Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis

Abstract

Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30-eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30-eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.

Figure 1. eVP30 binds eNP with high specificity.

(a) Constructs of eNP (top) and eVP30 (bottom) used in this study. (b) Summary of in vitro pull-down-based binding results for a series of truncation constructs for eNP. +, binding; −, no binding. (c) Size exclusion chromatography coupled to multi-angle light scattering results for eVP30_110–272 (black; molecular weight (MW) 38.7±0.030 kDa), eNP_600–739 (red; MW 16.8±0.030 kDa) and eVP30_110–272-eNP_600–739 complex (green; MW 39.6±0.030 kDa). The calculated MW for monomeric eVP30_110–272, dimeric eVP30_110–272 and eNP_600–739 are 18.7, 37.3 and 17.03 kDa, respectively. The calculated MW for a 1:1 complex of eVP30_110–272–eNP_600–739 is 35.7 kDa. (d) Representative ITC binding isotherm for the 1:1 complex between eVP30_110–272 and eNP_600–739. K_D=3.75±1.7 μM. (Also see Supplementary Fig. 1). Error represents s.d.

Figure 2. A peptide derived from eNP binds eVP30 using the same interface despite different VP30 complexes in the asymmetric unit.

Cartoon (top) and surface (bottom) representations of the following complexes are shown: (a) 2:2 ratio of eVP30 (green) and eNP (magenta). (b) 4:4 ratio of eVP30 (green) and eNP (magenta). (Also see Supplementary Fig. 1).

Figure 3. eNP binds within a highly conserved narrow groove within eVP30 via a combination of hydrophobic and electrostatic interactions.

(a) eVP30 residues (green) involved in interactions with eNP are shown in stick representation (blue) and labelled. The eNP peptide is shown in stick representation (purple). (b) Stick representation of the eNP peptide residues 602–612 (purple) with a 2σ electron density map (sigma-weighted 2Fo-Fc; blue mesh). Orientation is 45° rotated from the orientation in a. (c) LigPlot⁺ representation of the protein–protein interactions between eVP30 (green) and eNP (purple). Protein side chains are shown as ball and sticks. Hydrogen bonds are shown as green dotted lines. Spoked arcs represent non-bonded contacts and the length of the arc represents the extent of the interaction. (Also see Supplementary Fig. 3).

Figure 4. Effects of eNP interface residue mutations on eVP30 binding.

(a) FP competition assay measuring binding between eNP_590–739 WT and mutants and FITC-labelled eVP30BP. Error bars represent s.d. of at least three experiments. (b) Immunoprecipitation (top) and corresponding western blots (bottom) of mutant HA-eNP with WT Flag-eVP30. (c) FITC–eVP30BP binding with WT or mutant eVP30_110–272 measured by FP. Error bars represent s.d. of at least three experiments. (d,e) Co-immunoprecipitation of WT HA-eNP full length with WT or mutant FLAG-eVP30 full length. IP, immunoprecipitation; IB, immunoblot; WCE, whole-cell extract; E, empty vector.

Figure 5. Interaction between eVP30 and eNP observed in the crystal structure is not strictly required for viral RNA synthesis.

MGA was performed using (a) WT and select interface mutants of eVP30 (50, 100, 200 ng) and (b) WT and select interface mutants of eNP (125, 250, 500 ng) (western blots represent the expression of eNP at 500 ng). Representative western blots are shown. Errors represent s.d. of at least three experiments.

Figure 6. Additional residues at the eVP30 and eNP interface may be important for viral RNA synthesis and transcription.

(a) VP30 interface mutants with WT (dark grey bar) or stem-loop 5′UTR mutant MG template (light grey bar). (b) The MG assay was performed in the presence of either GFP control plasmid or plasmid consisting of GFP-eVP30BP (used at 50, 500 ng). The effect of GFP-eVP30BP (500 ng) was examined on the WT-MG. Representative western blots are shown. Errors represent s.d. of at least three experiments.

Figure 7. eVP30BP inhibits VP30-dependent viral RNA synthesis.

(a) WT MG or 5′UTR mutant MG template (b) in the presence of VP30 interface mutants. The corresponding western blots for each MG assay is shown below each chart. (Also see Supplementary Fig. 4). The MG data and representative western blots are shown. Errors represent s.d. of at least three experiments.