Fig 1: The internal fusion loop of GP is critical for TLR4 activation(A) The pre-treatment of stable hTLR4/MD2/CD14 HEK293 cells with RSLA reduces TLR4 activation by GP and LPS equally, suggesting a similar mechanism. The cells were pre-incubated with RSLA at concentrations of 100 ng/μL, 300 ng/μL, and 1 μg/μL for 1 h pre-induction, followed by LPS concentrations of 10 and 100 ng/μL and 100 and 500 ng/μL of GP for 6 h, and NF-κB activity was assessed.(B) Sequence analysis of GP according to the Kyte and Doolittle scale for hydrophobicity reveals five regions of high hydrophobicity.(C) Deletion of the internal fusion loop sequence, hit no. 4, in fully glycosylated GP diminished TLR4 activation as evident from NF-κB signaling assays. The cells were incubated with 100 ng/μL LPS and 500 ng/μL GP for 6 h, and activity was assessed. The negative control consisted of purified GP, treated with proteases for 1 h, and was followed by boiling for 20 min.The negative control was set to 1-fold change and the values normalized to it. Statistical significance was assessed using a one-way ANOVA test. Data presented as means of triplicates (A and C).
Fig 2: sGP1,2 binds TLR4/MD2 beyond the LPS binding pocket(A) Protein-protein contacts include residues from both TLR4 chains as well as MD2.(B) Two copies of Ebola GP1/2 glycoproteins crosslink TLR4/MD2 into a signaling competent complex.(C) GP1/2 displays additional contacts outside the hydrophobic cavity of MD2, to which the IFL binds, increasing the interface to also potentially include glycan-mediated contacts.
Fig 3: The IFL of Ebola glycoprotein GP1/2 docks in the LPS binding site of the TLR4/MD2 dimer(A and B) The best docking pose for IFL (A) overlaps perfectly with LPS binding site of MD2 (B). Both are depicted in yellow spheres.(C) Trimeric prefusion GP1,2 as observed in crystal structure 3CSY (Lee et al., 2008).(D) Monomeric sGP1, 2 in the same orientation as observed in the trimer.(E) IFL deletion mutant (marked with asterisk) maintains the overall fold of GP1/2 but is unable to crosslink TLR4/MD2 into a signaling competent complex.(F) TLR4-MD2 can be crosslinked by one or two sGP1,2 molecules (second binding site not represented).(G) Close-up view of the IFL inside MD2 hydrophobic cavity. Structural representations are generated with The PyMOL Molecular Graphics System, v.2.5.2, Schrödinger.
Fig 4: PcrV-induced stimulation of glycolysis augments tumor-associated macrophage (TAM)-mediated tumoricidal activity. To induce TAMs in vitro, bone marrow-derived macrophages (BMDMs) were cultured in 10% FBS/DMEM containing 20% (v/v) LLC cell culture supernatant for 24 h and then primed or not with PcrV (10 μg/ml) for another 24 h For the induction of an M1-like phenotype, TAMs were pulsed with LPS (10 ng/ml) and IFNG (10 ng/ml) for 24 h For the inhibition of glycolysis, TAMs were pretreated with 2-deoxy-d-glucose (2-DG; 2 mM) for 2 h followed by exposure to PcrV for another 24 h (A) Assay for the extracellular acidification rate (ECAR). (B) Measurement of the lactic acid level in the culture supernatant. (C) Assay for ATP levels in cell lysates. (D) The NO level in the culture supernatant was detected using Griess reagent. (E) IL12 p40/70 and TNFA levels in the culture supernatant were analyzed by ELISA. BMDMs pretreated with 2-DG (2 mM, 2 h) were primed with PcrV (10 μg/ml) for 24 h and then co-cultured with LLC cells for another 24 h (F) Detection of NO level in the culture medium. (G, H) The apoptosis of LLC cells was detected by FACS. Data were expressed as means ± SD and analyzed by unpaired Student’s t-test. **p < 0.01 and ***p < 0.001. Oligo, oligomycin; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; LLC, Lewis lung cancer; FACS, fluorescence-activated cell sorting.
Fig 5: Shedding of GP leads to a cascade of inflammatory reactions mediated by TLR4The sequential effects of EBOV infection and the role of GP on the host’s body and immune system are depicted. Following infection, the virus replicates without activating innate immunity. Viral particles are released along with the spike GP, a target for endogenous TACE. After cleavage, shed GP (blue) activates TLR4, leading to local inflammation. The inflammatory reaction causes vascular leakage induced by endothelial cell infection. GP diffuses into the bloodstream, migrates, and activates remote cells from the original site of infection. This system-wide activation leads to the escalating activation of cytokines, formation of a cytokine storm, and finally to often fatal hemorrhagic fever and multiple organ failure.
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