233. (b) Sequence conservation in VP24 between ebola- and marburgviruses. Sequence conservation is mapped as navy (completely conserved) to red (least conserved). A hydrophobic cavity in Face 1 and a polar cavity in Face 3 are indicated by arrows. Least conserved regions are clustered around Face 2. Sequence identity is calculated using Homolmapper . Figures are illustrated using SUDV_1–233.

Figure 4. Key sites of VP24.

(a) The hydrophobic cavity at the base of Face 1. Leucines 57, 75, 79, 198, and 221 are completely conserved across the filoviruses. Electrostatic surface calculated with APBS is shown inset, with Y172 and M71 drawn in magenta. (b) Polar cavity at the base of Face 3. Residues Y186 and A187 are conserved across all ebolaviruses except SUDV. Residues P77, T193, K206, and M209 are conserved among ebolaviruses and the remaining residues are conserved across both ebola- and marburgviruses. Inset is calculated with APBS. Color scale ranges from −6kT/z (bright red) to +6kT/z (dark blue). (c) Residues important for virulence are colored navy; residues important for binding karyopherin 〈1 are colored cyan. Figures are illustrated using SUDV_1–233.

Figure 5. Results of deuterium exchange mass spectrometry analysis of SUDV_1–233 in complex with STAT1_1–683.

(a) Exchange results are mapped onto SUDV_1–233. Peptidic segments colored blue exhibit slowing of H/D exchange kinetics, while segments colored red exhibit an increase in H/D exchange kinetics. Segments colored white exhibit no change. Segments colored grey were not observed in these experiments. (b) Conservation map of SUDV_1–233. Circles indicate sites of change in H/D exchange upon binding to STAT1_1–683. Green arrows denote the position of the Face 3 cavity. Deuterium exchange mass spectrometry of (c) SUDV_1–233 alone and (d) in complex with STAT1_1–683 illustrated as sequence representations. Each block indicates peptidic regions defined by overlapping peptides and is consist of three time points (10 s, 100 s, and 1000 s). Deuteration levels at each time point are illustrated as blue (<10% deuteration) to red (>90% deuteration). Grey indicates residues that are not observed. Box indicates regions of either speeding up or slowing down of deuteration upon binding to STAT1_1–683. The average standard deviation of deuterium incorporation is 1.5% between replicates.