Fig 1.
Conservation of NP and VP30 interactions.
A) Sequences of filovirus NP 600–617 (EBOV numbering) were aligned with Clustal Omega [19]. Residues identical to EBOV are colored green and similar residues are in yellow. B) Sequences of filovirus VP30 C-terminal region were aligned with Clustal Omega [19]. Those amino acids of the VP30 CTD that are visible in the EBOV VP30-NP complex crystal structure are colored blue. VP30 amino acids within 5 Å of EBOV NP 602–614 are indicated in purple.
Fig 2.
Cross-species binding of filovirus VP30 and NP.
ITC experiments were carried out using EBOV, SUDV or MARV VP30 CTD (ebolavirus 139–288 or Marburgvirus 146–281) and EBOV, SUDV or MARV NP peptide (ebolavirus 600–617 or Marburg virus 552–569) fused to T4 lysozyme. Equilibrium dissociation constants (KDs) are presented in micromolar and are the average of three replicates. Species-matched pairs are indicated in orange and intra-genus pairs in blue.
Fig 3.
The NP 600–620 peptide is essential for mini-replicon activity.
Mammalian cells were transfected with plasmids encoding the Ebola virus RNA synthesis components NP, VP35, VP30 and L, along with a model viral mini-replicon encoding firefly luciferase. Full-length NP was exchanged with either NP 1–600 or 1–620 in the presence or absence of VP30. A full-length NP mutant, in which the conserved residues from 605–612 had been scrambled to 605-PVARPYAP-612, was also included. Negative controls for the assay included transfections lacking the viral polymerase (-L) or the mini-replicon RNA (-3E5E-ffLuc) and a no-DNA mock transfection (-DNA). A) Data is shown as the percent luciferase activity compared to the wild-type positive control (left-most sample) and is the average of three independent experiments. B) Western blot for NP, VP35 and VP30 of cleared cellular lysates transfected with the mini-replicon components. All truncated and mutant NPs expressed at levels similar to wild-type NP.
Table 1.
X-ray diffraction data collection and processing statistics.
Fig 4.
Filovirus NP binds to VP30 in a shallow hydrophobic cleft.
A) In the crystal structure, EBOV VP30 CTD forms a dimer of globular domains (cartoon). NP 602–614 binds in a shallow cleft on the VP30 globular domain distal to the dimeric interface (sticks). B) and C) The NP-binding site on VP30 is mostly hydrophobic. D) MARV VP30 CTD also forms a dimer of globular domains and binds MARV NP peptide in the same location as EBOV. E) and F) The MARV NP binding site is also hydrophobic and contains many similar interactions with EBOV. The MARV NP peptide turns N-terminal of P558 to present a different conformation.
Fig 5.
A previously determined EBOV VP30 CTD structure clashes with the NP peptide.
Previously determined VP30 CTD structures as well as the VP30 CTD-NP peptide structures described here were structurally aligned using PyMol [27]. The EBOV NP peptide (602–614) bound to VP30 CTD is in yellow and shown as sticks. NP peptide binding is incompatible with the conformation of the VP30 CTD in 2I8B.pdb [11].
Table 2.
EBOV VP30 mutations to the NP-binding site alter the affinity of the interaction.
Fig 6.
Co-immunoprecipitation of VP30 viral interaction partners.
Immunoprecipitation of HA-tagged VP30 and VP30 mutants with A) NP or B) VP35. Immunoprecipitation of VP30 with C) FLAG-tagged VP35 1–80 and monomeric NP or D) with FLAG-tagged VP35 80–340 and oligomeric NP-RNA complexes.
Fig 7.
Localization of VP30 to NP inclusions.
eGFP-EBOV NP was cotransfected with HA-tagged EBOV VP30 or VP30 mutants DNA constructs into 293T cells. VP30 was detected with a mouse anti-HA primary antibody and a goat anti-mouse, Alexa Fluor 647-conjugated secondary antibody. Nuclei were stained with Hoechst 33342. EBOV NP forms punctate inclusions in transfected cells.
Fig 8.
Mutation of the VP30 NP-binding site alters replicase activity.
A) Mini-replicon activities are presented as a percentage of the firefly luciferase activity observed with wild-type VP30. Dissociation constants observed in ITC experiments are listed for each mutant. B) Western blot for NP, VP35 and VP30 of cleared lysates from cells transfected with the mini-replicon system, confirming similar levels of expression compared for VP30 mutants to wild-type.
Fig 9.
Analysis of RNA levels shows the VP30-NP interaction to be a general modulator of RNA synthesis activity.
RNA from mini-replicon transfected cells was reverse transcribed using primers specific for vRNA (blue), cRNA (red) or mRNA (green). Quantitative PCR was performed with primers amplifying the firefly luciferase ORF common to all RNA types. VP30 mutants are indicated and are arranged from high affinity (left) to low affinity (right) binders. Wild-type (WT) and negative controls (-L, -3E5E-ffLuc, -DNA) are presented for comparison. Data are normalized to the wild-type control samples.
Fig 10.
The gradient of VP30-NP interaction affinities shows two phases of RNA synthesis activity.
Increasing affinity (decreasing KD) of the VP30-NP interaction (blue) from wild-type (WT), as assessed by multiple assays (see Table 2, Figs 4 and 7), represses viral RNA synthesis activity (pink) (Figs 8 and 9) while mildly decreasing affinity activates viral RNA synthesis. A transition occurs beyond the affinity of the VP30 D202R mutant (dashed line) such that further reductions in affinity result in diminished RNA synthesis activity reflecting the essential nature of the VP30-NP interaction to the RNA synthesis complex.